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THESIS
SUBMITTED TO
UNIVERSITY OF LUCKNOW
FOR THE DEGREE OF
2014
By
Doctor Of PhilosophyIN
BIOCHEMISTRY
&
DEPARTMENT OF BIOCHEMISTRY
UNIVERSITY OF LUCKNOW
LUCKNOW- 226007 (INDIA)
STUDIES ON BLOOD LYMPHOCYTE EXPRESSION
PROFILES OF MIXED FUNCTION OXIDASES AS
BIOMARKER OF EXPOSURE TO DIESEL
EXHAUST PARTICLES
Ankita Srivastava
DEVELOPMENTAL TOXICOLOGY DIVISIONCSIR-INDIAN INSTITUTE OF TOXICOLOGY RESEARCH
LUCKNOW – 226001
Dedicated to My Loving Mother
CERTIFICATE
This is to certify that the research work presented in this thesis entitled
“Studies on blood lymphocyte expression profiles of mixed
function oxidases as biomarker of exposure to diesel exhaust
particles” submitted to the Department of Biochemistry, University
of Lucknow, Lucknow in fulfillment of the award of the degree of
Doctor of Philosophy embodies the original research work carried out
by Mrs. Ankita Srivastava, under our joint supervision and has not
been submitted in part or full for the award of any degree or diploma of
this or any other University. It is further certified that he has fulfilled all
the requirements for the degree, regarding the nature and prescribed
period of work.
Dr. U.N. Dwivedi Professor
Department of Biochemistry University of Lucknow Lucknow-226007
Dr. D. Parmar Deputy Director and Head
Developmental Toxicology Division CSIR-Indian Institute of Toxicological Research
Lucknow-226001
ACKNOWLEDGEMENT
I fail to gather words to express my deep sense of gratitude and heartfelt
gratefulness to my supervisor and Mentor Dr. Devendra Parmar, Head,
Developmental Toxicology Division, IITR, Lucknow, a researcher of an
incomparable power of observation and a person of profound intellect. His
creative and expert guidance, constant encouragement, incessant discussion,
attention to detail untiring help, and the kindness which he always bestowed
on me have paved way for the successful completion of this endeavor. His
deep patience, caring ways and humane nature have gone a long way in
helping me to achieve my goal.
I feel privileged in expression of my deep gratitude to my University supervisor
Prof. UN Dwivedi, Department of Biochemistry, Lucknow University, Lucknow,
whose constant enlightened guidance, encouragement, untiring help, and the
kindness which he always bestowed on me have paved way for the
successful completion of this endeavor. It may not be an exaggeration to say
that without his helping attitude, innovative ideas and moral support it was
impossible to carry out this work.
I am also grateful to Prof. R.K. Mishra, Head, Department of Biochemistry
Lucknow University, Lucknow, for providing unflinching cooperation during the
course of this study.
I wish to convey my sincere thanks to Dr. K. C. Gupta, Director, IITR, for
providing the excellent research facility in the institute. With sincere
reverence, I am also thankful to the former director of IITR, Lucknow, Dr.
Ashwani Kumar, for giving his kind approval to carry out my research work at
this institute.
I feel fortunate to be associated with Dr. A.K. Agrawal (Scientist Emeritus), Dr.
V.P. Sharma, Dr. Alok Dhawan, Dr. V.K. Khanna, Dr. M.P. Singh, Dr. A.B.
Pant, Dr. Chetna Singh, Dr. Sanghamitra Bandyopadhyay, Dr. Alok K.
Pandey, Dr. Rajnish Chaturvedi, Dr. Sanjay Yadav, Scientists, IITR and
thankful for their luminary guidance, unflinching cooperation and helpful
suggestions during the course of this investigation. It is mentionable that their
practical advice and a good sense of humor made the lab a pleasant place to
work in.
I am glad to place on record my thanks to all worthy colleagues and friends,
Ashu, Parag, Arvind, Madhu, Anwar, Munindra, Sunistha, Saurabh, Amit,
Anshuman, Shailendra, Abhishek, Hasan, Ankita, Nilay, Prabhat, Dinesh,
Parul, Vivek, Ritesh, Ashutosh, Rajendra, Ajay, Rajesh, Shikha, and late
Yogesh.
My thanks are also due to Mr. B.S. Pandey and Mr. Sohan Lal for their
technical assistance and Mr. Alok for his help in day-to-day works of the lab.
I am also thankful to Defence Research & Development Establishment,
Gwalior for their financial support and to all the patients and controls that have
been enrolled and participated in my study to make it a fruitful event.
This thesis would not have been possible without the support of my brother
and mother. They have a tender loving heart and exceptional academic and
managerial skills from whom I have learnt a lot. I am grateful indebted, for all
the love and support they have provided me throughout my life.
Words will be inadequate to express thanks to my husband Dharmendra and
daughter Ashvikaa who contributed significantly with their love, affection and
moral supports.
(Ankita Srivastava)
(i)
TABLE OF CONTENTS
PAGE
NO.
Certificate
Acknowledgements
List of Tables v-vi
List of Figures vii-vii
List of Symbols and Abbreviations ix-x
Introduction xi-xiii
CHAPTER 1: REVIEW OF LITERATURE 1-42
1.1 Components of the MFO system 2
1.2 Cytochrome P450s 4
1.2.1 CYP1 family 7
1.2.2 CYP2 family 8
1.2.3 CYP3 family 9
1.2.4 CYP4 family 10
1.3 Regulatory mechanisms for Xenobiotic metabolizing
CYPs
11
1.4 Glutathione S-Transferase 17
1.5 Modulation of activity of MFOS: Possible markers of
toxicity
21
1.6 Expression profiles of CYPs in peripheral blood
lymphocytes
22
1.7 Biomarkers for monitoring toxicity of vehicular
emissions including diesel exhaust particles
30
1.7.1 Composition and physicochemical
properties of DEP
31
1.7.2 Ambient & occupational DEP exposure 32
1.7.3 DEP deposition & clearance 33
1.7.4 Epidemiological Studies 33
1.7.5 Role of CYPs in pulmonary toxicity by DEP
exposure
35
1.7.6 Evidence of oxidative stress induced by
DEP via ROS generation
37
1.7.7 ROS mediated by transition metals present
on DEP
39
1.7.8 Inflammatory response to DEP exposure 40
1.7.9 Mechanistic pathway for inflammation after 40
(ii)
DEP exposure
1.8 Peripheral Blood Lymphocytes: A tool for predicting
toxicity of DEP
41
CHAPTER 2: Gene expression profiling of candidate genes in
peripheral blood lymphocytes for predicting toxicity of
diesel exhaust particles
43-60
2.1 INTRODUCTION 43
2.2 MATERIALS AND METHODS 45
2.2.1 Chemicals 45
2.2.2 Animals and treatment 45
2.2.3 RNA Extraction 46
2.2.4 TaqMan Low Density Array (LDA) Analysis 46
2.2.5 Statistical Analysis 47
2.3 RESULTS 48
2.4 Discussion 54
CHAPTER 3: Similarities in diesel exhaust particles induced
alterations in expression of cytochrome P-450 and
glutathione S-transferases in rat lymphocytes and
lungs
61-95
3.1 INTRODUCTION 61
3.2 MATERIALS AND METHODS 62
3.2.1 Chemicals 62
3.2.2 Animals and treatment 63
3.2.3 Isolation of blood lymphocytes 63
3.2.4 Preparation of microsomes 63
3.2.5 Protein estimation 64
3.2.6 EROD and MROD assay 64
3.2.7 PROD assay 65
3.2.8 NDMA(d) assay 65
3.2.9 GST assay 65
3.3 GSH assay 66
3.3.1 Lipid peroxidation assay 66
3.3.2 RNA isolation 67
3.3.3 Semi-quantative RT-PCR analysis 67
3.3.4 Quantitative Real time-PCR (RT-PCR)
analysis
67
3.3.5 Immunoblot analysis 68
(iii)
3.3.6 Immunocytochemistry 68
3.3.7 Statistical analysis 69
3.4 RESULTS 69
3.4.1 Effect of DEP on drug metabolizing
enzymes in rat lungs
69
3.4.2 Effect of DEP on drug metabolizing
enzymes in rat PBL
71
3.4.3 Effect of DEP on lipid peroxidation and
total glutathione (GSH) content
73
3.4.4 Effect of DEP on protein expression of
CYP1A1/1A2 isoenzymes
74
3.4.5 Effect of DEP on protein expression of
CYP2E1
75
3.4.6 Immunocytochemical localization of CYP
1A1/1A2 in PBLs after DEP treatment
76
3.4.7 Immunocytochemical localization of
CYP2E1 in PBLs after DEP treatment
77
3.4.8 Semi-quantitative Reverse Transcriptase
(RT)-PCR analysis
77
3.4.8.1 mRNA expression of CYP1A1 78
3.4.8.2 mRNA expression of CYP1A2 80
3.4.8.3 mRNA expression of CYP1B1 81
3.4.8.4 mRNA expression of transcription factors
(AhR and Arnt)
82
3.4.8.5 mRNA expression of CYP2E1 84
3.4.8.6 mRNA expression of GST isoforms
(GSTPi,GSTM1 and GSTM2)
84
3.4.9 Quantitative Real-time PCR (qRT-PCR)
studies in rat lung
86
3.5 Quantitative Real-time PCR (qRT-PCR)
studies in PBL
87
3.5.1 Quantitative mRNA expression of GST
Isoforms in rat lung
88
3.5.2 Quantitative mRNA expression of GST
Isoforms in rat PBL
89
3.6 DISCUSSION 90
(iv)
CHAPTER 4: Similarities in DEP induced alterations in Xenobiotic
metabolizing enzymes and DNA damage in blood
derived and lung derived cell lines
96-115
4.1 INTRODUCTION 96
4.2 MATERIALS AND METHODS 97
4.2.1 Particle preparation and characterization 97
4.2.2 Cell culture and treatment 97
4.2.3 Exposure 98
4.2.4 MTT assay 98
4.2.5 Comet assay 99
4.2.6 Quantitative Real Time-PCR (qRT-PCR)
Analysis
100
4.2.7 Enzymatic analysis 100
4.2.8 Immunocytochemical analysis 100
4.2.9 Determination of ROS 100
4.3 Statistical analysis 101
4.4 RESULTS 101
4.4.1 Particle Characterstistics 101
4.4.2 Cytotoxicity 101
4.4.3 DEP induced DNA damage in A549 and
IM9 cells
103
4.4.4 Effect of pretreatment of CYP1- modifiers
on DEP mediated induction of CYP mRNA,
protein expression and associated enzyme
activity
104
4.4.5 Involvement of reactive oxygen species in
DEP induced toxicity
108
4.4.6 Role of CYP1A1 in DEP mediated DNA
damage
109
4.5 DISCUSSION 111
SUMMARY
REFERENCES
LIST OF PUBLICATIONS
116-121
122-153
154
(v)
LIST OF TABLES
TABLE
NO.
TITLE PAGE
NO.
1.1 Reactions performed by mixed function oxidase system 2
1.2 Relative mRNA expression of human xenobiotic metabolizing
CYPs and their transcription factors in hepatic and extrahepatic
tissues
6
1.3 CYP induction and prototypic inducers 11
1.4 Classification of Human GSTs. 19
2.1 DEP induced alterations in mRNA expression of candidate
genes in lungs and PBL
49-50
2.2 Relative quantification of candidate genes in lungs and PBL of
control and DEP treated rats
51-53
3.1 Effects of transtracheal instillation of DEP on the activity of
drug metabolizing enzymes in lungs
71
3.2 Effects of transtracheal instillation of DEP on the activity of
drug metabolizing enzymes in peripheral blood lymphocytes.
72
3.3 Effects of transtracheal instillation of diesel exhaust particles
on GSH content and lipid peroxidation in lungs and peripheral
blood lymphocytes
74
3.4 Effects of transtracheal instillation of DEP on the relative
mRNA expression of CYP isoenzymes, AhR and ARNT in rat
lungs
87
3.5 Effects of transtracheal instillation of DEP on the relative
mRNA expression of CYP isoenzymes, AhR and ARNT in rat
PBL
88
3.6 Effects of transtracheal instillation of DEP on the relative
mRNA expression of GST isoenzymes in rat lungs
89
3.7 Effects of transtracheal instillation of DEP on the relative
mRNA expression of GST isoenzymes in rat PBL
90
(vi)
4.1 Cytotoxicity, as assessed by MTT assay in various
concentrations of DEP in A549 cells
102
4.2 Cytotoxicity, as assessed by MTT assay in various
concentrations of DEP in IM9 cells
102
4.3 Cytotoxicity, as assessed by MTT assay in various
concentrations of MC in A549 & IM9 cells
102
4.4 Cytotoxicity, as assessed by MTT assay in various
concentrations of α-NFin A549 & IM9 cells
103
4.5 Effect of diesel exhaust particles on DNA damage as assessed
by comet assay in A549 and IM9
104
4.6 Effects induced by 3-MC, DEP and α-NF on the relative mRNA
expression of CYP 1A1 and 1B1 in A549 and IM9
105
4.7 Effects induced by 3-MC, DEP and α-NF on the EROD activity
in human A549 and IM9 cell line
106
4.8 Reactive oxygen species production (ROS) by 3-MC, DEP,
MC+DEP and MC+DEP+ α-NF in cultured A549 and IM9 cell
line
109
4.9 Genotoxic effect induced by CYP1A1 inducer 3-MC and diesel
exhaust particles in A549 cells with and without CYP1A1
inhibitor alpha naphthoflavone
110
(vii)
LIST OF FIGURES
FIGURE
NO.
TITLE PAGE
NO.
1.1 Classical scheme of phase I and phase II Drug metabolizing
enzymes (DMEs)
3
1.2 The Cytochrome P-450 Catalytic Cycle 5
1.3 Induction of CYP family members mediated by AhR 12
1.4 Induction of CYP2 Family members by CAR 14
1.5 Induction of CYP3 family members mediated by PXR 16
1.6 Induction of CYP4 family members mediated by PPAR 17
1.7 Composition of DEP 31
3.1 Western blot analysis of rat lung protein with anti CYP1A1/1A2 75
3.2 Western blot analysis of rat lung proteins isolated from control
and DEP treated rats with polyclonal antibody raised against rat
liver CYP2E1
76
3.3 Immunocytochemical detection of CYP1A1/CYP1A2 in rat blood
lymphocytes isolated from control and DEP treated rats (15
mg/kg)
76
3.4 Immunocytochemical detection of CYP2E1 in rat blood
lymphocytes isolated from control and DEP treated rats (15
mg/kg)
77
3.5 Ethidium Bromide-stained agarose showing β-Actin mRNA in
lung and lymphocyte of rats pretreated with Diesel Exhaust
Particles
78
3.5.1 Ethidium Bromide-stained agarose showing CYP1A1 mRNA in
lung and lymphocytes of rats pretreated with DEPs
79
3.5.2 Ethidium Bromide-stained agarose showing CYP1A2 mRNA in
lung and lymphocytes of rats pretreated with DEPs
80
3.5.3 Ethidium Bromide-stained agarose showing CYP1B1 mRNA in
lung and lymphocytes of rats pretreated with DEPs
81
(viii)
3.5.4 Ethidium Bromide-stained agarose showing AhR mRNA in lung
and lymphocytes of rats pretreated with DEPs
83
3.5.5 Ethidium Bromide-stained agarose showing ARNT mRNA in
lung and lymphocytes of rats pretreated with DEPs
83
3.5.6 Ethidium Bromide-stained agarose showing CYP2E1 mRNA in
lung and lymphocytes of rats pretreated with DEPs
84
3.5.7 Ethidium Bromide-stained agarose showing GSTpi mRNA in
lung and lymphocytes of rats pretreated with DEPs
85
3.5.8 Ethidium Bromide-stained agarose showing GSTM1mRNA in
lung and lymphocytes of rats pretreated with DEPs
86
3.5.9 Ethidium Bromide-stained agarose showing GSTM2 mRNA in
lung and lymphocytes of rats pretreated with DEPs
86
4.1 Size distribution and zeta potential of diesel exhaust particles in
DMEM medium
101
4.2 Immunocytochemical detection of CYP1A1 in A549 cell line
isolated from (A) control, (B) α-NF, (C) DEP,(D) MC (E)
MC+DEP(F) MC+DEP+α-NF treated cells
107
4.3 Immunocytochemical detection of CYP1A1 in A549 cell line
isolated from (A) control, (B) α-NF, (C) DEP,(D) MC (E)
MC+DEP(F) MC+DEP+α-NF treated cells
108
(ix)
LIST OF SYMBOLS AND ABBREVIATIONS
AHH : Aryl hydrocarbon hydroxylase
AhR : Aryl hydrocarbon receptor
Arnt : Aryl hydrocarbon nuclear translocator
BAD : Bcl2-associated agonist of cell death
BBC3 : Bcl-2-binding component 3
BCIP : 5-Bromo 4-chloro-3-indolyl phosphate
Bcl2 : Bcl2-associated X protein
BID : BH3 interacting domain
BSA : Bovine serum albumin
CAR : Constitutive androstane receptor
Casp 1 : Caspase1
Casp 3 : Caspase3
c-FOS : FBJ murine osteosarcoma viral oncogene homolog
CCL2 : Chemokine (C-C motif) ligand 2
CCL5 : Chemokine (C-C motif) ligand 5
CYP : Cytochrome P450
DMSO : Dimethyl sulfoxide
DTT : Dithiothreitol
EDTA : Ethylene diamine tetraacetic acid
ER : 7-Ethoxyresorufin
EROD : 7-Ethoxyresorufin O-deethylase
FOSl1 : FOS like antigen 1
GST : Glutathione S-transferase
HIF1α : Hypoxia inducible factor,alpha
ICAM1 : Intercellular Adhesion Molecule 1
IL : Interleukin
LDA : Low density array
MAPK : Mitogen activated protein kinase
MC : 3-Methylcholanthrene
mRNA : Messenger RNA
MFO : Mixed function oxidase
MgCl2 : Magnesium chloride
MT : Metallothionein
NADH : Nicotinamide adenine dinucleotide
NADPH : Nicotinamide adenine dinucleotide phosphate
NBT : Nitro blue tetrazolium
NDMA-d : N-nitrosodimethylamine demethylase
NOS2 : Nitric oxide synthase 2
OD : Optical density
(x)
OGG1 : 8- Oxoguanine glycosylase
OH : Heme oxygenase
PAH : Polycyclicaromatic hydrocarbon
PARP : Poly ADP ribose polymerase
PB : Phenobarbital
PBREM : Phenobarbital responsive enhancer element
PBL : Peripheral blood lymphocytes
PBMC : Peripheral blood mononuclear cell
PBS : Phosphate buffered saline
PCB : Polychlorinated biphenyls
PCN : Pregnenolone 16α-carbonitrile
PCNA : Proliferating cell nuclear antigen
PCR : Polymerase chain reaction
PDCD8 : Programmed cell death 8
PMSF : Phenyl methyl sulfonyl chloride
PPARα : Peroxisome proliferator activated receptor α
PR : Pentoxyresorufin
PRDX2 : Peroxyredoxin
PTGS2 : Prostaglandin-endoperoxide synthase 2
PXR : Pregnane X receptor
qRT-PCR : Quantitative real time-polymerase chain reaction
ROS : Reactive oxygen species
RT-PCR : Reverse transcriptase-polymerase chain reaction
SDS-PAGE : Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Sod : Superoxide dismutase
TBS : Tris buffered saline
Tp53 : Tumor protein 53
TCDD : 2,3,7,8-tetrachlorodibenzo- p-dioxin
TGF-β : Transforming growth factor beta
Top2A : Topoisomerase 2A
VCAM1 : Vascular cell adhesion protein 1
XME : Xenobiotic metabolizing enzyme
XRE : Xenobiotic response element
α-NF : α-naphthoflavone
β-NF : β-naphthoflavone
μl : Micro litre
μM : Micro Molar
Introduction
(xi)
INTRODUCTION
The mixed function oxidase systems (MFO) are a battery of enzymes
including primarily, the cytochrome P450s (CYPs), which are embedded in the
smooth endoplasmic reticulum of the cells. CYP enzymes are a superfamily
of hemeproteins that serve as terminal oxidases in the mixed-function oxidase
system and catalyzes the introduction of an oxygen atom into lipophilic
substrates during the first phase of steroid, fatty acid, or xenobiotic
metabolism including drugs, toxins, and carcinogens. A major function of CYP
catalyzed reactions is to convert a compound into a more polar metabolite
that can be easily excreted directly by the organism or conjugated with
endogenous substrates, catalysed by phase II enzymes into more polar
excretable metabolites. Glutathione S-transferase (GSTs) are the major
phase II enzymes that play a critical role in providing protection against
reactive electrophilic species and products of oxidative stress.
The use of diesel engines has been steadily increasing because of fuel
efficiency and low levels of carbon dioxide emissions. However, diesel
engines emit 30-100 times more particulate matter than other engines,
making diesel exhaust particles (DEP) one of the major components of
airborne particulate matter in urban and industrialized areas.DEP exposure
may be a serious health risk to exposed individuals in both environmental and
occupational settings. DEPs are constituents of particulate matter (PM2.5) in
the atmosphere and are involved in pulmonary disorders. Basically, DEPs are
carbon based particles containing approximately 30% by weight various
organic compounds, including polycyclic aromatic hydrocarbons (PAH), nitro-
aromatic compounds, quinones and aldehydes and heterocyclic compounds,
adsorbed onto a carbonaceous core. PAHs present in DEPs are known to
induce the expression of certain CYPs, involved in their metabolism, in rat
lungs and liver. Similar increase in the expression of specific forms of GSTs
has been reported after exposure of DEP in the target tissues. Inhalation and
intratracheal instillation of DEP and organic extracts of DEP (OE-DEP) have
been shown to cause lung inflammation, aggravation of asthmatic symptom,
lung cancer, and electrocardiographic alterations. Studies have also shown
that the organic component of DEP, which generates ROS through interaction
(xii)
with microsomal enzymes, is involved in pulmonary inflammation. DEP
exposure is reported to lead to DNA damage and tumour induction through
the production of 8-hydroxyguanosine (8-OHdG).
In recent years, there has been a significant interest in developing
assays that can be used as indicators/ biomarkers of the extent and
persistence of effects caused by exposures to toxic agents. As blood is easily
available, it has been used to investigate many biochemical and genetic
biomarkers. In several human and animals studies, effects on biomarkers in
blood cells have been shown to be associated with known or suspected
exposure to genotoxic carcinogens. Smoking related PAH-DNA adducts in
human lymphocytes are a good dosimetric exposure marker and these have
been shown to be higher in lymphocytes than the other blood cells. The
induction of genes whose products function in detoxification processes is a
well-characterized cellular response to chemical challenge. Advantage has
been taken of this response by developing molecular assays that can detect
inducible gene expression in peripheral blood lymphocytes, which possess a
full complement of genes, whose products are involved in the metabolism and
detoxication of xenobiotics. Lymphocytes have been shown to express
several members of CYP and GST and gene family. Although CYPs have
been used as a biomarker of susceptibility with the individuals having variant
genotypes to be at greater risk to the toxicity of carcinogens, interest has
recently been centered to develop and validate CYP and GST mRNA
expression profiles as a biomarker to predict exposure of the environmental
toxicants and their effects.
Studies were therefore initiated to develop the expression profiles of
CYPs and GSTs in peripheral blood lymphocytes as a surrogate to monitor
tissue expression of these enzymes in rats exposed to DEPs, which are
known to affect the expression of the carcinogen metabolizing CYPs and
GSTs in rat lung. Studies were therefore planned with the following objectives:
i. Using taqman low density array (TLDA) based Real time PCR (RT-PCR) of
pathway focussed genes, identify the DEP responsive genes in freshly
prepared peripheral blood lymphocytes and lungs in rats exposed to different
doses of DEP.
(xiii)
ii. Characterize the expression of PAH-metabolizing CYPs and GST isoenzymes
in freshly prepared blood lymphocytes isolated from rats exposed to DEP and
investigate similarities or differences, if any, in blood lymphocyte enzymes
with tissue (lung) enzyme.
iii. Explore the role of PAH-metabolizing CYPs in the DEP induced toxicity in
blood and tissue derived cell lines.
Investigation of the similarities and differences, if any, in the blood
lymphocyte expression profiles of drug metabolizing enzyme with tissue
enzymes will help in identifying the suitability of use of blood lymphocytes as a
possible biomarker for predicting exposure and toxicity of diesel exhaust
particles.
Chapter 1
Review of Literature
1
CHAPTER 1
REVIEW OF LITERATURE
The Mixed Function Oxidase (MFO) system is a battery of enzymes located in
the smooth endoplasmic reticulum of the cells and that performs different
functionalisation reactions (Gram, 1973; Parke, 1975; Andrews et al., 1976;
Ioannides and Parke, 1987) (table 1.1). Though MFOs are present in almost all
the mammalian tissues, liver is primarily the tissue that exhibit maximum activity
of the MFOs (Krishna and Klotz, 1994; Pelkonen et al., 2008). MFO catalyzes
the oxidation, and reduction of numerous endogenous and exogenous
substances of widely diverse chemical structure. In addition, they are also
involved in the biosynthesis of cholesterol, steroid hormones, bile acids and the
oxidative metabolism of fatty acids, lipophilic drugs and other chemical (Chen et
al., 1999; Shou et al., 1997; Lee, 1998). MFOs are involved in the oxidation of a
wide range of substrates by incorporation of oxygen into the substrate. Since
MFOs are involved in the incorporation of only one of the two atoms of molecular
oxygen, the corresponding enzymes are categorised as monoxygenases and the
reaction being known as MFO reaction or monooxygenase reaction (Sato et al.,
1978). The MFO reaction can also be represented by the following equation:
In this reaction, RH represents an oxidisable substrate and ROH, the
hydroxylated metabolite. As can be seen from the above reaction, reducing
equivalents (derived from NADPH + H+) are consumed and only one atom of the
molecular oxygen is incorporated into the substrate (generating the hydroxylated
metabolite), whereas the other oxygen atom is reduced to water (the reaction is
actually a hydroxylation rather than a genuine oxidation).
MFOs are primarily concerned with detoxification involving the formation of more
polar, readily excretable metabolites (Ioannides et al., 1984, 1987; King et al.,
1999). Paradoxically, however, the same mixed-function oxidase system can
lead to the formation of more toxic and reactive intermediates, a process known
as "metabolic activation or bioactivation”. Activation of mixed function oxidase
enzymes by various agents has been shown to lead to toxic effects because of
2
the formation of mutagenic or carcinogenic metabolic intermediates (Ioannides et
al., 1984, 1987; Sheweita, 2000).
Table 1.1: Reactions performed by mixed function oxidase system
Reaction Substrate
Aromatic hydroxylation Lignocaine
Aliphatic hydroxylation Pentobarbitone
Epoxidation Benzo(a)pyrene
N-Dealkylation Diazepam
O-dealkylation Codeine
S-dealkylation 6-methylthiopurine
Oxidative deamination Amphetamine
N-oxidation 3-methylpyridine
2-acetalaminofluorene
S-oxidation Chlorpromazine
Phosphothionate oxidation Parathione
1.1 Components of the MFO system
MFO system contains many enzyme including cytochrome P450 (CYP),
NADPH-cytochrome P450 reductase, cytochrome b5, and NADH-b5 reductase.
CYPs constitute the major component of MFO system that are involved in the
metabolism and detoxification of majority of drugs and chemicals (Oliw et al.,
1982). Another member of MFO system is NADPH-P450 reductase which
contains one molecule of FAD and FMN per mole of apoprotein (Yamano et al,
1989). CYP accepts its reducing equivalents from the flavoprotein as follows:
This reaction requires the presence of Mg++ (Peters et al., 1970)
The reductase acts as a transducer i.e. moving reducing equivalents sequentially
on to CYP, one electron at a time. The redox state of the flavoproteins during
oxidation is not known. However it is believed that FAD accepts the reducing
equivalents from NADPH + H+ and FMN donates them to CYP (Poulos et al.,
1992).
3
MFOs also contain cytochrome b5 and NADH- b5 reductase. Cytochrome b5
(b5) and NADH-b5 reductase (b5R) are ubiquitous electron transport proteins. In
endoplasmic reticulum, cytochrome b5 plays important roles in maintenance of
normal cellular functions by transferring electrons to microsomal desaturase
enzymes that synthesize unsaturated fatty acids, plasmalogens, and cholesterol
(Vergeres and Waskell, 1995). NADH-b5 reductase is responsible for
transferring electrons from NADH to b5 in these reactions (Yamazaki et al.,
1996), although NADPH can also be used as the electron donor in some cases.
The major enzymes of the MFO system which are involved in Phase I metabolic
processes are superfamilies of the members of terminal monoxygense of the
MFO system i.e. CYPs. The CYP system ranks first among the Phase I
detoxification system in terms of catalytic versatility and for the drug candidates
or xenobiotics it detoxifies or activates. Phase I reactions involves direct enzyme
mediated changes of molecules, like oxidation, reduction and hydrolytic
cleavages (Fig.1.1). While the major function of CYP catalyzed reactions is to
convert a compound into a more polar metabolite that can be either excreted
directly by the organism or be conjugated by phase II enzymes and eliminated
from the body. The phase II enzymes consist superfamily of enzymes including
glutathione S-transferases (GST), sulfotransferases (SULT) and UDP-
glucuronosyltransferases (UGT) NAD(P)H:quinone oxidoreductase (NQO) or
NAD(P)H menadione reductase (NMO), epoxide hydrolases (EPH) and N-
acetyltransferases (NAT) (Xu et al., 2004).
Figure 1.1: Classical scheme of phase I and phase II Drug metabolizing
enzymes
4
1.2 CYTOCHROME P450s (CYPs)
CYPs represent a superfamily family of heme-containing monooxygenases
which are crucial for the oxidative, peroxidative and reductive metabolism of a
wide variety of xenobiotics, environmental pollutants as well as endogenous
substrates such as cholesterol, bile acids, fatty acids, prostaglandins and
leukotrines (Anzenbacher and Anzenbacherová, 2001; Guengerich, 2001;
Danielson, 2002; Shimada, 2006; Tamási et al., 2011). CYP enzymes are
expressed ubiquitously in different life forms and are found in animals, plants,
fungi and bacteria and believed to have originated from an ancestral gene that
existed over 3 billion years ago (Nelson et al.,1999; Danielson, 2002). In
eukaryotes, CYPs are majorly localized in endoplasmic reticulum while some are
in mitochondrial membrane of cells (Gonzalez, 1990; Omura, 1999; Lewis, 2001;
Sullivan et al., 2008).
The term „cytochrome P450‟ was coined in 1962 for a coloured substance in the
cell because of its unusual spectral properties which displayed a typical
absorption maximum of the reduced CO-bound complex at 450 nm (Omura and
Sato, 1962). The CYP enzymes often referred to as heme thiolate proteins are
the monooxygenases (mixed function oxidases) that catalyze the incorporation
of an oxygen atom into the substrate and converts them into more water soluble
products (Coon et al., 1998; Vaz, 2001; Yan and Caldwell, 2001; Coon, 2005).
Figure 1.2 summarizes the schematic mechanism of action by CYP, where Fe
represents the heme/iron atom at the active site, RH the substrate, RH(H)2 a
reduction product, ROH a monooxygenation product, and XOOH a peroxy
compound that can serve as an alternative oxygen donor (Porter and Coon,
1991). In this cycle, a substrate binds to the low-spin ferric enzyme, effectively
displacing the water ligand that is coordinated to the central heme and changing
the complex to a high spin-state. As a result of this shift from a low spin to a high
spin state, the complex has a greater reduction potential and is more easily
reduced than the original ligated heme. An oxygen molecule then binds to the
heme centre, forming an oxy-heme complex. The reduction of this complex,
followed by two subsequent protonations and heterolysis of the O-O bond results
in the formation of the original heme enzyme ligated with water and an
oxygenated substrate product (Groves and McClusky, 1978). Dissociation of
ROH then restores the P450 to the starting ferric state (Porter and Coon, 1991).
5
Figure 1.2: The Cytochrome P-450 Catalytic Cycle (Porter and Coon, 1991)
CYPs are notable both for the diversity of reactions that they catalyze, substrates
they metabolize and the wide variety of chemicals which can induce their
expression. CYPs are involved in reactions as diverse as hydroxylation, N-, O-
and S- dealkylation, sulfoxidation, epoxidation, deamination, desulphuration,
dehalogenation, peroxidation, and N-oxide reduction (Table 1.1). Despite the
diversity of reactions catalysed by this enzyme system, all CYPs comprise a
similar structure with amino acid sequence variation. Individual isoforms has
evolved by repeated gene duplications (Nebert et al., 1991; Degtyarenko and
Archakov, 1993; Danielson, 2002). To add uniformity to CYP enzyme
classification, a nomenclature system was adapted in 1996 (Nelson et al., 1996).
Based on amino acid homologies, the CYP superfamily has been classified into
several families and subfamilies (Nebert et al., 1989; Nebert et al., 1991; Nelson
et al., 1996). The CYP proteins with 40% or greater sequence identity are
included in the same family (designated by Arabic number), and those with 55%
or greater identity in the same subfamily (designated by a capital letter) where
the individual genes are numbered arbitrarily. To illustrate this naming system,
CYP2C9*1*2 is taken as an example. In this isozyme designation, CYP is the
standard abbreviation for mammalian cytochrome P450. Families of the isozyme
6
which share greater than 40% protein sequence homology with each other are
designated by the first number following the cytochrome P450 designation e.g.,
2. Subfamilies which share greater than 55% homology with each other are
differentiated by the letter following the family designation e.g., C. Single
members of subfamilies represent a particular gene and are designated by the
number following the subfamily description e.g., 9. An asterisk and a number
following the member description designate an allele e.g., *1 and *2. The *1
allele for CYP2C9, and most others, is known as the wild type and denotes
normal enzyme activity.
TABLE 1.2: Relative mRNA expression of human xenobiotic metabolizing
CYPs and their transcription factors in hepatic and extrahepatic tissues
Gene Small
Intestine
Kidney Lung Placenta Liver
CYP1A1 + + +++ ++/+ ++
CYP1A2 - - +/- +/- +++
CYP1B1 + ++/+ ++/+ + +
CYP2A6 - - ++/+ +/- +++
CYP2A13 +/- +/- + - +++
CYP2B6 ++/+ ++/+ +++ +/- +++
CYP2C9 ++ +/- +/- +/- +++
CYP2C19 ++ +/- +/- +/- +++
CYP2D6 ++/+ + + ++/+ +++
CYP2E1 ++/+ + +++/++/+ + +++
CYP3A4 +++ + +/- +/- +++
CYP3A5 +++/++ ++ +++/++ + +++/++
CYP3A7 +/- + +/- +/- +
PXR ++ +/- - - +++
CAR +/- ++ - +/- +++
FXR +++/++ ++ +/- ++/+ +++
HNF4α +++/++ +++ +/- - +++/++
AhR + ++ +++/++ +++ ++
PPARα ++ ++ + + +++/++
+++: high expression; ++: moderate expression; +: low expression; -:
undetectable expression; NA: data not available (Pavek and Dvorak, 2008).
Of the total CYPs, those found in families 1-4, which are considered as
xenobiotic /drug metabolizing enzymes (XMEs / DMEs) (Waxman, 1999;
7
Ingelman-Sundberg, 2001).The relative hepatic expression of CYPs and the
extrahepatic expression in relation to liver for major xenobiotic metabolizing
CYPs and their transcription factors in intestine, kidney, lung and placenta is
summarized in table 1.2
CYPs that metabolize majority of the xenobiotics belong to the CYP1, 2, 3 and to
a lesser degree, CYP4 families. CYPs present in these gene families are known
to play the important role in hepatic as well as extrahepatic metabolism and
biotransformation. A common feature of these four CYP families is that their
transcription is induced upon xenobiotic challenges to experimental animals and
humans (Table.1.3).
1.2.1 CYP1 family: CYP1 family typified by CYP1A1, CYP1A2 and CYP1B1
are mainly involved in the metabolism of PAHs e.g. MC and halogenated
aromatic hydrocarbons (HAHs) e.g. TCDD. Expression of CYP1A can be
elevated 100 fold or more in liver and many extrahepatic tissues following
exposure to TCDD, 3-MC or other PAHs (Lewis, 1996; Kondraganti et al., 2002).
These CYPs have also shown to be induced by substantial variations between
individuals from a variety of modulating factors, including genetic
polymorphisms, age, gender, disease status, pharmacotherapy, and dietary
factors such as smoking (Willey et al., 1997; Zevin and Benowitz, 1999; Nebert
and Russell, 2002; Vrzal et al., 2004). Importantly, they all are active in the
metabolism of PAHs into intermediates that can bind to DNA and, if the damage
goes unprepared, may produce mutations involved in neoplastic transformation
(Riddick et al., 2003; Shimada and Fujii-Kuriyama, 2004; Baird et al., 2005;
Shimada, 2006).
CYP1A1 is a major extrahepatic CYP enzyme (Obligacion et al., 2006; Casarett
et al., 2008; Thelen and Dressman, 2009). CYP1A1 has been shown to mainly
catalyse the metabolism of relatively large flat- structured aromatic
hydrocarbons. CYP1A1 is not found to be constitutively expressed and is
expressed only after exposure to the inducers (Nebert, 1989; Penman et al.,
1994; Crespi et al., 1997; Bofinger et al., 2001) and exhibits a broad tissue
distribution (Kalow, 1991; Galván et al., 2005; Ito et al., 2007; Uno and Osada,
2011). CYP1A2 constitutes about 13% of the total CYP content and is mainly a
hepatic enzyme (Raunio et al., 1998; Imaoka et al., 1996). Substrates and
8
inhibitors for CYP1A- includes phenacetin, ethoxyresorufin, methoxyresorufin,
caffeine, α- naphthoflavone, furafylline etc (Tucker et al., 2001; Rendic, 2002;
Brandon et al., 2003). Heterocyclic and aromatic amines, certain nitroaromatic
compounds or aflatoxin B1 (which may, however, also be present in
contaminated food) are activated by CYP1A2 (Eaton et al., 1995).
Similar to CYP1A1, CYP1B1 is also mainly an extrahepatic CYP expressed in
almost every tissue, including kidney, prostrate, mammary gland, and ovary
(Sutter et al., 1994, Shimada et al., 1996, Tang et al., 1996, 2000). In general,
basal expression of CYP1B1 is higher compared to CYP1A1 (Shimada et al.,
1996; Eltom et al., 1998). It has been suggested that CYP1B1 is overexpressed
in tumours (Rochat et al., 2001; Gibson et al., 2003; Tokizane et al., 2005).
These findings demonstrate that even the CYPs classified as “xenobiotic-
metabolizing” enzymes may have important functions in modulating growth and
differentiation.
1.2.2 CYP2 family: The CYP2 family, the largest and most diverse of the CYP
family, comprises CYP2A, CYP2B, CYP2C, CYP2D and CYP2E (Nelson et al.,
1996; Omiecinski et al., 1999; Du et al., 2004; Cribb et al., 2005; Gresner et al.,
2007). CYP2B isoenzymes are one of the important CYPs that are primarily
involved in the metabolism of foreign chemicals and drugs including
amphetamines and benzodiazepines and are inhibited by metyrapone (Yang et
al., 1998; Martignoni et al., 2006; Kapoor et al., 2007). CYP2B1/2B2, the major
CYPs belonging to CYP2-families, are primarily members expressed in rats
whereas in humans, the CYP2B family includes CYP2B6 and 2B7. CYP2B6 is
expressed in the liver and in some extrahepatic tissues, whereas CYP2B7
mRNA expression was detected in lung tissue (Mimura et al., 1993; Czerwinski
et al., 1994; Muangmoonchai et al., 2001; Martignoni et al., 2006). CYP2B1 is
generally much more catalytically active than CYP2B2. Both are expressed
constitutively in the liver and extrahepatic tissues such as small intestine and
lungs (Lindell, 2003). The CYP2B1 and CYP2B2 proteins exhibit 97% amino
acid similarity (14 substitutions of 491 amino acids) and have distinct
chromatographic and electrophoretic properties (Waxman and Azaroff, 1992).
CYP2B6 expressed in human appears to bioactivate 6-aminochrysene and the
antineoplastic drugs such as cyclophosphamide and ifosfamide (Huang et al.,
9
2000; Wang and Tompkins, 2008). CYP2B1 is non-constitutive and highly
inducible by PB while CYP2B2 is constitutive but it also moderately induced by
PB (Lindell et al., 2003; Caron et al., 2005) and PB-like inducers [e.g.
chlorpromazine, phenytoin, dichlorodiphenyltrichloroethane, 1,4-bis[2-(3,5-
dichloropyridyloxy)]benzene (TCPOBOP) and polychlorinated biphenyls] (Gervot
et al., 1999, Sueyoshi and Negishi, 2001).
CYP2E1: The CYP2E1 enzyme has been studied extensively due to its role in
the metabolism of ethanol and also as an activator of chemical carcinogens
(Lieber, 1997). CYP2E1 activates some tobacco specific nitrosamines (TSNA)
such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-
nitrosonornicotine (NNN) (Yamazaki et al., 1992; Hecht, 1999; Kushida et al.,
2000). Most of the over 70 substrates demonstrated are small and hydrophobic
compounds (Ronis et al., 1996), including only a few pharmaceuticals, such as
paracetamol, chlorzoxazone, enflurane, and halothane (Omicienski et al., 1999).
Disulfiram is a clinically used inhibitor of CYP2E1 (Frye and Branch, 2002).
About 7% of the liver P450 content consists of CYP2E1 (Shimada et al., 1994,
Imaoka et al., 1996). It is also expressed in lung and brain (Raunio et al., 1995a).
In the liver, CYP2E1 metabolize substrates such as ethanol, acetone, acetol,
benzene, pyridine etc. Its activity towards acetone and acetol oxidation suggests
that CYP2E1 is involved in the pathway of gluconeogenesis during the fasting
state (Novak and Woodcraft, 2000; Lieber, 2005). During chemical metabolism,
CYP2E1 generates reactive oxygen species (ROS) such as superoxide anion,
hydrogen peroxide and a powerful oxidant such as 1-hydroxyethyl free radicals
which have been implicated as the causative agents in alcoholic liver diseases
(ALD) (Butura et al., 2009; Jarvelainen et al., 2000). This feature, combined with
the ability of CYP2E1 to convert dioxygen into reactive oxygen radicals which
can promote lipid peroxidation suggests important toxicological implications for
this enzyme. It is also involved in the metabolic activation of many hepatotoxins
such as acetaminophen or hepatocarcinogens such as nitrosamines (Kushida et
al., 2000; Prescott, 2000).
1.2.3 CYP3 family: CYP3 family has only one subfamily i.e. CYP3A. CYP3A
subfamily is one of the most abundant CYP present in the human liver. CYP3A1,
10
CYP3A2, CYP3A9, CYP3A18 and CYP3A23 are expressed in rats and CYP3A4,
CYP3A5, CYP3A7 and CYP3A43 in humans (Nelson et al., 1996). CYP3A
genes are expressed in liver and extrahepatic tissues. CYP3A2 is one of the
major CYP expressed constitutively in rat liver. CYP3A4 is apparently the most
important CYP enzyme for drug metabolism in humans. This is because of its
amount in the liver, (which may be increased by induction to more than 60%),
and it participation in the metabolism of the majority of drugs with known
metabolic pathways. CYP3A enzymes catalyses the metabolism of steroid
hormones and bile acid 6β-hydroxylation reactions. These isoforms also
metabolise a large number of drugs, including erythromycin, cyclophosphamide,
dapsone, lidocaine, midazolam and nifedipine among others (Wilkinson, 1996).
They also activate procarcinogens, including aflatoxin B1 and its fungal derived
relatives (Santacroce et al., 2008). Both glucocorticoids, like dexamethasone
and antiglucocorticoids, like pregnenolone 16α-carbonitrile (PCN) induce these
enzymes at the transcriptional level (Schuetz et al., 1998). While the rat and
human CYP3A genes are all inducible by dexamethasone, the antiglucocorticoid
PCN is an efficacious CYP3A inducer in the rat but not in humans. By contrast,
rifampicin, the antibiotic is an excellent CYP3A inducer in human but not in the
rat (Waxman, 1999).
1.2.4 CYP4 family: CYP4A, CYP4B and CYP4F are the three subfamilies in
CYP4 gene family. CYP4A isoforms (CYP4A1, 4A2, 4A3) are responsible for the
oxygenation of fatty acids, including arachidonic acid and other eucosanoids and
xenobiotics like phthalate esters and other chemicals termed as peroxisome
proliferators. CYP4B isoforms catalyse metabolism of xenobiotics like 2-amino
fluorene, carbontetrachloride, aflatoxin, etc (Xu et al., 2003; Guenrich, 2003).
CYP4F1 and CYP4F2 are expressed in rat and human livers respectively (Chen
and Hardwick, 1993). CYP4F3 is expressed in human leucocytes (Kikuta et al.,
2004). The inductive response of CYP4A has been relatively well characterized.
Both the endogenous compounds and peroxisomal proliferators have been
shown to bind to peroxisome proliferator activated receptor α (PPAR α) and
activate the CYP4A gene transcription (Meredith et al., 2003; Raucy et al.,
2004). The tissue distribution of PPARα, namely liver > kidney > heart > other
11
tissues mirrors the peroxisome proliferators chemical (PPC) responsiveness of
these tissues.
1.3 Regulatory mechanisms for Xenobiotic metabolizing CYPs: Induction
of majority of CYPs induced by due to xenobiotics is usually tissue-specific,
rapid, dose-dependent, and reversible upon removal of inducers. Because of the
ability of drugs and environmental chemicals to modulate the expression of CYP
and/ or stabilization of CYP, they can play a significant role in increasing the rate
of metabolism of foreign compounds to detoxified products or in some cases to
reactive intermediates. Several mechanisms of regulation, including
transcription, translation, and posttranslational modification are involved in the
induction of CYPs (Aguiar et al., 2005). Induction of majority of CYPs induced by
xenobiotics generally involves participation of orphan nuclear receptors (Table
1.3).
Table 1.3: CYP induction and prototypic inducers
CYPs Prototypic inducers CYPs
CYP1A1,CYP1A2,CYP1B1 PAH,TCDD CYP1A1,CYP1A2,CYP1B1
CYP2B1,CYP2B2 Phenobarbital CYP2B1,CYP2B2
CYP2E1 Ethanol, Isoniazid CYP2E1
3A1,3A2, 3A23 Dexamethasone 3A1,3A2, 3A23
4A1,4A2,4A3 Clofibrate 4A1,4A2,4A3
Major mechanism of CYP induction through classical inducers like PB, MC, PCN
and dexamethasone is due to increase in mRNA expression at transcription level
(Okey, 1990; Waxman and Azaroff, 1992; Dogra et al., 1998). Potent inducers of
the CYP1 family include PAHs, such as α-naphthoflavone or halogenated
aromatic hydrocarbons eg. TCDD. These inducers invoke their transcription via a
specific receptor protein, termed the Ah receptor, AhR (Savas and Jefcoate,
1994). The Aryl hydrocarbon receptor (AhR) is a ubiquitous cytosolic protein
that responds to planar aromatic ligands by forming an activated complex with
two molecules of the molecular chaperone hsp90 and the X-associated protein 2
(Ma and Whitlock, 1997; Carver et al., 1998). Upon ligand binding the AhR
undergoes a conformational change and translocates to the nucleus (Fig 1.3).
Once nuclear translocation has occurred, hsp90 is released from the Ah
12
receptor, either at the translocation stage or upon dimerisation with another
bHLH partner, such as aryl hydrocarbon nuclear translocator (ARNT).
This heterodimeric Ah receptor/ARNT complex binds to xenobiotic-responsive
elements located upstream of the CYP1A1 and CYP1B1 genes and promotes
their transcription (Fig. 1.3).
Figure 1.3: Induction of CYP family members mediated by AhR
Constitutive androstane receptor (CAR), pregnane X receptor (PXR/SXR) and
Peroxisome proliferator activated receptor (PPAR) belongs to same nuclear
receptor gene family (NR1) and catalyze the induction of CYP2B-, 2E1, 3A and
4A isoenzymes. These receptors share a common heterodimerization partner,
retinoid X receptor (RXR) and are subject to cross-talk interaction with other
nuclear receptor and with a broad range of other intracellular signaling pathways
(Honkakoski et al., 1998; Kliewer et al., 1999; Waxman, 1999; Burk et al., 2004).
These receptors are called orphan nuclear receptors as their endogenous
ligands are not yet known. There is substantial evidence that CAR and PXR/SXR
regulate the majority of CYP enzymes in the hepatocyte (Moore et al., 2000).
Ligand binding usually activates nuclear receptors and CYP isoforms often
catalyze both formation and degradation of these ligands (Honkakoski and
13
Negishi, 2000). CYPs also metabolize many exogenous compounds, some of
which may act as activators of nuclear receptors and disruptors of endocrine and
cellular homoeostasis (Honkakoski and Negishi, 2000). Thus CYP genes are
uniquely positioned to respond to both endogenous and exogenous signals by
changes in CYP gene expression, and to modulate the strength and duration of
these signals and even to form new signaling molecules through CYP-mediated
metabolism (Honkakoski and Negishi, 2000). These signaling molecules may
then exert their function via the ligand-dependent nuclear receptors. These
nuclear receptors have relatively low specificity and affinity for their ligands so
that a wide range of structurally diverse chemicals can activate them and thus
comprise a broad response mechanism to xenobiotics (Honkakoski and Negishi,
2000).
CAR has been identified as the mediator of induction of CYP2B genes by
Phenobarbital (PB), the classical inducer of drug metabolism (Honkakoski et al.,
1998, Kawamoto et al., 1999; Wang and Negishi, 2003; Pustylnyak et al., 2005).
CAR is a novel orphan nuclear receptor, which was originally characterized as a
constitutive activator of retinoid acid response elements (RARE). CAR acts
differently than the more traditional receptors. Interestingly, CAR resides in the
cytoplasm of hepatocytes, where it is unable to affect gene transcription. On
exposure to PB, CAR translocates from the cytoplasm into the nucleus, where it
forms a heterodimer with the retinoid X receptor (RXR). As shown in fig. 1.4, the
binding of the CAR-RXR heterodimer to nuclear receptor-binding sites in the 5‟-
flanking sequence of the CYP2B genes results in activation of a 51-base pair
phenobarbital responsive enhancer module (PBREM) of the distal promote and
increased transcription (Sueyoshi et al., 1999; Zelko and Negishi, 2000; Swales
and Negishi, 2004). The PBREM sequences are conserved in the mouse, rat,
and human CYP2B genes (Sueyoshi et al., 1999; Zelko et al., 2001; Rencurel et
al., 2005)
14
Figure 1.4: Induction of CYP2 Family members by CAR
In addition to PB, PBREM is capable of responding to various PB-type inducers
while neither 3- MC (CYP1A1 inducer), dexamethasone (CYP3A inducer), nor
clofibrate (CYP4A inducer) activates the PBREM (Honkakoski et al., 1998;
Sueyoshi and Negishi, 2001). Thus, PBREM has emerged as a versatile
response element that is capable of responding specifically to PB and various
other PB-type inducers. It has been proposed that CAR is deactivated in vivo by
endogenous inverse agonist steroids related to androstanol, thus suppressing
CYP2B1/CYP2B6 transcription. This suppression is overcome by agonist binding
to CAR, which abolishes the inhibitory inverse agonist from CAR leading to the
induction of CYP2B1/CYP2B6 (Waxman, 1999; Rushmore and Kong, 2002;
Wang and LeCluyse, 2003).
PXR/ SXR mediates the induction of CYP3A4 in humans (Bertilsson et al., 1998;
Blumberg et al., 1998; Lehmann et al., 1998) and CYP3A7 (Pascussi et al.,
1999). Initially, it was supposed that the inducibility of CYP3A by dexamethasone
was regulated by the glucocorticoid receptor (GR). However, studies have
shown that the nuclear receptor distinct from GR termed as PXR which is
strongly activated by steroids related to pregnenolone, antiglucocorticoids like
15
PCN and other inducers like dexamethasone bind to and transactivate the
CYP3A gene (Kliewer et al., 1998). Characterisation of a human-PXR (hPXR)
indicate that this receptor as well as mouse-PXR (mPXR) heterodimerises with
RXR and efficiently transactivates either DR3 elements, present in CYP3A rat
genes or ER6 elements, present in human (Figure 1.5) (Pascussi et al., 2000;
Goodwin et al., 2002; Kliewer et al., 2002). However, mPXR and hPXR share
only ~75% amino acid sequence in their COOH-terminal ligand binding domain
region as compared to 96% identity between their DNA binding domains and this
may apparently result in significant differences in ligand binding specificities.
hPXR, but not in mPXR, is highly activated by xenochemicals that preferentially
induce human CYP3A genes such as rifampicin, while mPXR but not hPXR
exhibits the strong response to PCN that characterizes mouse CYP3A gene
induction. Thus, the species dependent ligand specificity for CYP3A induction
seen in vivo can be explained by corresponding ligand specificity of each
species of PXR. Other CYP3A inducers and PXR activators include
antihormones belonging to several steroid classes, the organo-chlorine
pesticides trans-nonachlor and chlordane and various nonplanar chlorinated
biphenyls (Lehmann et al., 1998, Schuetz et al., 1998). Interestingly, PXR can
also be activated by PB (Lehmann et al., 1998) suggesting that the effects of PB
on liver CYP genes may be mediated by multiple receptors (eg. CAR for CYP2B
and PXR for CYP3A). The receptors crosstalk with other nuclear receptors or
transcription factors controlling various signaling events thus providing novel
insights into connection of intermediate metabolism with xenobiotic (Willy et al.,
1995; Urquhart et al., 2007; Pascussi et al., 2008; Pavek and Dvorak, 2008).
16
Figure 1.5: Induction of CYP3 family members mediated by PXR
The PPARα mediates the induction of CYP4A (Johnson et al., 1996; Qi et al.,
2000). Upon binding to the ligand, PPARα forms a heterodimer with RXR, which
is activated by 9-cis-retinoic acid (Figure 1.6). The ligand bound heterodimer of
PPARα and RXR binds to a regulatory DNA sequence known as the peroxisome
proliferator response element (PPRE) and enhances the stimulation of CYP4A
by peroxisome proliferator chemicals (PPCs) (Aldridge et al., 1995; Palmer et al.,
1998). PPARα null mice have been shown to be resistant to the induction of
genes encoding CYP4A. Besides PPARα, two other isoforms PPARδ and
PPARγ are also known to exist, which have overlapping but distinct ligand
specificities. Though these receptors do not regulate most of the CYP4A genes
in the liver, they may play some role in regulating PPARα dependent expression
of some CYP4A genes as found in PPARα-null mice (Lemberger et al., 1996;
Honkakoski and Negishi, 2000; Vega et al., 2000; Mandard et al., 2004).
17
Figure 1.6: Induction of CYP4 family members mediated by PPAR
1.4 GLUTATHIONE S-TRANSFERASE (GSTs)
GST isoenzymes (EC: 2.5.1.18) are ubiquitously distributed in prokaryotes
and eukaryotes organisms (Hayes and Pulford, 1995). GSTs catalyse the
conjugation of reduced glutathione (GSH) with compounds that contain an
electrophile centre through the formation of a thio ether bond between the
sulphur atom of GSH and the substrate (Chasseaud, 1979; Mannervik, 1985). In
addition to conjugation reactions, a number of GST isoforms exhibit other GSH
dependent catalytic activities such as reduction of hydroperoxides (Coles and
Ketterer, 1990) and isomerisation of unsaturated compounds (Benson et al.,
1977; Habig and Jacoby, 1980). Apart from catalyzing conjugation reaction,
GSTs also perform noncatalytic function such as sequestering of carcinogens,
intracellular transport of hydrophobic ligands and modulation of signal
transduction pathways (Adler et al., 1999; Cho et al., 2001). Under certain
conditions, conjugation with GST could result in activated metabolites and
increased toxicity (Chen et al., 2000; Kong et al., 2000; Rushmore and Kong,
2002). GST activities have been widely used as potential biomarkers for the
monitoring of environmental pollution (Shailaja and D'Silva, 2003; Cunha et al.,
2007). The substrates of GSTs include halogenonitrobenzenes, arene oxides,
18
quinones, and α, β-unsaturated carbonyls (Hayes et al., 2005). GSTs also
metabolize several endogenous molecules, such as prostaglandins (Bogaards et
al., 1997), steroids (Barycki and Colman, 1997), and the histidine metabolite
urocanic acid (Shimizu and Kinuta, 1998).
GSTs exhibit broad and overlapping substrate specificity (Mannervik and
Danielson, 1988), which makes it difficult to identify and characterize individual
isoforms based solely on catalytic properties. Three major families of proteins
exhibit glutathione transferase activity. Two of these, the cytosolic and
mitochondrial GSTs, comprise soluble enzymes. The third family comprises
microsomal GSTs and is now referred to as membrane-associated proteins in
eicosanoid and glutathione metabolism. Cytosolic and mitochondrial GSTs share
some structural similarities.
Cytosolic GSTs represent the largest family. Mammalian cytosolic GSTs are all
dimeric with subunits of 199–244 amino acids in length. Seven classes of
cytosolic GSTs are recognized in mammalian species, designated alpha (a), mu
(m), omega (o), pi (p), sigma (s), theta (t), and zeta (z) (Coggan et al., 1998;
Hayes and pulford, 1995; Board et al.,1998) (table 1.4) . Other classes of
cytosolic GSTs, namely beta, delta, epsilon, lambda, phi, tau, and the „„U‟‟ class,
have been identified in non mammalian species. In rodents and humans,
cytosolic GSTs within a class typically share > 40% identity and those between
classes share < 25% identity. The mammalian mitochondrial class kappa (k)
GSTs are dimeric with subunits of 226 amino acids. Mouse, rat and human
possess only a single kappa GST (Hayes and Pulford, 1995; Hayes et al., 2005).
Mouse and human alpha-class GSTA4 and mu-class GSTM1 can also associate
with mitochondria and membranes (Gardner and Gallagher, 2001; Raza et al.,
2002; Robin et al., 2003). The fact that microsomal GSTs do not share any
sequence identity with the cytosolic enzymes suggest that they evolved
separately (Hayes and Pulford, 1995).
19
Table 1.4: Classification of Human GSTs.
Superfamily Class Enzymes Substrate
Soluble Alpha GSTA1
GSTA2
GSTA3
GSTA4
CDNB;7-chloro4-nitrobenzo-2 oxa-1,3 diazole; Δ5-
androstene-3,17 dione
CDNB;7-chloro4-nitrobenzo-2 oxa-1,3 diazole;
cumene hydroperoxide
Not determined
Ethacrynic acid; 4-hydroxynonenal; 4-
hydroxydecenal
Soluble Mu GSTM1 CDNB;trans4-phenyl-3buten-2 one;aflatoxin B1
epoxide; trans-stilbene
GSTM2 CDNB;1,2dichloro4-nitrobenzene; aminochrome
GSTM3 CDNB; H2O2
GSTM4 ND
GSTM5 CDNB
Soluble Pi GSTP1 CDNB
Soluble Sigma GSTS1 PGD2 synthase
Soluble Theta GSTT1 Dicloromethane; dibromomethane
GSTT2 1-menaphthyl sulphate; Cumene hydroperoxide
Soluble Zeta GSTZ1 Dichloroacetate; fluoroacetate; malelyacetoacetate
Soluble Omega GSTO1 Thioltransferase;CDNB
Soluble Kappa GSTK1 ND
MAPEG Microsomal
MGST1
CDNB; 7-chloro4-nitrobenzo-2 oxa-1,3 diazole;
Cumene hydroperoxide
MGST1
like 1
PGE2 synthase
MGSTII CDNB; leukotriene C4 synthase; 5-HPETE
MGSTIII leukotriene C4 synthase; 5-HPETE
LTC4S leukotriene C4 synthase
FLAP 5-lipoxygenase activating
protein
To date, 16 cytosolic and 6 microsomal GSTs have been identified. The alpha-
class GSTs are basic proteins, class mu GSTs are neutral proteins, and
members of the pi class are acidic (Hoensch et al., 2002). Less is known about
the remaining classes. In humans, GSTA1, GSTA2, GSTM1, GSTP1, GSTT1,
20
and GSTT2 appear to be the most abundant cytosolic transferases (Hoensch et
al., 2002).
Alpha GST (GSTA): GSTA share approximately 11-12 kb length containing
seven exons (Hayes, 1995). Alpha class GSTs such as GSTA-1 leads to
isomerisation of Δ5andosterone -3, 17 Dione. GSTA2 plays a role in reduction of
cumene peroxide and GSTA4 leads to conjugation of 4-hydroxynonenal with
GSH.
GST Mu (GSTM): GST M is approximately 5kb in length and their isoforms such
as GSTM1 leads to conjugation of trans-stilbene oxide. GSTM2 leads to
conjugation of CDNB with GSH (Ross et al., 1993).
GST Pi (GSTP): Pi GST is about 3kb long containing seven exons and are less
complicated than alpha and mu class multigene families. GSTP leads to
conjugation of benzo(a) pyrene diol epoxide with GSH (Watson et al., 1998).
GST theta (GSTT): GSTT are about 3.7 kb and containing 5 exonic regions
(Board et al., 1998), GSTT1 leads to the conjugation of 1, 2 epoxy-3-(p-
nitrophenoxy) propane with GSH while GSTT2 leads to conjugation of 1-
menaphthyl sulphate with GSH.
GST Zeta (GSTZ): The Zeta class GST spans 10.9 kb and is composed of 9
exons (Blackburn et al., 1999). This class leads to isomerisation of
maleylacetoacetate.
Biological mechanisms responsible for controlling the expression and regulation
of various GST isoforms are complex. A number of well-known responsive
elements, such as glucocorticoid response element, xenobiotic response
element, and antioxidant responsive element or electrophile responsive element
(ARE/EpRE), are thought to mediate transcriptional regulation of GSTs (Van
Bladeren, 2000). However, little is known about which GST isoforms are induced
by the various factors that are known to induce phase I enzymes. Recent studies
have shown that the physiological regulation of biotransformation enzymes may
be under the control of transcription factors that are responsive to an assortment
of endogenous and exogenous activators (Savas et al., 1999; Staudinger et al.,
2001; Xie and Evans, 2001).
21
1.5 MODULATION OF ACTIVITY OF MFOS: POSSIBLE MARKERS OF
TOXICITY
MFO enzyme systems are known to exhibit broad substrate specificity
and are induced by variety of chemicals and endobiotic compounds. However,
CYPs, the major component of MFO system in addition to exhibiting broad
substrate specificity, also show substrate selectivity which can be effectively
exploited in clinical studies and diagnostics for identifying chemical
classes/drugs to which humans are exposed occupationally. Subfamilies of
CYPs like CYP1, CYP2 and CYP3 play a central role in metabolism of chemicals
such as PAHs. The mRNA expression of principal members of CYP1A family is
CYP1A1, CYP1A2 and CYP1B1 is highly inducible in many organs by PAHs,
such as MC and Benzo(a)pyrene B(a)P, and halogenated aromatic
hydrocarbons, such as TCDD (Lewis, 1996; Kondraganti et al., 2002). The
expression of CYPs belonging to CYP2A, 2B & 2C subfamilies is induced
several fold by phenobarbital (PB) and a large number of structurally unrelated
chemicals termed `PB-like‟ inducers (Waxman and Azaroff, 1992; Xiong et al.,
2002; Bae et al., 2004). The role of CYP2A subfamilies in the metabolic
activation of N-nitrosamines, including a number of tobacco nitrosamines, and of
1, 3-butadiene is well established (Kamataki et al., 2002). CYP2A6 is the most
efficient of the isoforms studied, followed by CYP2E1, in catalysing the
bioactivation of N-alkylnitrosamines, such as dimethylnitrosamine, and of cyclic
nitrosamines, such as N-nitrosopiperidine (Kamataki et al., 2002). Ethanol and
other substrates of CYP2E1 increase the expression of CYP2E1 several fold in
the liver and other extra hepatic tissues in experimental animals as well as
humans (Song et al., 1990; Raucy, 1995; Dey et al., 2002, 2005). Likewise the
3A subfamily can be induced effectively by macrolide antibiotics (rifampicin) and
synthetic steroids such as dexamethasone or by pregnenolone-16α-carbonitrile
(PCN) (Waxman and Azaroff, 1992; Lewis, 1996; Cui et al., 2005). Similarly,
induction of 4A is induced by Clofibrate and other peroxisome proliferators in
hepatic and extrahepatic tissues (Meredith et al., 2003; Raucy et al., 2004).
Expressions of the various CYPs have been broadly used as marker to identify
environmental exposure. CYP1A1 and 1B1 have also been shown as a
biomarker of exposure in a population who are occupationally exposed (eg.
workers at factory) or accidentally exposed (eg. exposure of dioxin like
22
compounds) to PAHs (Cosma et al., 1992; Spencer et al., 1999; Toide et al.,
2003; Lemm et al., 2004; van Duursen et al., 2005; Mandal, 2005; Hu et al.,
2008). CYP1A1 has been shown to be a biomarker of acute inhalation exposure
of diesel exhaust particles (DEP) and may be implicated in an accelerated
production of ROS and the subsequent aggravation of lung injury (Takano et al.,
2002; Totlandsdal et al., 2010). The induction of CYP1A has also been widely
used as a biomarker of exposure to environmental induced polychlorinated
biphenyls (PCB) mixtures in diverse species including birds, fish and mammals
(Rifkind et al., 1984; Goksoyr et al., 1991; Smolowitz et al., 1992; Rattner et al.,
1993; Letcher et al., 1996).
Thus, the potential of different chemicals to induce the expression of different
CYPs to different extent be exploited to develop CYP expression profiles as a
biomarker to distinguish chemical exposure or adverse effects of drugs. It has
been shown that CYPs are expressed in peripheral blood lymphocytes and can
be used as a biomarker in the monitoring of susceptible individuals or subgroups
with exposure to environmental toxicants
1.6 EXPRESSION PROFILES OF CYPs IN PERIPHERAL BLOOD
LYMPHOCYTE
In recent years there has been an interest to develop non-invasive
assays that can be used as a biomarker to predict exposure to environmental
chemicals. Lymphocytes have advantages for use in the development of least-
invasive assays to screen human population for toxicant exposure and the
applicability of these cell types as indicative of toxicant exposure has been well
documented (Lucier & Thompson, 1987, Harris CC, 1989). Due to easy
availability and life span of about several years, lymphocytes are used to
investigate many cytogenetic and biochemical biomarkers (Kriek et al., 1998). In
addition, alterations in the PBL due to smoking and environmental pollution are
much larger than in the other blood cells (Savela and Hemminki, 1991;
Grzybowska et al., 1993). Exposure of humans to PAH is thought to be the
contributing factor to the incidence of lung cancer in smokers compared to non
smokers (Tang et al., 1995) Smoking related PAH-DNA adducts in human
lymphocytes are a good dosimetric exposure marker and these have been
shown to be higher in lymphocytes than other blood cells.
23
Lymphocytes possess full complement of genes whose products are responsible
for the metabolism and detoxification of xenobiotic compounds. Peripheral blood
lymphocytes (PBL) have been shown to express several members of xenobiotic
metabolizing enzymes, involved in the toxification and detoxification of PAHs.
Phase I enzymes such as members of the CYP gene family and phase II
enzymes such as GST gene families are expressed in both animal and human
lymphocytes, though gene expression in lymphocytes is not always
representative of expression in other tissues. (Raucy et al., 1997, 1999; Vanden
Heuvel et al., 1993; Rumsby et al., 1996; Haas et al.,2005; Hannon-Fletcher and
Barnett, 2008).
Using cultured human blood lymphocytes, Hoffbauer and Goedde, (1972) firstly
reported the presence of aryl hydrocarbon hydroxylase (AHH) activity, though
at a very low level in human blood lymphocytes. Measurable amounts of AHH
activity was reported in monocyte cultures, mouse peritoneal macrophages and
in granular leucocytes (Busbee et al., 1972; Ptashne et al., 1974; Bast et al.,
1976; Gelboin et al., 1976; Lake et al., 1977; Burke et al., 1977). Further,
induction of AHH activity in blood lymphocytes by MC and TCDD demonstrated
the responsiveness of lymphocyte CYPs (Burke et al., 1977). A good correlation
was observed between AHH activity in pulmonary alveolar macrophages and its
inducibility in lymphocytes. Further studies showed that the MC or TCDD
inducible CYP associated with AHH activity in human lymphocytes was in fact a
P448 like CYP isoform (CYP1A1/1A2) and the MC induced lymphocytes
catalyzed the O-deethylation of ethoxyresorufin, an activity specific for P-448 in
liver (Burke and Mayer, 1975; Burke et al., 1977). Low ethoxyresorufin O-
deethylase (EROD) activity was found in lymphocytes, which might indicate the
total absence or the presence of very low levels of CYP in the untreated cells.
Kouri et al., (1974) suggested that no constitutive AHH activity was present in
human lymphocytes. Burke et al., (1977) reported that the hydroxylation of
benzo(a)pyrene was carried out in lymphocytes which exhibited poor metabolic
capacity for O-deethylation of ethoxyresorufin. Bast et al., (1976) also reported
the presence of benzo(a)pyrene hydroxylating ability of CYP in unstimulated
monocyte cultures. Since benzo(a)pyrene hydroxylation is primarily catalyzed by
CYP1A1 in liver, these studies indicate that CYP1A1 might be present in
uninduced lymphocytes, although in insignificant levels (Aoyama et al., 1989).
24
Pretreatment with MC may have induced the levels of CYP1A1 in lymphocytes
thereby resulting in significant benzo(a)pyrene hydroxylation activity in human
lymphocytes. Radioimmunoassay using monoclonal antibody have also shown
that CYP1A1 expression in lymphocytes increases significantly after treatment
with benz(a)anthracene, another CYP1A1 inducer (Song et al., 1985). Further,
Clark et al., (1992) compared the sensitivity of mitogen activated human and
murine lymphocytes to TCDD by analyzing the dose-response relationships of
lymphocytes for induction of EROD activity and found that humans are at least
as sensitive to rats to CYP1A1 enzyme induction produced by transcriptional
activation of the CYP1A1 gene. They also demonstrated that humans exhibit
interindividual differences in their responsiveness to TCDD and related
compounds by quantifying EROD activity in lymphocytes from different
individuals and this interindividual variation could be attributed to the genetic
differences found in CYP1A1. Krovat et al., (2000) were able to detect low levels
of CYP1A1 mRNA in fresh human lymphocyte cultures using a Quantitative
Competitive Reverse Transcriptase Polymerase Chain Reaction (QC RT-PCR)
assay.
Cultured lymphocytes requires mitogen stimulation that causes lymphocytes to
proliferate resulting in the activation of several cell signaling pathways and
increases the gene transcription such as expression of PAH-metabolizing CYPs
and associated transcription pathways( Kouri et al., 1979; Hukkanen et al., 1997;
Raucy et al., 1999). Wide differences were reported in lymphocyte AHH activity
in different laboratories. Further variations observed in the AHH activity in blood
lymphocytes in different laboratories could partly be attributed to culture
conditions and mitogenic stimulation.
Freshly prepared lymphocytes offers advantage than cultured lymphocyte as
surrogate cells as they are easily available in a least invasive way and can be
seen as reflecting the overall state of the organism as they circulate through the
whole body (Collins et al., 2008)
a) CYP1A1 in freshly prepared blood lymphocytes: As opposed to
measuring CYP1A1 induction in cultured mitogenised cells, Omiecinski et al.,
(1990) reported for the constitutive CYP1A1 mRNA expression in non cultured
adult human lymphocytes. Vanden Heuvel et al., (1993) reported CYP1A1
mRNA expression in freshly prepared cultured human blood lymphocytes by
25
sensitive reverse transcriptase polymerase chain reaction (RT-PCR) studies and
were also able to measure EROD activity in these cells. Petushkova et al.,
(1996) demonstrated P450 1A1 and P450 2B1/2B2 dependent O-dealkylation of
7-ethoxyresorufin (ER) and 7-pentoxyresorufin (PR) in non stimulated,
uninduced human lymphocytes. Fung et al., (1999) demonstrated the presence
and inducibility of CYP 1A1 in freshly isolated rat blood lymphocytes. P450 1A1,
but not P450 1A2 was detected by Western blot analysis of lymphocytes from
untreated rats and archetypal inducers of CYP 1A1 like -naphthoflavone,
cigarette smoke and pyridine were effective in the inducing the levels of P450
1A1. Krovat et al., (2000) also compared the expression pattern of CYP1A1 in
fresh lymphocytes with human blood cell lines. Low levels of interindividual
variation existed, and the mRNA profile was essentially conserved across
different established human blood cell lines and highly analogous to the basal
expression patterns identified in freshly isolated lymphocytes.
Previous studies from our laboratory also reported the catalytic activity and
expression of CYP1A in rat blood lymphocytes (Dey et al., 2001; Saurabh et al.,
2010). Freshly prepared, unstimulated rat blood lymphocytes were found to
catalyze CYP1A1 dependent 7-ethoxyresorufin-O-deethylase (EROD).
Pretreatment with MC or β-naphthoflavone (β-NF), the CYP1A inducers, resulted
in significant induction in the activity of lymphocyte EROD suggesting that like
the liver enzyme, EROD activity in lymphocytes is inducible and this induction is
mediated by the MC inducible isoenzymes of CYP. That this increase in the
activity of EROD could be primarily due to the increase in the expression of
CYP1A1 isoenzymes was demonstrated by RT-PCR and western
immunoblotting studies indicating an increase in the expression of CYP1A1 in
blood lymphocytes after MC pretreatment. Significant inhibition of the EROD
activity in lymphocytes isolated from MC pretreated rats, by anti-CYP1A1/1A2
and α-naphthoflavone further provided evidence that the CYP1A1/1A2
isoenzymes are involved in regulating the activity of EROD in blood lymphocytes
(Dey et al., 2001; Saurabh et al., 2010). Saurabh et al., (2010) have reported
basal expression of CYP1A2 in lymphocytes which was several fold lower than
liver. Significant increase in the mRNA expression of CYP1A2 as well as AhR
and Arnt in lymphocytes following pretreatment with 3-methylcholanthrene (MC)
have demonstrated that responsiveness is retained in the blood lymphocytes,
26
though the magnitude of increase is several fold lower when compared to liver.
This increase in the mRNA expression was found to be associated with an
increase in the protein expression of CYP1A2 in blood lymphocytes. Further,
CYP1A2 expressed in blood lymphocytes catalyzed the O- dealkylation of 7-
methoxyresorufins (7-MR), though the reactivity was several fold lower in
lymphocytes when compared to the liver enzyme.
b) CYP1B1 in blood lymphocytes: Significant mRNA expression of PAH
metabolising CYP1B1 was reported in workers at the PAHs work site (Hanaoka
et al., 2002). Various studies have also suggested that elevated CYP1B1 gene
expression was associated with occupational exposure to PAH/dioxins (Hanaoka
et al., 2002; Toide et al., 2003; Tuominen et al., 2003; Baccarelli et al., 2004).
Dassi et al., (1998) demonstrated the presence of CYP1B1 mRNA in blood
mononuclear cells through a competitive RT-PCR assay. A substantial overlap in
the expression of CYP1B1 mRNA was found in non smokers and smokers
indicating that smoking does not seem to be potent in inducing CYP1B1
transcription in mononuclear cells. Baron et al., (1998) also reported the mRNA
expression of CYP1B1 in human blood monocytes and macrophage subsets.
This observation was confirmed by Northern blot analysis, immunoblotting and
immunohistochemical studies. His data further indicated that this enzyme is the
main CYP in human monocytes and monocyte-derived macrophages under
constitutive conditions and that it is not induced by benzanthracene (BA), which
binds to Ah receptor, suggesting another regulation of this mRNA expression in
these cells. Nguyen et al., (2000) were also able to detect CYP 1B1 mRNA
expression through DNA array and RT-PCR in PBL of healthy human population.
c) CYP2A in blood lymphocytes: Various Studies have demonstrated mRNA
expression of CYP2A6 in blood lymphocytes of healthy humans and in cancer
patients using RT PCR and DNA array studies (Nguyen et al., 2000; Furukawa
et al., 2004; Siest et al., 2008. PBL isolated from healthy individuals has further
shown similarities in the mRNA expression of CYP2A6 with the tissue enzyme.
(Oscarson, 2001; Pitarque et al., 2001; Wang et al., 2006; Ingelman-Sundberg et
al., 2007; Di et al., 2009). As observed with the mRNA expression, there was
marked variation in the CYP2A6 protein levels and its dependent COH activity in
PBL isolated from healthy controls. The variation in mRNA expression may be
attributed to genetic polymorphisms of CYP2A6 in humans (Camus et al., 1993;
27
Nakajima et al., 2004). Most of the functionally important polymorphic alleles of
CYP2A6 are reported to either result in abolished activity (*2, *4, *5 and *20) or
reduced activity (*6, *7, *10, *11, *12, *17, *18 and *19) of CYP2A6 (Pitarque et
al., 2001; Kamataki et al., 2005; Malaiyandi et al., 2005). As seen with human
lymphocytes CYP2A isoforms such as CYP2A1, CYP2A2 and 2A3 were
expressed in rat lymphocytes (Sharma et al., 2012). Further Sharma et al.,
(2012) reported significant protein expression of CYP2A6 and its associated
catalytic activity in PBL and found similarities in the regulation of blood
lymphocyte CYP2A with the tissue enzyme.
d) CYP2B in blood lymphocytes: Human CYP2B6 which has an approximately
80% of sequence homology with rat CYP2B1 have also shown to express in
freshly isolated peripheral blood mononuclear cells (Hukkanen et al., 1997;
Furukawa et al., 2004). RT-PCR and western blotting analysis have also
supported the increase in the expression and level of CYP2B6 in human and
CYP2B1 in rats after the exposure to their specific inducers (Baron et al., 1998;
Hannon-Fletcher and Barnett, 2008; Saurabh et al., 2012). Saurabh et al., 2012
also demonstrated increase in the expression of CYP2B1 and 2B2 isoenzymes,
its associated transcription factor and nuclear receptor CAR in PBL of rats
exposed to PB and suggested that mechanisms similar to that observed in the
tissues exist in blood lymphocytes
e) CYP2E1 in blood lymphocytes: Several studies have shown that freshly
isolated blood lymphocytes of human and laboratory animal express measurable
levels of CYP2E1 mRNA and protein (Raucy et al., 1997, 1999; Song et al.,
1990; Soh et al., 1996; Haufroid et al., 2003, Dey et al., 2002, 2005; Sharma et
al., 2012). Expression of CYP2E1 mRNA and protein in the blood lymphocytes is
known to be influenced by the same factors that affect the concentration of
tissue enzymes including exposure to xenobiotics and certain physiological
states (Song et al., 1990; Raucy et al., 1995; Soh et al., 1996)
A positive correlation was obtained between the elevated levels of CYP2E1, as
evidenced by immunoblot analysis and the levels of haemoglobin A1, a
metabolic indicator in diabetic subjects. Raucy et al., (1995) reported the
presence of immunochemically detectable CYP 2E1 in freshly isolated rabbit
lymphocytes and neutrophils. They found that the in vivo administration of
ethanol caused a 2 to 10 fold induction of lymphocyte P450 2E1 depending on
28
the blood alcohol concentration (BAC). A good correlation was obtained
between the extent of fold induction in lymphocyte microsomes and BAC. Again,
the induction observed in lymphocytes and neutrophils was similar to the
increases observed for inducers in other extrahepatic tissues like kidney and
bone marrow. Immunoblot analysis of the enzyme present in the leukocyte
revealed the presence of a single band with similar mobility to that present in
liver, but whether the protein band was 2E1 or 2E2 could not be concluded.
Various studies have demonstrated the expression of P450 2E1 in human blood
lymphocytes (Raunio et al., 1998; Nguyen et al., 2000; Krovat et al., 2000).
Finnstrorm et al., (2001) found 1000-fold lower expression level of CYP2E1
mRNA in peripheral blood lymphocytes of alcoholic liver disease patients when
compared to liver of patients suffering from the disease. Almost 27-fold inter-
individual variation of CYP2E1 expression in peripheral blood lymphocytes of
alcoholic liver disease patients was also observed when compared to liver
enzyme, which shows 18-fold variation.
Studies from our laboratory have shown that ethanol pre-treatment significantly
induced the expression of CYP2E1 in rat blood lymphocytes (Dey et al., 2002,
2005; Sharma et al., 2012). They have demonstrated that CYP2E1 expressed in
blood lymphocytes was catalytically active and functional as demonstrated by
CYP2E1 dependent N-nitrosodimethylamine demethylase (NDMA-d) activity and
NADPH dependent lipid peroxidation in blood lymphocytes. As observed with the
enzyme activity, pre-treatment with ethanol resulted in 3-4 fold increase in
CYP2E1 dependent lipid peroxidation in lymphocytes. The NDMA-d exhibited
monophasic pattern of enzyme activity and ethanol pre-treatment resulted in a
significant increase in the affinity of the substrate concomitant with 2- fold
increase in the apparent Vmax. In vitro inhibition studies using specific inhibitors
such as DMF hexane and DMSO and anti-CYP2E1 for CYP2E1 catalyzed
reactions resulted in significant inhibition of NDMA-d and basal (NADPH) and
CCl4 supported lipid peroxidation in lymphocytes. Inhibition of NDMA-d and
CCl4 supported lipid peroxidation under in vitro conditions by these inhibitors
indicate the involvement of CYP2E1 in catalysing the activity of NDMA-d and
NADPH dependent lipid peroxidation in blood lymphocyte (Dey et al., 2002,
2005; Sharma et al., 2012). Recent study from our laboratory has also shown
the expression of ethanol metabolizing CYP2E1 in freshly prepared human blood
29
lymphocytes (Khan et al., 2011). Significant increase in the CYP2E1 mRNA and
protein expression was observed in the freshly prepared blood lymphocytes
isolated from non-cholestatic alcoholic liver cirrhotic patients when compared to
non-alcoholic controls and non-alcoholic cirrhotic patients. This increase in blood
lymphocyte CYP2E1 expression was associated with an increase in CYP2E1
dependent enzyme activity in blood lymphocytes isolated from alcoholic liver
cirrhotic patients when compared to non-alcoholic controls and non-alcoholic
cirrhotic patients (Khan et al., 2011). Haufroid et al., (2003) further reported that
expression of CYP2E1 in hepatitis C patients was 7000-fold lower level in
peripheral blood lymphocytes than in the liver. Finnstrom et al., (2001) found
1000-fold lower expression level of CYP2E1 mRNA in peripheral blood
lymphocytes of alcoholic liver disease patients when compared to liver of
patients suffering from the disease. Almost 27-fold inter-individual variation of
CYP2E1 expression in peripheral blood lymphocytes of alcoholic liver disease
patients was also observed when compared to liver enzyme which shows 18-fold
variation.
f) CYP3A in blood lymphocytes: Mahnke et al., (1996) provided the first
immunochemical evidence for the presence of CYP3A in rat leukocyte
microsomes. They, however reported that CYP3A was not present constitutively
in white blood cells. In vivo administration of prototypic CYP3A inducers
(dexamethasone, clotrimazole, phenobarbital, pregnenolone 16α-carbonitrile) led
to the increased expression of CYP3A, although the induction observed in
lymphocytes was several fold (upto 1000 fold) lower than that observed in liver.
Janardan et al., (1996) demonstrated the presence of CYP3A5 mRNA and a
protein that was recognized by an anti-CYP3A polyclonal antibody but failed to
detect CYP3A5 activity in peripheral blood cells. Later on, Baron et al., (1998)
suggested that CYP 3A can also be estimated from PBL with some experimental
manipulation. Krovat et al., (2000) reported the presence of very low abundance
of CYP3A4 mRNA in fresh human lymphocytes and human blood cell lines,
which was also confirmed by Western blot analysis. Nguyen et al., (2000) also
found a weak expression of CYP3A5 mRNA in human blood mononuclear cells
using DNA-array. Dey et al., (2006) reported significant mRNA expression of
CYP3A1 along with associated protein and enzyme activity in control rat blood
lymphocytes. Dexamethasone resulted in 3–4-fold increase in the activity of
30
erythromycin demethylase (EMD) in freshly isolated peripheral blood
lymphocytes. This increase in the enzyme activity was found to be associated
with an increase in the rate of the reaction and affinity of the substrate towards
the enzyme. Significant inhibition of the EMD activity on in vitro addition of
ketoconazole, a specific CYP3A inhibitor in liver and polyclonal antibody raised
against rat liver CYP3A have suggested that EMD activity in blood lymphocytes
is catalyzed primarily by CYP3A isoenzymes. Pretreatment with dexamethasone
was found to significantly increase the expression of CYP3A protein in freshly
isolated rat blood lymphocytes, as observed with liver. Furthermore, several fold
increase in CYP3A mRNA expression following pre-treatment with
dexamethasone showed similarities in the regulation of CYP3A isoenzymes in
rat blood lymphocytes with the liver enzyme (Dey et al., 2006).
Kikuta et al., (1998) isolated and sequenced cDNA of CYP4F3, which catalysis
hydroxylation of leukotriene B4, from human polymorphonuclear leukocytes.
Other CYPs reported to be present in blood lymphocytes include CYP2B6/7,
CYP 4A11, CYP2D6, CYP2J2, CYP2F1 etc. (Krovat et al., 2000; Nguyen et al.,
2000). The occurrence of these CYP mRNA does suggest the possibility of using
this more readily available tissue as an indicator of changes in the status of
certain tissue enzymes.
1.7 BIOMARKERS FOR MONITORING TOXICITY OF VEHICULAR
EMISSIONS INCLUDING DIESEL EXHAUST PARTICLES
Air quality crisis has been attributed to vehicular emissions in both
developed and developing countries and contribute to about 40-80% of total air
pollution (Ghose et al., 2005). Vehicle emissions are responsible for 70% of the
country‟s air pollution and have increased 8 folds over a period of over 20 years.
Air pollution from vehicle exhaust has worsened in India and is ranked among
top ten most polluted areas of the world (Blacksmith Institute). Motor vehicle
emissions are also the main source of fine and ultrafine particulate matter which
are supposed to be the major air pollutant of the atmosphere. Urban areas
exhibit both the highest level of pollution and largest target of impact on human
health (Goyal and Sidhartha, 2003). India has 23 major cities of over 1 million
people and ambient air pollution exceeds the WHO Standards in many of them
(Gupta et al., 2002). In India, diesel vehicles account for 6% of total vehicles
31
(Khillare et al., 2005). A major constituent of urban air pollution is diesel exhaust,
a complex mixture of gases, chemicals and particles (Schuetzle, 1983; Draper,
1986; Singh et al., 2004). Symptoms like chronic cough, wheezing and
breathlessness have been reported on exposure to diesel exhaust particles
(Chabra et al., 2001).
Diesel exhaust particles (DEPs) are the important and toxic component of
ambient particulate matter to which majority of people are exposed on a daily
basis. DEPs account for a large portion of ambient fine and ultrafine particles,
and have been historically used as a surrogate to measure human exposure to
diesel exhaust emissions (Ris, 2007; US EPA 2002; Wichmann, 2007). The
majority of these particles tend to be found in the greatest concentration within
the immediate vicinity of busy streets or highways (Corfa et al., 2004; Cyris et al.,
2004). DEPs emitted from diesel engines have 100 times more particles than
modern gasoline engines (Riedl & Diaz-Sanchez, 2005). Despite the
unfavourable impacts to our environment and human health, diesel is the
primary source of fuel for mass transportation in the developed as well as
developing countries based on increased efficiency and endurance of diesel
engines in comparison to gasoline engines (Krivoshto et al., 2008).
1.7.1 Composition and physicochemical properties of DEP
DEPs comprise of a carbonaceous core to which organic and inorganic
compounds, such as polycyclic aromatic hydrocarbons (PAHs), nitro and
oxygenated derivatives of PAHs (ketones, quinines and diones), heterocyclic
compounds, aldehydes, aliphatic hydrocarbons and heavy metals are adsorbed
(Schuetzle, 1983; Draper, 1986) (fig1.7).
FIGURE 1.7: Composition of DEP
32
Over 450 different compounds have been identified in diesel exhaust with 40
recognized hazardous pollutants (lnadera, 2006; Annesi-Maesano et aI., 2007).
Major PAH components of DEP include phenanthrenes, fluorenes,
naphthalenes, fluoranthrenes, and pyrenes (Nel et al., 1998). An important
physical characteristic of diesel particles is that they are very small in size.
Diesel exhaust particles contribute to ambient airborne particulate matter and
can exist in the range of coarse (PM10, particulates of an aerodynamic diameter
of less than or equal to 10 µm), fine (PM2.5, particulates of an aerodynamic
diameter of less than or equal to 2.5 µm), ultrafine (diameters below 0.1 µm or
100 nm), and nanoparticles (diameters less than 50 nm). However majority of
DEPs are fine (2.5-0.1 mm) or ultrafine (0.1 mm) particles and make up of large
particulate components in DEP. It has been postulated that because smaller
particles have a greater relative surface area, they should carry proportionally
more chemicals and have greater biologic effects (Li et al., 2002; Oberdoster,
2002).
1.7.2 Ambient & Occupational DEP Exposure
DEP concentrations in ambient air are employed as a measure of
exposure to diesel emissions. DEP mass in ambient particulate matter is variable
approximating zero in rural areas to 35% of PM10 in urban areas. In urban
areas, where people spend a large portion of their time outside in close proximity
to major roadways, DEP concentrations average 1.6-2.4 μg/m3, and as high as
4.0 μg/m3 in some locations, unlike rural environments with relatively low
ambient DEP concentrations (0.6-0.74 μg/m3) (Hesterberg et al., 2009; Ris,
2007; US EPA, 2002). Furthermore, in urban environments off-road sources of
DEP contribute roughly twice the amount of PM, as compared to on-road
sources. While current air quality regulations and improvements in diesel engine
technology have resulted in national decreases in ambient DEP concentrations,
occupational monitoring indicates that DEP levels remains high, 100-400 μg/m3,
for miners, railroad workers, public-transit workers, airport crew, mechanics,
dock workers, and truck drivers (Hesterberg et al., 2009; Ris, 2007; US EPA,
2002).
33
1.7.3 DEP Deposition & Clearance
It is estimated that approximately 30% of inhaled DEPs is deposited in
the respiratory tract (Horvath et al., 1988). DEP is deposited throughout the three
general regions of the respiratory tract: the extrathoracic, tracheobronchial, and
the alveolar. The regional deposition of DPM depends upon the size and mass of
the particles. The largest (above around 4 μm) and smallest (below around
0.002 μm) particles tend to deposit in the extrathoracic region (US EPA, 2002),
DEP in the 0.005 μm size range deposits in the tracheobronchial region in
addition to the extrathoracic region (Oberdorster, 2002; Riedl, 2005). Particles
between approximately 0.2 μm and 0.002 μm are generally deposited in the
alveolar region of the respiratory tract, through diffusion (US EPA, 2002).
Particles deposited within the respiratory tract may be cleared through
mechanical processes (mucociliary transport and macrophage phagocytosis) or
by dissolution.
Mechanical processes are generally responsible for the clearance of the
carbonaceous core of DEP, while dissolution is generally the clearance
mechanism for the adsorbed organics (Adamson, 1978, 81). In the extrathoracic
and tracheobronchial regions, DEP is generally cleared by mucociliary transport
(the movement of mucous in which the DEP is deposited towards the larynx, by
rhythmic beating of the cilia lining the respiratory tract (Felicetti et al., 1981).
Within the alveolar region, macrophage phagocytosis is the primary mechanical
clearance mechanism (Warheit et al., 1988). Alveolar macrophages engulf DEP
particles, and then are removed through mucociliary transport or through the
lymphatic system. DEP particles are also cleared through endocytosis by
alveolar lining (Type I) cells, and translocation to the lymph nodes (White and
Garg, 1981).
1.7.4 Epidemiological Studies
Epidemiological studies have demonstrated that airborne PM, of which
DEPs is a major contributor, are responsible for causing respiratory mortality and
morbidity (Pope et al., 2006; Robinson et al., 2010). A positive correlation
between elevated levels of PM in ambient air and increase respiratory mortality
and morbidity was associated in high risk groups (Dockery et al., 1993, 1994;
Pope et al., 1995). Increased PM has been associated with pulmonary and
34
cardiovascular diseases and cancer (Pope et al., 2002; Brunekreef, 2002).
Numerous studies have shown associations between increased symptoms of
cough, bronchitis, asthma, and chronic obstructive pulmonary disease (COPD)
to increases in the concentration of air pollutants including DEP (Jaffe et al.,
2003; Riedl and Diaz-Sanchez, 2005).
In addition to epidemiological studies, human controlled exposure studies have
also been used to investigate the health effects of pollutants including diesel
exhaust. Compared to epidemiological studies, human exposure studies enable
the direct effect on humans to be studied under well define PM exposure
concentrations and durations, and in the absence of confounding exposures to
other air pollutants. The clinical effects of short-term DEP exposure have been
explored in both healthy subjects as well as individuals with inflammatory lung
diseases like asthma and COPD. Exposure of healthy subjects to short-term
DEP of relatively high concentrations results in pulmonary and systemic
inflammatory responses characterised by increase in cell adhesion molecules on
the pulmonary endothelium (ICAM-1, VCAM-1) and recruited cells (LFA-1) as
well as increases in inflammatory chemokines (IL-8, GRO-a, IL-5) without
corresponding change in pulmonary function (Salvi et al., 1999, 2000; Pourazar
et al., 2004; Stenfors et al., 2004).
However, individuals with mild to moderate asthma yielded conflicting results in
both controlled and non-controlled exposure studies. Asthmatics exposed to
lower levels of DEP for 1 hr showed significant increases in pulmonary levels of
the pro-inflammatory cytokine IL-6 as well as significant increases in both airway
resistance and hyper-responsiveness (Nordenhall et al., 2001). However, in
another study exposure of asthmatic patients to DEP for 2hr showed showed no
induction in inflammatory cytokine (Stenfors et al., 2004). In this study, neither
healthy individuals nor asthmatic individuals demonstrated any effect of diesel
exhaust on pulmonary function indices. Finally, there was a lack of inflammation
following exposure of patients with mild-to moderate asthma to diesel exhaust
(100mg/m3) for 2h (Behndig et al., 2011). In contrast, healthy individuals showed
increased neutrophil numbers, myeloperoxidase, and IL-6 in the bronchial wash
and submucosal neutrophils on biopsy 18 h after the same exposure.
Further, a recent panel study of 60 participants with mild to moderate asthma
demonstrated adverse respiratory effects when exposed for 2hr to real-world
35
traffic emissions. The results showed significant reductions in lung function and
increased inflammatory changes in sputum as compared to asthmatics that
walked in a pollution free area (McCreanor et al., 2007). In in vitro investigation,
exposure to low doses of DEP caused an increased release of proinflammatory
mediators from collected asthmatic bronchial epithelial cells, whereas higher
DEP doses decreased the release of inflammatory mediators (Bayram et al.,
1998).
Epidemiological, controlled human exposure and in-vitro cell investigation do not
definitively confirm a relationship between DEP exposure and worsening of
asthma. Most of these studies have employed levels which are significantly
lower than the threshold exposures of 300mg/m3 observed for an inflammatory
response in healthy volunteers (Ghio et al., 2012). However, it was expected that
asthmatic individuals would be a sensitive population. Some research suggests
a decreased sensitivity among asthmatic individuals to diesel exhaust and DEPs
relative to healthy individual ((McCreanor et al., 2007).
1.7.5 Role of CYPs in pulmonary toxicity by DEP exposure:
Several studies have shown that DEP exposure indeed alters the CYP
enzymes including both family 1 and 2 members in the lung (Table 1.9). Takano
et al., (2002) showed that instillation of intratracheally DEP in mice resulted in a
dose dependent increase in the expression of CYP1A1 at both the mRNA and
protein levels. In comparison, carbon black (CB) particles did not induce the
expression of this enzyme. The increased expression of CYP1A1 was thought to
facilitate ROS generation and subsequent aggravation of lung injury. The
induction of CYP1A1 by DEP was also reported in cultured human bronchial
epithelial cells (16HBE). This effect was attributed to the chemical component of
DEP (Bonvallot et al., 2001). Hatanaka et al., 2001 further demonstrated that the
CYP family 1 isoenzymes including CYP1A1, CYP1A2, and CYP1B1 were all
induced after DEP exposure. In fact, in rats exposed to 0.3 or 3 mg/m3 DEP (12
h/day, for 4 weeks), elevated mRNA levels of CYP1A1 and CYP1B1 and
increased 7-ethoxyresorufin O-deethylase (EROD) activity were found not only in
the lung, but also in the liver, when compared to the control rats. In addition, the
expression of CYP1A2 was increased by DEP exposure in the liver (but not in
the lung). In kidney, where the individual isozymes were not detected, DEP
36
exposure significantly increased the total CYP protein. These family 1 enzymes
were shown in an earlier study to activate the genotoxicity of DEP extract and its
major component, 1-nitropyrene, in a SOS/umu assay using Salmonella
typhimurium TA1535/pSK1002 (Yamazaki et al., 2002). Using the same assay,
Hatanaka et al., (2001) demonstrated that the lung, liver, and kidney microsomes
from rats exposed to 0.3 mg/m3 of DEP all exhibited increased capacity to
activate the genotoxic effect of 1-nitropyrene, 1-aminopyrene, and the DEP
extract. Rengasamy et al., (2003) have shown that the induction of CYP1A1 in
the rat lung by DEP exposure was both dose- and time dependent.
Intratracheally instilled DEP (5, 15, or 35 mg/kg) resulted in increased CYP1A1
protein and EROD activity at 1 day post-exposure, but the enzyme level declined
with time and returned to control level at five days post-exposure. CB did not
induce CYP1A1 protein or activity, suggesting that the induction of CYP1A1 is
mediated through the organic component. However, both DEP and CB particles
resulted in sustained and dose dependent decrease in the CYP2B1 protein and
activity of enzyme pentoxyresorufin O-dealkylase (PROD) activity for seven
days. In experiments where rats were exposed repeatedly to low dose of DEP,
there was no significant increase in CYP1A1, but a sustained decrease in
CYP2B1 in lung microsomes even at 10 days after the last exposure dose.
These results show that the organic and the particulate components of DEP
respectively induce CYP1A1 and suppress CYP2B1 expression in the rat lung.
DEP or CB exposure also altered a number of other enzymes in the pulmonary
system. The GST and catalase activities were down-regulated by DEP as well as
by CB at one and seven days post-exposure. The quinone reductase activity
was induced by DEP but was not affected by CB exposure and neither DEP nor
CB affected the activity of NADPH-cytochrome P450 reductase (Rengasamy et
al., 2003). Further Sagai et al., (1993) also showed that DEP inhibited GST and
other phase II enzymes including superoxide dismutase, and glutathione
peroxidase. The inductive effect of DEP on quinone reductase activity, that
prevent the formation of benzo(a)pyrene quinone-DNA adducts, generated by
CYP1A1 and P450 reductase, could be attributed to electrophiles and phenolic
antioxidants, which may be present in the DEP-derived chemicals, induce
quinone reductase activity (Perestera et al., 2000; Joseph et al., 1994). The
reduction of CYP2B1, GST, and catalase by DEP or CB may have resulted from
37
a central mechanism mediated through particle-induced oxidative stress in the
lung. Although certain compounds such as toluene and xylene are known to
inhibit CYP2B1 activity (Furman et al., 1998, Verschoyle et al., 1993), these
molecules are not present in the CB particles. Instead, CYP2B1 activity may be
modulated by nitric oxide (NO), which binds to the reactive heme-iron center and
thereby inhibits the enzyme activity (Ferrari et al., 2001). In addition to its effect
on enzyme activity, NO has also been shown to regulate the expression of
CYP2B1/2 at the mRNA and protein levels (Khatsenko et al., 1997) Since both
DEP and CB induce alveolar macrophage (AM) production of NO, (Yang et al.,
2001) it is likely that NO is involved in the down-regulation of CYP2B1 while GST
is inactivated by the presence of H2O2 through disulfide linkage between reactive
cysteine sulfhydryl residues (Shen et al., 1991). Catalase, on the other hand,
controls the H2O2 level in vivo. A decrease in catalase activity by DEP or CB
could increase cellular H2O2, thus resulting in lowered GST activity. Studies have
shown that DEP inhibit the activity of catalase isolated from various cell types
(Mori et al., 1996). This would support the concept of a H2O2-mediated
inactivation of GST. On the other hand, DEP or CB particles also induce alveolar
macrophage (AM) production of superoxide and H2O2 that could also lower the
level of catalase and GST enzymes.
1.7.6 Evidence of oxidative stress induced by DEP via ROS generation
The production of ROS is believed to play important role in the primary
cytotoxic effects of diesel exhaust particles (DEP) (Knaapen et al., 2004). ROS,
such as superoxide, hydrogen peroxide, and hydroxyl radical, are reactive with
proteins, lipids, and DNA, leading to cellular damage (Hiura et al., 1999; Li et al.,
2002; Kumagi et al., 1997; Sagai et al., 1993). Increased formation of hydroxyl
radicals have been detected by non invasive electron spin resonance
spectroscopy in the lungs of DEP instillated mice (Han et al., 2001). Production
of superoxide (O2- and hydroxyl radicals(OH) from organic extracts of DEP was
reported in vitro studies (Kumagai et al.,1995; Sagai et al.,1993). In vivo and in
vitro studies have reported that an increased amount of ROS is generated in
cells upon exposure to DEP and other air pollutants. For example, exposure of
16-HBE bronchial epithelial cells to DEP induced the production of ROS, as
detected by fluorescent probes, as well as gene expression of the phase I
38
(cytochrome P-450 1A1) and phase II (nicotinamide adenine dinucleotide
phosphate quinone oxidoreductase-1) xenobiotic metabolizing enzymes.
The induction of oxidative stress through ROS generation is a characteristic of
exposure to DEP (Xiao et al., 2003; Li et al., 2002). Oxidative stress develops
when there is an imbalance between the production of ROS and the availability
of anti-oxidant defences (McCord, 2000). Oxidative stress is considered a main
mechanism of DEP-induced toxicity and inflammation (Li et al., 2010; Schwarze
et al., 2013). DEP-induced ROS-formation may activate redox-sensitive
transcription factors such as NF- B, Nrf2 and AP-1 and signaling molecules
such as MAPK involved in regulation of pro-inflammatory genes IL-4, IL-6, IL-8
and TNF-α, as well as chemokines and adhesion receptors (Bonvallot et al.,
2001; Pourazar et al.,2005; Schwarze et al., 2013). Further evidence for a key
role of oxidative stress in the upregulation of proinflammatory cytokines has
been demonstrated by the capacity of antioxidants to reduce both NF-κB
activation and cytokine release from cells challenged with DEP (Hashimoto et
al., 2000; Bonvallot et al., 2001; Li et al., 2002).
Studies have shown that organic fraction of DEP induces oxidative stress
through ROS generation (Dellinger et al., 2001). Many studies have reported that
organic compounds of DEP such as PAH and quinones are desorbed from DEP
and become available to bind the cytosolic aryl hydrocarbon receptor (AhR) and
Nrf2 transcription factors and induce the expression of phase I and phase II
xenobiotic metabolizing enzymes (Bonvallot et al., 2001; Rengasamy et al.,
2003; Takano et al., 2002., Srivastava et al., 2013; Baulig et al., 2003;
Totlandsdal et al 2010; Gualtieri et al., 2011). Organic compounds of DEP on
metabolism mediates generation of ROS and reactive PAH quinones. A role of
PAH is supported by the correlation between the PAH content of fine and
ultrafine particles and their ability to induce oxidative stress in macrophage (Li et
al., 2002). Quinones are known to generate oxidative stress by redox cycling.
They are suspected to be responsible for the production of O2- and OH radicals
detected in methanol extracts of DEP. Redox cycling quinines undergo one
electron reductions by NADPH P450 reductase (NQO1) to form semiquinones
(Monks et al., 1992). These semiquinones can be recycled to the original
quinones leading to the formation of O2-. The detoxification of quinones can
occur by two electron reduction performed by phase II enzyme NADPH quinone
39
oxidoreductase (NQO1). In addition, phase I enzymes such as CYP1A1 elicit
inflammation through CYP1A1 mediated ROS generation which activates
transcription factors and release of cytokines (Baulig et al., 2003; Schwarze et
al., 2013).
ROS generated by DEPs leads to oxidative DNA damage DNA damage. PAH
associated with diesel exhaust are genotoxic, forming PAH-DNA adducts and
resulting in mutation and DNA strand breakage (Li et al., 2006). Mutagenic DNA
adduct, 8-hydroxydeoxyguanosine (8-OH-Gua) which is responsible for
carcinogenesis was found to be increased in the mouse and rat lung after DEP
exposure (Ichinose et al., 1997; Nagashima et al., 1995; Tsurudrome et al.,
1999; Risom et al., 2003). Following DEP exposure, mRNA expression of
OGG1 (8-oxoguanine DNA glycosylase, an enzyme involved in repair of 8-OH-
Gua) and ERCC1(excision repair cross complementary 1), an enzyme involved
in nucleotide excision pathway was elevated in lungs and liver of DEP exposed
rats(Tsudrome et al.,1999; Dybdahl et al., 2003; Risom et al., 2003).
1.7.7 ROS mediated by transition metals present on DEP
On their surface, particles may contain soluble transition metals such as
iron, copper, chromium and vanadium that can generate ROS through Fenton
type reactions and act as catalysts by Harber–Weiss reactions (Halliwell, 1999):
•O2− + H2O2−Fe→ •OH + OH− + O2
In the Fenton reaction, ferrous iron (Fe2+) reduces hydrogen peroxide (H2O2)
with the formation of hydroxyl radical and oxidation of ferrous iron to ferric iron
(Fe3+). This reaction can recycle by reductants such as superoxide anions,
glutathione and ascorbic acid by reducing Fe3+ to Fe2+. The hydroxyl radical
(•OH) is extremely reactive (reaction rate constant usually above of 108M−1
s−1), which implicates that it attacks any biological molecules at diffusion
distance (Halliwell, 1999). Several studies have shown that iron and other
transition metals leaching from particles or by their presence on particle surfaces
play a role in the generation of ROS in biological systems (Han et al., 2001, Ghio
et al., 2000). It has been recently suggested that DEP contain functional groups
at the surface with the capacity to complex host iron, whereby iron accumulates
and oxidative stress is induced (Han et al., 2001, Ghio et al., 2000). This is in
accordance with in vitro studies demonstrating that DEP generate superoxide
40
anions which can lead to hydrogen peroxide and hydroxyl radicals without any
biochemical or biological activation (Dellinger et al., 2001; Sagai et al., 1993).
1.7.8 Inflammatory response to DEP exposure
Inflammation is considered a key step in the development of health
effects associated with DEP exposure (Schwarze et al., 2013). Many studies
have examined the effects of DEPs and reported numerous alterations in
inflammatory endpoints. However, in vivo studies has major limitations such as
large differences in dosimetry between animals and humans, employment of
relatively high exposure levels, routes of administration which are not
physiological in the human and large differences in sensitivity between species.
Consequently, direct extrapolations of the results from animal investigation to
humans are never ideal. Inflammatory effects of diesel exhaust and DEPs in in
vivo studies involve significant induction of airway inflammation characterised by
increased number of eosinophils, neutrophils, and lymphocytes in the BAL as
well as increased expression of proinflammatory cytokines (e.g. IL-1β, TNF-α,
MIP-1α, TARC, and keratinocyte chemoattractant) (Ma and MA, 2002; Riedel
and Diaz-sanchez, 2005; Ghio et al., 2012).
Similarly, proinflammatory effects of DEPs have also been examined by in vitro
studies employing airway epithelial cells, nasal epithelial cells, alveolar
macrophages, mast cells, and cell lines (Scwarze et al., 2013). The results of
this investigation also support a proinflammatory capacity of DEPs. However,
there are numerous limitations of in vitro cultured cell studies such as
employment of unrealistic doses and the physiologic relevance of the cell type
used. Inflammatory effects of diesel exhaust and DEPs in cultured cells are as
follows: increased reactive oxygen and nitrogen species generation; augmented
tyrosine kinase activity and cell signaling, activation of transcription factors;
increased RNA, protein expression, and release of proinflammatory mediators,
increased RNA and protein expression of adhesion molecules, attenuation of
ciliary beat frequency (Scwarze et al., 2013).
1.7.9 Mechanistic pathway for inflammation after DEP exposure
Particle exposure causes oxidant generation possibly resulting from
electron transport by organic compounds and a disruption of iron homeostasis. A
41
cascade of reactions follows, including cell signaling by kinases, transcription
factor activation, and inflammatory mediator release which culminates in
inflammation.
An early event in the cellular response to DEPs is phosphorylation dependent
cell signaling (Pourazar et al., 2005). In vivo and in vitro studies demonstrated
that DEP exposure induced the activation of the mitogen-activated protein (MAP)
kinase cascade (ERK, p38, and Jun kinases) which represents intracellular
signaling network by which DEP mediates specific biological effects (Li et al.,
2009). Following exposure of DEP to human respiratory epithelial cell line, MAP
kinase pathways (i.e.ERK1/2 andP38) were triggered leading to the activation of
the nuclear factor NF-kB (Morano et al., 2002; Amara et al., 2007). Reactive
oxygen species were implicated in the response because DEPs induced an
increase in intracellular hydroperoxides, while antioxidants inhibited the
activation of MAP kinases as well as NF-kappa B and cytokine release. In
addition to kinase cascades, various nuclear transcription factors also control the
activity of genes involved in inflammation (Li et al., 2002; Xiao et al., 2003).
Exposure to diesel exhaust was associated with kinase phosphorylation (phos-
JNK and phos-p38) and a nuclear accumulation of transcription factors (Nrf2,
NF-kappaB, and AP-1) (Li et al., 2004; Pourazar et al., 2005). Such translocation
of transcription factors leads to an increased expression of proinflammatory
mediators whose genes have binding sites for these transcription factors in their
promoter regions eventually causing inflammation (pulmonary and systemic).
1.8 Peripheral Blood Lymphocytes: A tool for predicting toxicity of DEP
As mentioned earlier in the review, the xenobiotic metabolizing enzymes
expressed in freshly prepared PBL may prove to be very useful for monitoring
toxicity of DEPs. Though the mechanism involved in toxicity of DEP is relatively
well characterized in the tissues such as lung, not much information is available
using sentinel tissues such as PBL. As the CYPs and GSTs that are involved in
the metabolic activation and detoxification are expressed in PBL, blood cells
could be exploited for such biomonitoring studies.
The candidate has therefore attempted to characterise the effects of DEP on the
expression of xenobiotic metabolizing enzymes in freshly prepared PBL. Studies
were also carried out by candidate to investigate similarities in the mechanism of
42
regulation of blood lymphocyte enzymes with the tissue enzymes after DEP
exposure. These studies will eventually demonstrate utility of using expression
profiles of candidate genes in PBL as a possible biomarker for predicting toxicity
of vehicular emissions for developing fingerprints of blood lymphocytes as a rapid
and sensitive tool for predicting exposure and toxicity of DEP.
Chapter 2
Gene expression profiling of candidate genes in peripheral
blood lymphocytes for predicting toxicity of diesel
exhaust particles
43
CHAPTER 2
Gene expression profiling of candidate genes in peripheral blood
lymphocytes for predicting toxicity of diesel exhaust particles
2.1 Introduction
Diesel exhaust particles (DEPs) are the major component of ambient
fine particulate matter (PM 2.5). DEPs are composed of particulate matter
(PM) and porous carbon nuclei to which vast amount of organic compounds,
such as polycyclic aromatic hydrocarbons (PAHs), nitroaromatic
hydrocarbons, heterocyclics, quinones, aldehydes, and aliphatic
hydrocarbons, as well as traces of heavy metals are adsorbed (Sen et al.,
2007). The small size of DEPs facilitates it’s penetration into the lungs leading
to their accumulation in bronchiolar and alveolar regions of lungs (Yu and Xu,
1987). It has been reported that polycyclic aromatic hydrocarbons (PAHs)
could be desorbed from DEP and bind to the cytosolic aryl hydrocarbon
receptor and induce gene expression of drug metabolizing enzymes (DMEs)
like CYP1A1 and CYP1B1(Jacob et al., 2011; Srivastava et al., 2012). Studies
have shown that DEP exposure induces the generation of reactive oxygen
species (ROS) which leads to oxidative stress resulting in a variety of toxic
manifestations including inflammatory response, DNA damage and apoptosis
(Kumagi and Shimojo, 2001; Li et al., 2002, 2006, 2008; Wan and Diaz-
Sanchez, 2007). It has been suggested that these adverse biological effects
induced by DEP are linked to their capacity to generate ROS (Wan & Diaz-
Sanchez, 2007; Li et al., 2008).
DNA microarrays have provided a mechanistic insight of the adverse
effects of DEP in lungs. The role of drug metabolizing enzymes, oncogenes,
inflammatory response genes and other stress related genes have been
identified in DEP induced pulmonary toxicity (Sen et al.,2007; Omura et al.,
2009). Likewise, the role of antioxidative enzymes such as hemeoxygenases
(HO-1 and HO-2), glutathione S-transferases P subunit (GST-P), thioredoxin
peroxidase (TDPX-2), NAD(P)H dehydrogenase and proliferating cell nuclear
antigen (PCNA) were demonstrated in DEP induced lung inflammation.
Induction of HO-1 and downregulation of transglutaminase-2 (TGM-2) were
44
identified as putative biological response markers of DEP exposure (Sen et
al., 2007).
Studies in our laboratory have shown that freshly prepared peripheral
blood lymphocyte (PBL) can be used as a surrogate to monitor alteration in
the tissue expression after exposure to various inducers of DMEs (Dey et al.,
2001, Saurabh et al., 2010., Sharma et al., 2012). Although very little
information is available on gene expression profiling after exposure to DEP,
DNA array studies using human (PBMCs) showed considerable effect of DEP
on biological processes such as inflammation and oxidative stress (Peretz et
al., 2007). DNA array studies have also shown the expression of various
DMEs in PBL isolated from humans (Nguyen et al., 2000; Siest et al., 2008).
Though gene expression profiling studies using microarray technology has the
potential to provide mechanistic insights, microarray technology has
limitations in detecting genes which have low basal expression and often
fails to identify the role of closely related isoenzymes in toxic manifestation
(Harbig et al., 2005). Further, variability in data is reported when same set of
experiment is performed using different array platforms (Gwinn et al., 2005).
Taqman low density array (TLDA) based RT-PCR, which requires no
further validation, has been used as an accurate and sensitive method to
identify & classify toxicants, based on their characteristic transcription profiles
(Goulter et al., 2006). Recent study from our laboratory using TLDA has
demonstrated similarities in the induction of DMEs and their associated
transcription factors in PBL and liver (Sharma et al., 2013). The present study
was now aimed to validate a sensitive bioassay using freshly prepared PBL
for monitoring the DEP induced alterations in the expression of selected
genes including DMEs, inflammatory molecules and those involved in DNA
damage & apoptosis. In our effort to develop blood lymphocyte expression
profiling as an alternate to monitor tissue expression, attempts were also
made to identify similarities in the alteration of these genes, in PBL and lungs
isolated from DEP treated rats.
45
2.2 Materials and Methods
2.2.1 Chemicals
DEP (Standard Reference material 2975) was procured from National
Institute of Standards and Technology, Gaithersburg, MD. Taqman low
density array (TLDA) plates of customized 96 genes array consisting of CYPs,
GSTs antioxidant enzymes, their respective transcription factors and genes
involved in inflammation, apoptosis and oxidative DNA damage, was procured
from Applied Biosystems, USA. High-capacity cDNA Reverse Transcription
Kit and Taqman Universal PCR Master Mix were also procured from Applied
Biosystems, USA.
2.2.2 Animals and treatment
Adult male albino wistar rats (6-8 week old) were procured from CSIR-
Indian Institute of Toxicology Research (IITR) breeding colony on campus and
raised on standard pellet diet and water ad libitum. DEPs were mixed with
sterile normal saline and the suspension was sonicated for five minutes using
an ultrasonic processor with a microtip at a frequency of 100 megahertz.
Animal care and experimentation was done in accordance with the policy laid
down and approved by the Animal Care Committee of the Centre. The
animals were divided into four groups, each containing six animals. The
animals in the control group were treated with normal saline while other group
of rats was treated with DEPs (7.5- or 15- or 30 mg/kg) body weight
respectively. For treatment, rats were anaesthetized with ketamine (75-100
mg/kg, i.p) and trachea was exposed by surgery on the ventral side of the
neck. A needle was inserted onto the wall of trachea through which particulate
suspension was instilled (2 ml/kg body weight) slowly onto the tracheal lumen.
The animals were anaesthetized twenty-four hours after the administered
dose. Blood was drawn from the heart and processed for the isolation of
lymphocytes. Rats were subsequently sacrificed and lungs were perfused with
ice cold normal saline to remove the blood.
46
2.2.3 RNA Extraction
Total RNA was extracted from whole blood isolated from control and
DEP treated rats by Mouse RiboPure-Blood RNA isolation kit (Ambion, USA)
and from lung by TRIzol reagent (Life Technologies, USA) according to
manufacturer’s protocol. The protocol utilising TRIzol reagent, a mono- phasic
solution of phenol and guanidium isothiocyanate, is an improvement to the
single-step RNA isolation method developed by Chomczynski and Sacchi,
(1987). During sample homogenization, TRIzol reagent maintains the integrity
of the RNA, while disrupting cells and dissolving cell components. Addition of
chloroform followed by centrifugation separates the solution into an aqueous
phase and an organic phase. RNA remains exclusively in the aqueous phase.
After transfer of the aqueous phase RNA is recovered by precipitation with'
isopropyl alcohol, dissolved in water and stored at -800C for further
processing.
2.2.4 TaqMan Low Density Array (LDA) Analysis
TaqMan LDA consist of 96 TaqMan Gene Expression Assays (Applied
Biosystems) preconfigured in a 384-well format and spotted on a microfluidic
card (4 replicates per assay). Each TaqMan Gene Expression Assay consists
of a forward and reverse primer at a final concentration of 900 nM and a
TaqMan MGB probe (6-FAM dye-labeled; Applied Biosytems) at final
concentration of 250nM. The assays are gene specific and have been
designed to span an exon-exon junction. The genes included in these
expression assays are phase I drug metabolizing CYPs (CYP1A, 1B1, 2A, 2B,
2C1, 2E1, 3A1 and 4B1), phase II enzymes and antioxidant enzymes such as
GSTs (A, M1, M2, M3, M5, P1, K1, O1, O2 and mGST), NQO1, aldehyde
dehydrogenase (ALDH1), catalase, superoxide dismutase (SOD1, 3),
glutathione peroxidase (GPx1), HO-1, HO-2, peroxiredoxin (PRDX-2),
metallothionein (MT-1a, MT-3). Genes involved in transcription factors and
signalling (AhR, Arnt,, Hif-1α, c-Fos, Jun, Fosl1,Tank, JAK-2, MAPK 8, 9, 10),
inflammatory genes (IL-1b, IL-4, IL-5, IL-6, IL-10, IL-12a, IL-12b, IL-13, CCL5,
CXCL1, CCL2, CSF2, TGF-β1, TGFBR1, HGF, Sftpa1, Sftpd, FIGF, ICAM1,
VCAM, SELE, NOS2, PTGS2 & Caspase1), DNA repair genes (PARP,OGG1,
PCNA & Top2A), apoptosis and oncogenes (Caspase 3, 6, BID, BAD, Bok,
47
Bcl2, Bcl2l1, Bcl2l2, BBC3, PDCD8, Tp53, Araf & Kras) & genes involved in
coagulation/others (TIMP-1,TGM-2, PDZK1& Hspa8). Each assay and its
assay ID number are available at docs.appliedbiosytems.com/pebiodocs/
00112893.pdf.
cDNA was synthesized by High-Capacity cDNA Reverse Transcription
Kit (RT) (Applied Biosystems, USA) as described by Shah et al., (2009). For
RT, the reaction mixture in 20 µl contained 10 µl of 2X master mix 2X RT
buffers (2 µl), 0.8 mM dNTP Mix (0.8 µl), 2X Random Primers (2 µl), RNase
Inhibitor (1µl), 1µl MultiScribe Reverse Transcriptase and 3.2 µl Nuclease-free
H2O] and an equal volume of diluted RNA sample. RT reaction was carried
out in a thermal cycler consisting of 1 cycle at 250C for 10 min, 370C for 120
min, 850C for 5 sec and 40C on hold. cDNA (180 ng RNA/sample) was then
added to the reaction mix containing 55µl 2X Taqman Universal PCR Master
Mix and the volume up to 110 µl with Nuclease-free H2O, mixed by inversion
and spun briefly in an Heraeus microcentifuge. After the cards reached room
temperature, 110µl of each sample were loaded into each of the 4 ports on
TaqMan LDA. The cards were placed in Heraus Custom Buckets (Applied
Biosytems) and centrifuged in a Heraus Multifuge for 1 min at 331Xg.
immediately following centrifugation, the cards were sealed with a TaqMan
LDA sealer (Applied Biosytem) to prevent cross-contamination. The final
volume in each well after centrifugation was less than 1.5 µl. The real time
RT-PCR amplifications were then run on ABI PRISM 7900HT Sequence
Detection System (Applied Biosytems) using SDS 2.3 software. Thermal
cycling conditions were as follow: 50°C for 2 min, 94.5°C for 10 min followed
by 50 cycles of 97°C for 30s and 59.7°C for 60s. Data analysis was done with
the help of software RQ Manager (version 1.2).
2.2.5 Statistical analysis
Students’t-test was employed to calculate the statistical significance
between control and treated groups. P < 0.05 was considered to be significant
when compared with the controls.
48
2.3 Results
RT-PCR based TLDA revealed that genes involved in drug metabolism
such as PAH responsive CYPs and GSTs were expressed in freshly prepared
lung and PBLs. However ∆Ct values revealed that magnitude of expression of
these CYPs and GSTs were lower in PBL when compared to the levels
observed in lungs (Table 2.1). Comparison of ∆Ct values revealed that basal
expression of CYPs in control rat lungs was in the order:
CYP2E1>CYP1B1>CYP1A1> CYP1A2 while in PBL the order of expression
of CYPs was CYP1B1>CYP1A2>CYP1A1>CYP2E1 (Table 2.1). Similarly the
basal expression of GST isoforms in rat lung was in the order:
GSTM1>GSTP2>GSTM2>GSTO1>GSTA5>GSTK1>GSTO2>GSTM5>GSTM
3, while the basal expression of these GSTs in PBL was:
GSTP2>GSTM1>GSTO1>GSTM5>GSTK1>GSTM3>GSTO2>GSTM2>GSTA
5.
As evident from ∆Ct values PBL was found to express other phase II
and antioxidant genes such as SODs, HO, PRDX2 and MT. However, the
magnitude of expression was less in PBL when compared to the lung. The
transcription factors such as AhR, Arnt, c-Fos, Jun, HIF-1α and signalling
molecules such as MAPKs were also found to be expressed in PBL with
lesser ∆Ct values compared to that seen in lungs (Table 2.1).
Likewise, genes involved in inflammation, apoptosis, DNA repair such as
IL1b, IL-6, IL-12a, IL-12b, TGF-β1 CCL2, CCL5, CSF2, ICAM1, VCAM, BID,
BAD, PDCD8, BBC3 Bcl2, Bcl2l1, Bcl2l2 PARP, OGG1, Top2A and PCNA
were also found to be expressed in PBL. Comparison of ∆Ct values have
shown that basal expression of these mRNA in PBL were lower when
compared to the lungs (Table 2.1).
RT-PCR based TLDA revealed that transtracheal instillation of different doses
(7.5- or 15- or 30 mg/kg) of DEP simultaneously altered the mRNA expression
of genes involved in xenobiotic metabolism, stress response, inflammation,
apoptosis and DNA repair (Tables 2.1& 2.2). The mRNA expression of PAH
responsive CYPs and associated transcription factors (AhR and Arnt) were
increased in the lungs isolated from rats treated with different doses (7.5- or
15- or 30 mg/kg) of DEP. The increase in the expression of PAH responsive
49
CYPs (CYP1A1, 1A2, 1B1 and 2E1) and AhR and Arnt in the lungs at the lower
doses (7.5- or 15 mg/kg) was found to be dose dependent.
Table 2.1: DEP induced alterations in mRNA expression of candidate
genes in lungs and PBL
∆∆Ct =# ∆ Ct values of target gene-endogenous control
Lung Lymphocytes
Gene Control DEP(7.5 mg/kg)
DEP(15 mg/kg)
DEP(30 mg/kg)
Control DEP(7.5 mg/kg)
DEP(15 mg/kg)
DEP(30 mg/kg)
CYPS (Phase I) CYP1A1 9.26 7.82 7.57 8.12 11.65 10.86 10.79 11.05
CYP1A2 9.97 9.21 8.99 9.36 10.66 10.17 9.88 10.39
CYP1B1 7.95 6.63 6.46 6.77 9.78 9.18 8.99 9.38
CYP2E1 6.55 5.29 5.05 5.43 11.85 11.23 10.90 11.44
GSTs (Phase II) GSTA5/YC2 9.98 9.04 8.99 9.17 23.73 23.46 23.32 23.65
GSTM1 5.73 4.78 4.24 5.07 9.34 8.53 8.25 9.01
GSTM2 6.65 5.96 5.33 6.26 17.79 17.25 16.8 17.46
GSTM3 11.90 11.39 11.04 11.63 15.16 14.91 14.82 14.96
GSTM5 11.17 10.39 9.95 10.55 12.30 11.90 11.24 12.18
GSTP2 5.77 4.31 3.89 4.83 7.19 6.32 5.75 6.54
GSTK1 10.21 9.84 9.52 9.19 12.77 12.50 12.37 12.68
GSTO1 9.78 9.37 8.53 9.53 9.30 9.09 8.91 9.18
GSTO2 10.82 9.89 9.46 10.45 16.50 15.90 15.42 16.17
mGST1 6.05 5.66 5.05 5.88 6.75 6.56 6.48 6.63
Antioxidant Enzymes SOD1 5.40 5.08 4.77 5.20 7.03 6.63 6.18 6.68
SOD3 9.25 8.68 8.19 8.77 17.15 16.82 16.79 16.95
PRDX-2 7.39 7.18 6.89 6.99 8.76 8.42 8.35 8.55
MT-1a 6.67 6.51 6.49 4.98 8.42 8.17 7.63 7.25
MT-3 15.19 13.97 11.98 11.62 19.49 18.78 17.75 17.61
OH-1 9.28 8.32 8.25 8.39 9.75 9.56 9.49 9.59
OH-2 9.63 7.76 7.68 7.86 9.37 9.17 9.13 9.21
Transcription factors/ Signaling AhR 9.65 8.95 8.73 9.04 12.20 11.96 11.86 12.00
Arnt 9.21 8.70 8.43 8.93 9.17 8.97 8.91 9.01
MAPK8 10.62 9.83 9.76 9.67 11.58 11.14 10.98 10.71
MAPK9 8.86 8.58 8.20 7.93 10.59 10.34 10.06 9.99
MAPK10 11.65 10.86 9.91 9.88 14.46 13.97 13.86 13.65
50
Values represent mean ± S.E. of 3 experiments. The threshold cycle value (Ct Values) of
each sample was normalized with Ct values of endogenous control [∆Ct]. Fold Change is
calculated from ∆∆Ct# value of each sample.
# ∆∆Ct = ∆Ct of Treated - ∆Ct of Control
c-Fos 9.20 7.50 7.03 6.78 10.58 9.89 9.58 9.43
Fosl1 15.91 14.99 14.92 12.99 16.69 15.82 15.69 15.41
Jun 6.07 5.28 5.14 4.95 10.09 9.40 9.09 8.98
HIF-1α 7.29 6.41 5.84 4.90 8.29 8.13 7.80 7.51
Inflammation IL-1b 10.59 9.11 8.98 8.22 11.73 10.94 10.45 10.23
IL-6 15.34 10.79 10.19 9.8 16.75 15.68 15.13 15.08
IL-12a 10.51 1.75 9.03 8.81 14.77 14.59 14.28 14.17
IL-12b 14.15 13.99 13.78 11.99 19.43 19.03 18.34 18.16
TGF-β 7.14 6.88 6.38 5.99 8.77 8.36 8.28 8.17
CCL2 9.99 8.22 7.57 7.55 12.26 12.06 11.90 11.72
CCL5 6.35 5.47 4.59 3.77 7.57 7.22 7.11 7.04
CSF2 11.82 10.82 10.67 10.38 17.71 17.51 17.37 17.31
ICAM1 6.33 5.33 4.98 4.92 9.35 9.02 8.95 8.75
VCAM1 8.40 8.00 7.54 7.45 19.80 19.62 19.53 19.22
PTGS2 10.11 8.83 8.62 8.56 11.84 11.71 11.66 11.65
NOS2 10.95 9.52 8.53 7.96 11.57 11.37 11.17 10.97
Casp1 9.33 8.27 8.22 7.95 10.62 10.13 9.75 9.60
Oncogene Tp53 6.83 5.87 5.33 4.98 7.98 7.26 7.23 7.17
DNA repair PARP 5.22 4.27 3.78 3.72 8.71 8.13 7.98 7.94
OGG1 10.15 8.77 8.60 8.45 11.73 11.06 10.94 10.86
Top2A 8.33 7.72 7.33 7.18 9.75 9.18 9.15 8.78
PCNA 9.36 8.73 8.24 8.13 9.69 9.48 9.34 9.07
Apoptosis Casp3 8.86 7.31 6.98 6.72 8.98 8.71 8.01 7.96
BID 7.44 5.60 5.37 5.04 7.82 7.62 7.22 7.11
BAD 8.93 8.68 7.96 7.93 11.26 10.86 10.66 10.48
Bcl2 10.56 9.68 9.53 9.33 10.93 10.73 10.49 10.33
Bcl2l1 7.12 6.47 6.05 5.89 9.08 8.90 8.87 8.39
Bcl212 7.86 7.41 7.14 6.97 13.79 13.70 13.45 13.26
PDCD8 8.87 8.84 7.98 7.84 10.29 10.17 9.96 9.72
BBC3 9.88 9.28 9.26 8.98 10.53 10.49 10.44 10.41
51
However, when the dose of DEP was increased to 30 mg/kg, a decline in the
magnitude of induction in CYPs was observed when compared to the lower
doses (7.5- or 15mg/kg). CYP1A1, CYP1B1 and CYP2E1 showed greater
magnitude of increase after treatment of DEP when compared to CYP1A2
(Table 2.2). Similar to that seen with lungs, a dose dependent increase in the
mRNA expression of CYP1A1, 1A2, 1B1& 2E1 was observed in PBL isolated
from rats treated with relatively lower doses (7.5- or 15 mg/kg) of DEP while a
lesser magnitude of increase was observed in rats treated with the highest dose
(30 mg/kg) of DEP. As seen with the alterations of CYPs in lungs, CYP1A1,
1B1& 2E1 showed greater increase when compared to CYP1A2 in PBL.
Though the similar pattern of increase in the mRNA expression of PAH
responsive CYPs was observed in PBL and lungs after treatment of DEP, the
magnitude of induction observed in PBL was lower than that observed in the
lungs (Table 2.2).
Table 2.2: Relative quantification of candidate genes in lungs and PBL of
control and DEP treated rats
Relative Quantification (Fold change)
LUNG Lymphocytes
Gene Name DEP(7.5 mg/kg)
DEP(15 mg/kg)
DEP(30 mg/kg)
DEP(7.5 mg/kg)
DEP(15 mg/kg)
DEP(30 mg/kg)
CYPs (Phase I) CYP1A1 2.71 ± 0.30 3.22±0.45 2.21±0.20 1.72±0.10 1.83±0.20 1.50±0.10
CYP1A2 1.69±0.16 1.97±0.25 1.52±0.11 1.40±0.10 1.71±0.20 1.20±0.10
CYP1B1 2.48±0.20 2.81±0.32 2.26±0.20 1.50±0.10 1.73±0.20 1.31±0.11
CYP2E1 2.40±0.20 2.81±0.41 2.16±0.22 1.52±0.10 1.92±0.25 1.32±0.10
GSTs (Phase II) GSTA5/ Yc2 1.91 ±0.60 1.98±0.50 1.75±0.70 1.20±0.20 1.31±0.30 1.05±0.10
GSTM1 1.93±0.20 2.82±0.43 1.59±0.15 1.74±0.10 2.11±0.23 1.25±0.10
GSTM2 1.61±0.15 2.48±0.36 1.31±0.11 1.44±0.17 1.97±0.3 1.23±0.10
GSTM3 1.40±0.11 1.80±0.20 1.20±0.17 1.18±0.20 1.26±0.20 1.14±0.10
GSTM5 1.71±0.20 2.31±0.30 1.52±0.17 1.32±0.1 2.06±0.28 1.07±0.17
GSTP2 2.73±0.24 3.60± 0.67 1.90±0.2 1.81±0.20 2.70±0.22 1.56±0.23
GSTK1 1.29±0.17 1.60± 1.2 1.05±0.13 1.20±0.20 1.31±0.30 1.06±0.10
GSTO1 1.32±0.90 2.37±1.0 1.19±0.50 1.15±1.0 1.31±1.0 1.07±0.20
GSTO2 1.89±0.2 2.55± 0.32 1.29±0.20 1.50±0.20 2.10±0.50 1.25±0.20
mGST1 1.3±0.17 1.99±0.30 1.11±0.20 1.13±0.20 1.20±0.20 1.08±0.10
Antioxidant Enzymes SOD1 1.24±0.17 1.53±0.22 1.14±0.27 1.31±0.1 1.79±0.1 1.27±0.15
52
SOD3 1.48±0.17 2.08±0.22 1.39±0.3 1.25±0.2 1.28±0. 3 1.14±0.2
PRDX2 1.16±0.17 1.42±0.19 1.32±0.17 1.25±0.1 1.30±0.1 1.15±0.1
MT-1a 1.10±0.15 1.13±0.17 3.20±0.37 1.18±0.15 1.70±0.2 2.23±0.3
MT-3 2.30±0.25 9.23±1.7 11.80±3.8 1.62±0.25 3.34±0.6 3.65±0.7
OH-1 1.93±0.23 2.04±0.25 1.84±0.22 1.13±0.1 1.19±0.2 1.11±0.2
OH-2 3.60±0.37 3.80±0.34 3.40±0.37 1.14±0.2 1.17±0.2 1.10±0.2
Transcription factors/ Signaling AhR 1.60±0.17 1.89±0.3 1.52±0.17 1.18±0.20 1.26±0.10 1.14±0.20
Arnt 1.40±0.17 1.71±0.23 1.20±0.17 1.14±0.1 1.19±0.10 1.11±0.20
MAPK8 1.70±0.17 1.81±0.20 1.92±0.22 1.35±0.2 1.51±0.20 1.82±0.40
MAPK9 1.20±0.12 1.58±0.34 1.90±0.23 1.18±0.2 1.43±0.2 1.51±0.32
MAPK10 1.70±0.17 3.30±0.50 3.42±0.41 1.40±0.2 1.51±0.1 1.74±0.2
c-Fos 3.20±0.40 4.47±1.40 5.30±0.71 1.60±0.2 2.00±0.25 2.21±0.32
Fosl1 2.01± 0.2 2.29 ±0.3 7.60± 2.30 1.89±0.23 1.99±0.27 2.40±0.20
Jun 1.71±0.17 1.90±0.70 2.17±0.27 1.60±0.18 2.00±0.2 2.14±0.2
HIF1a 1.84±0.22 2.73±0.2 5.24±0.68 1.11±0.2 1.40±0.2 1.70±0.3
Inflammation
IL-1b 2.78-±0.25 3.04±0.32 5.14±1.7 1.72±0.20 2.41±0.30 2.82±0.32
IL-6 23.00±6.20 35.00±10.0 46.00±17.0 2.08±0.27 3.07±0.32 3.17±0.50
IL-12a 1.75±0.17 2.79±0.4 3.20±0.27 1.12±0.10 1.40±0.30 1.50±0.22
IL-12b 1.11±0.17 1.29±0.17 4.47±0.17 1.30±0.12 2.10±0.20 2.40±0.22
TGF-β 1.19±0.12 1.69±0.25 2.20±0.27 1.31±0.15 1.40±0.10 1.51±0.23
CCL2 3.40±0.42 5.30±0.80 5.40±0.45 1.14±0.20 1.28±0.2 1.44±0.17
CCL5 1.83±0.19 3.39±0.43 5.97±0.42 1.27±0.20 1.37±0.11 1.43±0.3
CSF2 2.00±0.27 2.20±0.2 2.70±0.25 1.14±0.21 1.26±0.12 1.32±0.22
ICAM1 2.00±0.22 2.55±0.27 2.66±0.40 1.25±0.24 1.31±0.3 1.51±0.32
VCAM 1.30±0.15 1.80±0.20 1.90±0.16 1.12±0.23 1.20±0.23 1.49±0.23
PTGS2 2.41±0.26 2.79±0.3 2.91±0.29 1.09±0.12 1.13±0.22 1.14±0.12
NOS2 2.68±0.17 5.35±0.77 7.94±0.97 1.14±0.20 1.31±0.10 1.50±0.20
Casp1 2.07±0.21 2.14±0.22 2.58±0.21 1.40±0.2 1.81±0.23 2.02±0.3
Oncogene Tp53 1.94±0.27 2.84±0.2 3.58±0.43 1.64±0.20 1.67±0.30 1.74±0.24
DNA repair PARP1 1.91±0.17 2.70±0.20 2.81±0.37 1.49±0.20 1.65±0.30 1.69±0.2
OGG1 2.59±0.26 2.91±0.29 3.22±0.47 1.58±0.20 1.72±0.20 1.81±0.27
TOP2A 1.50±0.18 2.00±0.20 2.20±0.24 1.47±0.20 1.51±0.20 1.94±0.20
PCNA 1.54±0.18 2.16±0.70 2.30±0.29 1.15±0.20 1.27±0.12 1.53±0.20
Apoptosis Casp3 2.91±0.53 3.65±0.82 4.40±0.90 1.20±0.10 1.94±0.20 2.02±0.30
BID 3.50±2.7 4.18±1.20 5.24±1.0 1.14±0.20 1.51±0.10 1.63±0.11
BAD 1.19±0.17 1.96±0.24 1.99±0.29 1.31±0.20 1.50±0.20 1.71±0.20
Bcl2 1.83±0.17 2.04±0.20 2.32±0.22 1.14±0.20 1.34±0.22 1.50±0.15
53
Bcl2l1 1.56±0.17 2.09±0.34 2.34±0.20 1.12±0.20 1.15±0.32 1.60±0.30
Bcl212 1.36±0.17 1.65±0.19 1.85±01.7 1.06±0.20 1.25±0.20 1.43±0.40
PDCD8 1.03±0.14 1.85±0.23 2.03±0.17 1.08±0.20 1.25±0.16 1.47±0.15
BBC3 1.52±0.17 1.53±0.17 1.86±0.19 1.02±0.10 1.05±0.10 1.08±0.10
Values represent mean ± S.E. of 3 experiments. The threshold cycle value (Ct Values) of
each sample was normalized with Ct values of endogenous control [∆Ct].Fold Change is
calculated from ∆∆Ct# value of each sample.
#∆∆Ct = ∆Ct of Treated - ∆Ct of Control
As observed with CYPs, a dose related increase was observed in the
expression of phase II enzymes such as GSTs, SODs (SOD1&3), PRDX2, HO-
1& HO-2 in lungs after transtracheal instillation of DEP. The magnitude of
increase observed in the expression of phase II enzymes in lungs at lower
doses (7.5- or 15 mg/kg) was dose dependent while a lesser magnitude of
increase was observed in the expression of these genes in the animals
receiving highest dose (30 mg/kg) of DEP (Table 2.2). Similar to that seen with
lungs, the increase observed in the phase II genes in blood lymphocytes was of
lesser magnitude when compared to those in the lungs. Likewise, a dose
dependent increase in the mRNA expression of phase II genes such as GSTs,
SODs (SOD1&3), PRDX2, OH-1& OH-2 was observed in PBL isolated from rats
treated with relatively lower doses (7.5- or 15 mg/kg) of DEP while a lesser
magnitude of increase was observed in rats treated with the highest dose (30
mg/kg) of DEP (Table 2.2). In contrast, the mRNA expression of
metallothionein genes such as (MT-1a and MT-3) showed a dose dependent
increase in lungs and PBL isolated from rats treated transtracheally with
different doses of DEP. Further, a lesser magnitude of induction was
observed in the expression of MT-1a and MT-3 in lymphocytes when
compared to lungs (Table 2.2).
As evident from the tables 2.1 & 2.2, significant dose dependent
increase in the mRNA expression of transcription factors (HIF-1α, c-Fos, Fosl
& Jun) and signalling molecules such as MAPKs (MAPK8, MAPK9 &
MAPK10) was observed in lungs and PBL isolated from DEP treated rats. A
dose dependent increase in the mRNA expression of HIF-1α, c-Fos, Fosl1,
Jun, and MAPKs was observed in both, lymphocytes and lungs of DEP
treated rats. However the magnitude of increase in the expression of these
54
transcription factors and signalling molecules was lesser in lymphocytes
compared to lungs
TLDA data further demonstrated a dose dependent increase in the
expression of inflammatory response genes pertaining to cytokines,
chemokines, adhesion molecules and inflammatory enzymes in both
lymphocyte and lung isolated from DEP treated rats. A dose- dependent
increase was observed in the mRNA expression of interleukins such as IL-1 ,
IL-6, IL-12a, IL-12b and TGF-β1 in both PBL, and lungs isolated from rats
treated transtracheally with DEP (Table 2.2). The increase in the mRNA
expression of IL-6 was considerably higher than other interleukins in both
lungs and PBL isolated from different doses of DEP. However, the magnitude
of induction observed in lymphocytes was less when compared to the lungs.
Chemokines and adhesion molecules involved in inflammation, such as
CCL2, CCL5, CSF2, ICAM1, and VCAM also showed similar pattern of
induction in both lungs and PBL isolated from rats treated transtracheally with
DEP. Further, inflammatory enzymes like PTGS2 and NOS2 were also
induced dose dependently in lungs and lymphocytes of rats exposed to DEP.
Among the DNA repair enzymes, PARP, OGG1, Top2A and PCNA also
showed dose dependent increase in lungs and lymphocytes isolated from
DEP treated rats. As observed with drug metabolizing enzyme, though the
pattern of increase was similar to that seen in lungs, the magnitude of
induction was higher in lungs when compared to PBL. (Table 2.2)
Similarly, proapoptotic genes such as BID, BAD, PDCD8 and BBC3
and antiapoptotic genes such as Bcl2, Bcl2l1, Bcl2l2 also showed dose
dependent and similar pattern of induction at all the doses (7.5- or 15- or
30mg/kg) of DEP in both lymphocyte and lungs of DEP treated rats. Further,
protooncogene (Tp53) and apoptotic enzyme (Caspase 3) were also induced
dose dependently in lungs and lymphocytes of DEP treated rats. However,
the magnitude of alterations was lesser in PBL when compared to the lungs
(Table 2.2).
2.4 Discussion
The present study using low density array have shown that
transtracheal instillation of DEP induces a similar pattern of increase in the
55
expression of PAH responsive CYPs (CYP1A1, 1A2, 1B1, 2E1), the phase I
and GSTs, the phase II enzymes in both, lungs and PBMCs isolated from rats
treated with the different doses of DEP. A dose dependent increase in the
expression of SOD, HO-1, HO-2, PRDX2 and MT, the other phase II
enzymes, was also observed in both, lungs and PBMCs isolated from rats
treated with different doses of DEP. Previous studies including those using
DNA array have shown that exposure to DEP affects the expression of
various genes associated with with xenobiotic metabolism (CYP1A1, CYP1B1
and GSTs), inflammation, oxidative stress response (OH-1, metallothionein),
protooncogenes, DNA repair genes and apoptosis (Sato et al., 1999;
Reynolds and Richards, 2001; Yanagisawa et al., 2004; Verheyen et al.,
2004; Omura et al., 2009 & Koike et al., 2002, 2004). The oedematous
changes in the lungs after DEP exposure was associated with increase in the
expression of stress related genes (Reynolds and Richards, 2001). The stress
related genes such as metallothionein, heat-shock protein 47, and
inflammatory response genes like Serum amyloid A3, calcium binding
proteina-9 and lipocalin 2 were found to be induced which exacerbates acute
lung injury in mice (Sato et al., 1999; Yanagisawa et al., 2004). Based on the
differential expression of HO-1 and TGM-2, these genes were also identified
as possible biomarker of PM exposure (Koike et al., 2004).
The present alterations in the DMEs were associated with a dose dependent
increase in the expression of several inflammatory mediators like chemokines
(CCL2, CCL5), cytokines (IL-1β, IL-6, TGF-β and IL-12) and adhesion
molecules (ICAM1, VCAM) in both, lungs as well as PBMCs isolated from
DEP treated rats. Similar upregulation of several genes known to be involved
in inflammation and ROS generation including proinflammatory cytokines,
antiinflammatory cytokines, intercellular adhesion molecule and chemokines
has been reported in human umbilical vein endothelial cells (HUVEC) or lung
derived cell lines exposed to carbon black particles or DEP or in lungs
isolated from DEP treated animals (Sen et al., 2007; Park et al., 2011;
Schwarze et al., 2013). Microarray studies in HUVECs exposed to carbon
black particles revealed that several genes, known to be involved in vascular
inflammation such as ICAM1, IL-8, SELE, PTGS2, CCL2, were significantly
upregulated suggesting that induction of vascular inflammatory genes helps in
56
exacerbating atherosclerosis and ischemic heart disease (Yamawaki et al.,
2006). Takano et al., (2002) showed inflammatory response in mice after DEP
treatment was associated with increased local expression of IL-1β, ICAM-1
and chemokines such as MIP-1α, MCP-1 and KC. Saber et al., (2006)
reported increase in expression of inflammatory markers (IL-6, KC and CCL2)
in lungs of mice at different time points after DEP inhalation. Yokota et al.,
(2008) showed that particle component of DEP enhance myocardial oxidative
stress together with the upregulation of cytokines such as GMCSF, IL-1, IL-6
and CXCL2 in BALF and found strong correlation between cytokine levels and
increase in the risk of cardiovascular mortality and morbidity. However,
several fold increase observed in the present study in the mRNA expression
of IL-6 in the lungs of DEP treated rats could possibly be due to the use of
more invasive transtracheal method employing surgery for instillation of DEP
in rats (Zins et al., 2010).
As reported earlier (Totlandsdal et al., 2012; Srivastava et al., 2012),
the induction in the phase I and phase II DMEs observed at low doses (7.5- or
15 mg/kg) of DEP was found to be dose-dependent, while at higher dose (30
mg/kg), the magnitude of induction was less when compared to the lower
doses. The suppression of the increase observed in the expression of various
DMEs in the lungs and PBMCs isolated from animals receiving the highest
dose of DEP (30 mg/kg) could be possibly explained by the dose dependent
increase observed in the expression of several inflammatory mediators like
chemokines (CCL2, CCL5), cytokines (IL-1β, IL-6, TGF-β and IL-12) and
adhesion molecules (ICAM1, VCAM) in the lungs or PBMCs isolated from
DEP treated rats. Totlandsdal et al., (2010) have also earlier shown that DEP
exposure in Beas-2B cells induced both CYP1A1 and proinflammatory
response cytokines such as IL-6, IL-8 and COX-2. DEP induced
proinflammatory response seems to occur via activation of NF-kB and p38
differential pathways and was facilitated by CYP induction. Interestingly, the
increased expression of CYP1A1 was suppressed at higher doses, while the
proinflammatory genes continue to show increased expression of cytokines.
The lesser magnitude of induction has also been reported with methanolic
extract of DEP (containing higher concentrations of PAH) when compared to
native DEP and has been attributed to the increased formation of
57
proinflammatory molecules, leading to oxidative stress, which in turn has a
suppressive effect on CYPs (Totlandsdal et al., 2010, 2012).
Further, dose dependent increase observed in the expression of caspases,
particularly caspase1 in lungs, have suggested that exposure to DEP leads to
the activation of proinflammatory cytokines including IL-1β, which triggers the
recruitment of adhesion molecules, growth factors and chemokines involved
in the initiation and amplification of inflammatory response and generation of
ROS (Dinarello, 2009; Denes et al.,2012). Induction of proinflammatory IL-1β
expression, both dependent and independent of caspase activation was
demonstrated in DEP exposed mice while the involvement of caspase 1 in IL-
1β maturation was reported in murine models (Provoost et al., 2011).
Likewise, the dose dependent increase was observed in the expression of
inducible nitric oxide synthase (NOS2), which stimulate cellular generation of
ROS as well as reactive nitrogen speciesin both PBMCs and lungs after
exposure of different doses of DEP. A positive co-operation of iNOS in DEP
induced lung inflammation and CYP induced mutagen activation was reported
in rats treated with DEP (Zhao et al., 2006; Amara et al., 2007). This increase
in expression of NOS2 may also help in explaining the lesser magnitude of
increase observed in the lungs isolated from rats receiving the highest dose of
DEP (Zhao et al., 2006; Amara et al., 2007). Interestingly, similarities in the
dose dependent increase in the expression of various inflammatory molecules
were observed in both PBMCs and lung. Array studies using PBMCs have
earlier reported time dependent effects on inflammation and oxidative stress
after inhalation of DEPs in healthy individuals (Peretz et al., 2007).
Increase in the expression of transcription factors such as AhR, Arnt c-
Fos, Fosl1, Jun, HIF-1α and signaling molecules such as MAPK in lungs or
PBMCs isolated from DEP treated rats has provided support to the previous
studies demonstrating their role in the regulation of phase I and phase II
DMEs. In addition to the binding of PAHs present in DEPs with AhR which
then heterodimerizes with Arnt to induce the expression of CYP1-
isoenzymes, stimulation of MAPK has also been shown to be critical for the
induction of AhR dependent gene transcription of PAH-metabolizing CYPs
(Tan et al., 2002; Androutsopoulos et al., 2009; Murray, 2010). Vogel et al.,
(2005) reported that DEP particles and organic extract induced the expression
58
of CYP1A1 which was suppressed by co-treatment with AhR antagonist
suggesting that CYP1A1 induction was mediated by binding of PAH present in
DEP with AhR.
It has been shown that phase I and II enzyme induction is
mechanistically dependent on the activation of AP-1, which is a heterodimeric
complex of Jun and Fos (Zang et al., 2004). Likewise, HIF-1α is involved in
regulating PAH-mediated induction of CYP1A and 1B1 by activating hypoxia
responsive genes and decreasing the availability of Arnt (Chan et al., 1999;
Androutsopoulos et al., 2009). Xu et al., (2009) have earlier shown that
exposure to diesel exhaust in mice significantly increased HIF-1α and VEGF
thereby inducing angiogenesis and vasculogenesis. However Bradley et al.,
2013 showed that chronic exposure to DEP in urban population significantly
decreased the cardiac expression of hypoxia inducible factor-1α with increase
in the expression of AhR which mediates CYP induction.
DNA array studies have earlier reported alterations in the expression of
various stress related genes such as MAPK2, MAPK5, cyclin D1,
prothymosin-alpha, DNA topoisomerase and multidrug resistant protein in
lungs isolated from rats treated with DEP ((Sen et al., 2007)). Our data
demonstrating similarities in increase in the expression of MAPK, Jun and Fos
in PBMCs and lungs isolated from DEP treated rats have indicated that
mechanisms similar to that observed in lungs exist in blood cells. Recent
study from our laboratory has demonstrated similarities in the activation of
CYP2E1 and associated MAP kinase and c-Jun in PBMCs and liver after
exposure to ethanol (Sharma et al., 2012). Likewise, dose dependent
induction of HIF-1α, in both, lungs and PBMCs after DEP exposure have
suggested that the induction of hypoxia pathway is also reflected in blood
cells after DEP exposure (Xu et al., 2009).
That the DEP induced inflammation and oxidative stress is associated
with DNA damage was further demonstrated by the present study indicating a
dose dependent increase in the expression of various DNA repair enzymes
like DNA glycosylase (OGG1), DNA topoisomerase, poly ADP ribose
polymerase and PCNA in lungs and PBMCs isolated from DEP treated rats.
Previous study including DNA array studies have reported that exposure of
DEP leads to the upregulation of DNA repair genes, which are known to be
59
induced in response to increased generation of ROS, in lungs and liver of
DEP treated animals (Wan and Diaz-Sanchez, 2007; Sen et al., 2007). Dose
dependent increase in the expression of p53 mRNA, a proto-oncogene in
lungs and PBMCs have further indicated the existence of p53 dependent
pathway regulating the transcription of genes involved in DNA damage repair
pathways and apoptosis induced by DEP (Landwick et al., 2007).
Significant increase in the expression of various proapoptotic,
antiapoptotic genes as well as proto-oncogenes and signaling molecules in
lungs and PBMCs have suggested that DEP exposure leads to a cascade of
events culminating in apoptosis. Consistent with the previous reports that
DEP triggers mitochondrial apoptotic pathway (Hiura et al 1999, 2000), a dose
dependent increase was observed in the expression of various proapoptotic
proteins like BbC3, PDCD8, BID and BAD (Bcl2 family) as well as caspase 3
and transcription factors (like Jun and Fos) and MAPK in lungs and PBMCs
isolated from DEP treated rats. Likewise, increase in the expression of
antiapoptotic genes of Bcl2 family like Bcl2, Bcl2l1, Bcl2l2, though of a smaller
magnitude, on exposure of DEP in both PBMCs and lungs, have suggested
that imbalance between proapoptotic and antiapoptotic signals leading to
apoptosis, are reflected in PBMCs. DNA array studies have also earlier
demonstrated differential expression of proapoptotic and antiapoptotic genes
in blood cells of relapsing-remitting multiple sclerosis patients (Achiron et al.,
2007).
Though the present study has provided important insights into the
mechanism of occurrence of various cellular events associated with acute
toxicity of DEP, the study has limitations when compared to the exposure of
DEPs observed in real life situations. As compared to the single dose
instillation of DEP in the present study, humans are likely to be exposed
chronically to DEP (Corfa et al., 2004; Buzzard et al., 2009). The present
acute study may have limitations in differentiating between the chronic effects
of DEP reported with long term exposure of exhaust particles. Further, in
addition to the particles, humans are also exposed to gases and semi- volatile
compounds present in the diesel exhaust. The physicochemical properties of
DEP may also vary markedly depending on the engine type and maintenance
conditions.
60
The results of the present TLDA study demonstrating significant
increase in the expression of various DMEs, associated transcription factors,
inflammatory signalling molecules as well as proapoptotic and antiapoptotic
genes in rat lungs and PBMCs after exposure to DEP suggests that mRNA
expression profiles could be used to monitor these events, which are cross
linked in inducing toxic manifestations in rat lungs. Further, similarities in the
alterations in expression profiles of these genes in PBMCs with the lungs
suggest that low density array of these selected genes in blood cells has the
potential to be utilized as a preliminary screen to monitor DEP induced toxicity
in individuals exposed to vehicular emissions.
Chapter 3
Similarities in diesel exhaust particles induced alterations in expression of cytochrome P-450 and glutathione S-transferases in rat lymphocytes and lungs
61
Chapter 3
Similarities in diesel exhaust particles induced alterations in expression
of cytochrome P-450 and glutathione S-transferases in rat lymphocytes
and lungs
3.1 Introduction
In the preceding chapter it was shown that transtracheal instillation of
different doses of DEP to adult wistar rats was found to induce a similar
pattern of increase in the expression of polycyclic aromatic hydrocarbon
(PAH) responsive cytochrome P450s (CYPs), glutathione-S-transferases
(GSTs), the phase II enzymes and their associated transcription factors in
both, lungs and peripheral blood lymphocytes (PBL) at all the doses. Similar
to that seen in lungs, this dose related increase in the expression of drug
metabolizing genes was associated with the increase in the expression of
genes involved in inflammation such as cytokines, chemokines and adhesion
molecules in PBL isolated from rats treated transtracheally with DEP. The
expression of various genes involved in DNA repair and apoptotic genes were
also increased in a dose dependent manner in PBL and lungs.
Studies have shown that cytochrome P450s (CYPs) induced by DEPs
leads to bioactivation of diesel exhaust ingredients resulting in pulmonary
toxicity (Yamasaki et al., 2000). Organic components of DEP are known to
induce CYP1A1 enzymes which may lead to pulmonary inflammation (Zhao et
al., 2006; Totlandsdal et al., 2010). Intratracheal instillation or inhalation of
DEP has been reported to increase the pulmonary expression of CYP1A1 at
both mRNA and protein level in mice and rats (Sato et al., 2000; Hatanaka et
al., 2001; Ma & Ma, 2002; Rengasamy et al., 2003). Induction of CYP1B1 in
lung, liver and kidney was also reported along with CYP1A1 in rats exposed
to DEPs (Hatanaka et al., 2001). However, a decrease in the CYP2B1
enzyme activity was observed after DEP exposure in rat lungs (Rengasamy et
al., 2003). Organic extract of DEP also induces ROS generation through the
CYP system during the catalytic cycle (Puntarulo and Cederbaum, 1998).
DEPs are also known to affect the activity of pulmonary antioxidant enzymes
such as glutathione-S-transferases (GSTs) and glutathione (GSH) contents in
62
animals and humans exposed to these particles (Al-Humadi et al., 2002;
Hirano et al., 2003; Omura et al., 2009). Elevated levels of PAH-derived DNA
adducts have also been observed in lymphocytes of humans following DEP
exposure (Hemminki et al., 1994).
To further demonstrate suitability of using PBL as a surrogate to
monitor toxicity of DEP, the present study attempted to investigate similarities
and differences, if any, in the alterations in the expression of CYPs and GSTs,
involved in the toxicity of DEP in freshly prepared PBL and lungs isolated from
rats exposed to different doses of DEPs. Though previous studies from our
laboratory have demonstrated similarities in the regulation of xenobiotic
metabolizing CYPs with the tissue enzymes (Dey et al., 2001, 2006; Saurabh
et al., 2010, 2011; Sharma et al., 2011), there are reports that failed to find
correlation of CYP mRNA expression with their functional activity (Finnstrom
et al., 2001; Haas et al., 2005). The present study, therefore, also attempted
to correlate alterations in mRNA and protein expression of CYPs with the
catalytic activity of DEP responsive drug metabolizing enzymes in PBL
prepared from rats exposed to DEP.
3.2 Materials and methods
3.2.1 Chemicals
7-Ethoxyresorufin, 7-methoxyresorufin, 7-pentoxyresorufin, resorufin,
N-nitrosodimethyl amine (NDMA), thiobarbituric acid (TBA), histopaque1077,
poly-l-lysine, phenylmethyl sulfonyl fluoride (PMSF), NADPH, dithiothreitol
(DTT), protease inhibitor cocktail, bromophenol blue, goat anti-rabbit IgG-
alkaline phosphatase com- plex, 5-bromo-4-chloro-3-indolyl phosphate
(BCIP), nitrobluetetrazolium (NBT), acrylamide, bisacrylamide and other
chemicals used in SDS-PAGE were procured from Sigma–Aldrich, St. Louis,
MI, USA. Rabbit anti-rat cytochrome P450 1A1 (CYP1A1), CYP1A2 and
CYP2E1 were procured from Millipore Corp. (MA, USA). Fluorescent labeled
secondary antibody was purchased from Invitrogen. All other routine
chemicals were procured from SISCO Research Laboratories Pvt. Ltd., India
or E. Merck, India.
63
3.2.2 Animals and treatment
The suspension of DEP (National Institute of Standards and
Technology, Standard Reference material 2975, Gaithersburg, MD) was
prepared as mentioned in Section 2.2.2 of the preceding chapter. Adult male
albino Wistar rats (8 week old) were divided into five groups, each containing
six animals. Rats in these groups were treated with different doses of DEP
(3.75- or 7.5- or 15- or 30 mg/kg body weight) or normal saline (controls)
instilled transtracheally into the rats as described in Section 2.2.2 of Chapter
2. The animals were anaesthetized 24 h after the administered dose and
blood and lungs were isolated as mentioned in Chapter 2.
3.2.3 Isolation of lymphocytes
Lymphocytes were isolated from the blood by the method as described
in our earlier studies (Dey et al., 2001). In brief, 4.0 ml of whole blood was
diluted with 4.0 ml of phosphate buffered saline (PBS), pH 7.4, and carefully
layered over 2.0 ml of histopaque 1077. After centrifugation at 400 × g for 30
min at room temperature, the upper layer was discarded and the opaque
interface containing mononuclear cells was transferred into a clean centrifuge
tube. After repeated washing of the lymphocytes with PBS and
recentrifugation at 250 × g, the resulting pellet was resuspended in 0.5 ml of
PBS. The number of lymphocytes was counted using a haemocytometer and
the viability of the cells was assayed by the trypan blue exclusion test.
Approximately 2–3 × 106 cells were present in 0.5 ml lymphocyte suspension
drawn from control rats. The viability of these cells was above 95%.
Microsomes were also isolated from lymphocytes for western blotting studies
as reported earlier (Hannon- Fletcher et al., 2008).
3.2.4 Preparation of microsomes
Lungs were perfused with ice cold normal saline to remove the blood and
then excised removing the heart. These perfused lungs were homogenized in 4
volumes of ice cold 0.25 M potassium phosphate buffer, pH 7.25, containing
0.15 M KCL, 0.25 mM PMSF, 0.01 M EDTA and 0.1 mM DTT (dithiothrietol).The
resulting homogenization mixture was centrifuged at 14,000 x g for 20 minutes
and supernatant S9 fraction was taken (Parmar et al., 1998).This S9 fraction
64
was again centrifuged at 40,000 rpm for 60 minutes to separate the microsomes
and cytosolic fractions. The pellets were resuspended in microsomal dilution
buffer containing 0.1 M potassium phosphate buffer, pH 7.25, 20%(v/v) glycerol,
0.25 mM PMSF, 0.01 M EDTA and 0.1 M DTT and stored at -80ºC for analysis.
3.2.5 Protein estimation
Protein content of the sample was measured by Lowry et al., (1951).
Protein sample of a suitable volume was taken and diluted to 1 ml by distilled
water. To this sample 5 ml of alkaline copper reagent was added (2% copper
sulphate, 2% sodium potassium tartrate, 2% sodium carbonate in 0.2N sodium
hydroxide) was added. The solution was kept for 10 minutes at room
temperature. The final blue colour was developed by addition with 0.5 ml of
Folin- Ciocalteau reagent (1N). The final solution was kept for 30 minutes at
room temperature. The intensity of colour as a measure for concentration was
measured at 660 nm in a visible spectrophotometer. The amount of protein was
determined by comparing it with the standard protein curve of BSA.
3.2.6 EROD and MROD assay
The activity of 7-ethoxyresorufin-O-deethylase (EROD) and 7-
methoxyresorufin-O-deethylase (MROD) a catalytic marker of CYP1A1 and
CYP1A2 catalysed reactions respectively were determined in rat lungs and
freshly prepared peripheral blood lymphocytes by the method of Parmar et al.,
(1998). The reaction mixture in 1.25 ml contained 0.05 M Tris, pH 7.2, 0.025 M
MgCl2, 5 M methoxyresorufin (MR), 500 M NADPH and a suitable amount of
lung or lymphocyte protein. The reaction was started with NADPH and
incubated for 30 minutes at 37ºC in case of both lungs and lymphocytes.
Reaction was stopped with 2 ml methanol and the mixture was centrifuged at
2000 rpm for 7 minutes. Levels of resorufin in the supernatant was measured
using a Perkin Elmer LS55 Luminescence Spectrometer at excitation
wavelength of 550nm and emission wavelength of 585nm with a slit width on
10nm each and integration time of one second.
65
3.2.7 PROD assay
The activity of 7-pentoxyresorufin-o-deethylase (PROD), a catalytic
marker for CYP2B1/2B2 was determined in rat lungs and freshly prepared
peripheral blood lymphocytes by the method of Parmar et al., (1998). The
reaction mixture in 1.25 ml contained 50 mM Tris pH7.5, 25 mM MgCl2, 10 µM
pentoxyresorufin (PR), 50 µM NADPH and a suitable amount of lung or
lymphocyte microsomal protein. The reaction was started with NADPH and
incubated for 10 minutes at 37ºC in case of lungs and 30 minutes in case of
lymphocytes. The reaction stopped by adding 2 ml methanol. The resulting
mixture was centrifuged at 2000 x g for 10 minutes. Levels of resorufin in the
supernatant was measured using a Perkin Elmer LS55 Luminescence
Spectrometer at excitation wavelength of 550 nm and emission wavelength of
585 nm with a slit width on 10nm each and integration time of 1 second.
3.2.8 N-nitrosodimethylamine demethylase (NDMA-d) assay
N-nitrosodimethylamine demethylase (NDMA-d) activity was assayed
in blood lymphocytes and lung microsomes by a slight modification of the
method of Yadav et al., (2006). The assay mixture contained a suitable
amount of lymphocytes or lung microsomes, 70.0 mM Tris-HCl, pH 7.4, 10
mM semicarbazide, 14 mM MgCl2, 215 mM KCl, 1mM NADPH and 4 mM
NDMA in 1.0 ml final volume. The reaction mixture was incubated at 37oC for
30 minutes and the reaction was stopped by the addition of 0.1 ml of 25% zinc
sulphate and 0.1 ml of saturated solution of barium hydroxide. After
centrifugation at 2000 rpm for 10 minutes, 0.7 ml of the supernatant was
mixed with an equal amount of Nash reagent. The tubes were then incubated
at 70oC for 20 min and the HCHO formed was measured at 415 nm.
3.2.9 GST assay
The activity of GST was measured as described by Habig et al., (1964).
The reaction mixture 1ml contain 0.2 M phosphate buffer (pH 6.5), 1mM
reduced Glutathione (GSH), 1mM 1-chloro 2, 4-dinitrobenzene (CDNB) and
suitable amount of lung cytosol and lymphocyte. The increase in absorbance of
the GST conjugate was measured using UV spectrophotometer at 340 nm. The
specific activity was expressed as nanomoles conjugate per minute per
66
milligram protein.
3.3 GSH assay
GSH content was measured as described by Ellman et al., (1959) with
slight modification in lung tissue and blood. Lung tissue was homogenized in 0.2
M phosphate buffer and 5% TCA was added and mixture was kept at RT for 30
minutes. The resulting mixture was centrifuged at 2500 rpm/15min.To the
supernatant 0.01% DTNB reagent was added and incubated for 15 minutes.
The colour formed by the reaction of GSH and DTNB in buffer at the
supernatant side was evaluated on spectrophotometer in 412 wavelength and
the results were expressed as µmoles GSH formed/g tissue. In case of blood,
0.02 ml of blood was taken and then diluted with water and them 1 ml of 0.2M
phosphate buffer (pH 8) was added together with 0.01% DTNB. The
absorbance was measured on spectrophotometer after 1 hour and the results
are expressed as µmoles GSH formed/ml blood.
3.3.1 Lipid peroxidation assay
Lipid peroxidation was measured as described by Dey et al., (2002) in
blood lymphocytes and lungs isolated from DEP exposed rats.The reaction
mixture in 2.0 ml contained 200 µm NADPH, 0.2 M potassium phosphate
buffer and a suitable amount of lymphocyte protein. The reaction mixture was
incubated for 20 min at 37°C. After incubation, 30% TCA was added and
reaction mixture was centrifuged at 3000 rpm for 20 minutes. 1ml of
supernatant was mixed with equal amount of 0.67% TBA and kept in a boiling
water bath for 10 mim and the MDA formed was determined at 535 nm in
spectrophotometer. The MDA formed was expressed in nmolesMDA
formed/min/mg protein.
Lipid peroxidation in lung tissue was measured using the method
described by Satoh et al., (1978) with slight modification. 1ml of 10%
homogenate was incubated for 1 hour at 37°C and 10% TCA was added.
After centrifugation at 2500 rpm for 10 min, the resulting supernatant was
treated with 0.67% TBA and kept in boiling water bath for 10 minutes. The
colour produced was measured in spectrophotometer at 530 nm. The MDA
formed was expressed in nmoles MDA formed/hr/gm tissue.
67
3.3.2 RNA isolation
Total RNA was isolated from whole blood by TRIzol LS and from lung
by TRIzol reagent (Life Technologies, USA) according to the manufacturers’
protocol as described in Section 2.2.3 of Chapter 2.
3.3.3 Semi-quantative RT-PCR analysis
cDNA was synthesized essentially as described in study by Johri et al.,
(2006). For RT, the reaction mixture in 20 l contained 1X cDNA synthesis
buffer, 0.5U RNAse , 1mM dNTPs mix, 200 units of Revert Aid TM H Minus M-
MuLV Reverse Transcriptase (1 unit / µl ) of MBI Fermentas and the Oligo
(dT)20 primed mRNA from the previous step. RT reaction was carried out by
incubating the reaction mixture at 42oC for 60 min. The reaction was
terminated by incubating the mixture at 70oC for 10 min. 1µl of RNAse H was
then added to the cDNA and the mixture was incubated at 37o C for 20 min.
Reactions without RNA was also carried out which served as the negative RT
control. Prior to the amplification of CYPs, normalization was carried out with
β-actin, the housekeeping reference gene. The PCR reaction mixture for
CYP1A1, 1A2, 1B1, AhR, Arnt, GSTPi, GSTM1and GSTM2 in 50 µl l
contained 1X PCR buffer, 0.2mM dNTPs mix, 0.2 -0.4 µM of each CYP
primers, 2 µl cDNA and 1.5 U Taq DNA polymerase from MBI Fermentas,
USA. MgCl2 at the final concentration of 1.0-3.0mM. PCR products were
analyzed by agarose gel electrophoresis using VERSA DOC Imaging System
Model 1000 (Bio-Rad, USA). The densitometry was performed using Quantity
One Quantitation software of Bio-Rad.
3.3.4 Quantitative Real time-PCR (RT-PCR) analysis
For quantitative RT-PCR analysis (qRT-PCR), cDNA was synthesized
by High-Capacity cDNA Reverse Transcription Kit (RT) (Applied Biosystems,
USA) as described by Shah et al.,(2009) and has been described in Section
2.2.4 of Chapter 2. The sequence of primers used for CYP1A1, CYP1A2,
CYP1B1, and β-actin has been described by Baldwin et al., (2006). The
primers for GSTPi, GSTM1 and GSTM2 were procured from ABI
(Rn02770492_gH, Rn00755117_m1& Rn00598597_m1 respectively). The
PCR reaction mixture for CYP1A1, 1A2, 1B1, GSTM1, GSTM2, GSTP1 and
68
β-Actin in 20 μl contained 1X TaqMan Universal PCR Master Mix (Applied
Biosystems, Foster city, California, USA), 10 pM of each gene primer, 4 pM
of each gene probe, 2 μl cDNA and nuclease-free H2O. TaqMan assays for
each gene target were performed in triplicate on cDNA samples in 96- well
optical plates on an ABI 7900HT Fast Real-Time PCR System (Applied
Biosystems). PCR conditions were as follow: 50°C for 2 min, 95°C for 10 min
followed by 40 cycles of 95°C for 15 s and 60°C.
3.3.5 Immunoblot analysis
CYP1A1/CYP1A2 and CYP2E1 isoenzymes were identified by western
blot analysis in lungs of control and DEP treated rats as described by Towbin
et al., (1979). Prior to immunoblotting studies, lung microsomes were
solubilised in buffer containing 1mM dithiothreitol, 1mM EDTA, 0.2% emulgen
911 and 20% glycerol for one hour at 4oC. After solubilization, samples were
recentrifuged at 10,000 rpm for 20 minutes and used for immunoblotting
studies. The solubilised lung microsomal protein (100 µg) was subjected to
SDS-PAGE (3% acrylamide stacking gel and 7.5% acrylamide separating gel)
and processed for western blotting. The membranes after transfer were
incubated with primary antibody (1:500 dilutions) overnight at 37°C. The
membranes after washing were incubated with secondary antibody (alkaline
phophatase conjugated goat anti-rabbit) at 1:10,000 dilutions for 30 min at
room temperature. After washing the colour was developed using 5-bromo-4-
chloro- 3-indolyl phosphate (BCIP) and nitrobluetetrazolium (NBT).
Densitometric analysis of the bands was carried out using Quantity one
quantitation software version 4.3.1(Bio-Rad, Hercules, California, USA).
3.3.6 Immunocytochemistry
Freshly isolated PBL from control and DEP treated rats (1 × 106
cells/ml) were seeded on to PLL coated coverslips in culture media using
RPMI 1640 supplemented with 10% fetal bovine serum and 50 mM Hepes,
pH 7.4. Cells were then allowed to adhere for 24 h under high humid
environment in 5% CO2 − 95% atmospheric air at 37°C. After 24 h, media was
removed and cells were washed with PBS and fixed with 4%
paraformaldehyde at 37°C for 20 min, followed by washing with PBS three or
69
four times. Cells were further incubated with 0.5% H2O2 (w/v) in methanol for
1 h. After washing the cells with PBS, cells were incubated in blocking buffer
containing 0.02% Triton X100 and 0.1% BSA in PBS for 15 min. The cells
were then incubated with 1:100 dilution of polyclonal antibody of CYP1A1/1A2
or CYP2E1 (primary antibody). The cells were then washed with PBS and
incubated with anti-rabbit FITC labeled secondary antibody (1:1000 dilution)
for 1 h. The cells were then counterstained with DAPI (nuclear stain)
containing antifade. The cells were observed under fluorescence microscope
(Leica Qfluro Standard, Leica Microsystems Imaging Solutions Ltd.,
Germany). Experiments were performed at least three times, and on average
20 fields were evaluated for double blind scoring on each slide.
3.3.7 Statistical Analysis
Students `t’ test was employed to calculate the statistical significance
between control and treated groups. P<0.05 was considered to be significant
when compared with the controls.
3.4 Results
3.4.1 Effect of DEP on drug metabolizing enzymes in rat lungs
Transtracheal instillation of different doses of DEP resulted in
significant alterations in the activity of CYP dependent monooxygenases in rat
lungs 24 hours after the treatment (Table 3.1). As evident from the table, a
dose related increase in the activity of CYP1A1 and 1A2 dependent EROD
and MROD activity was observed in lung microsomes isolated from rats
treated with 3.75- or 7.5- or 15- or 30 mg/kg of DEP, with maximum increase
being observed in rats receiving the dose of 15 mg/kg of DEP. Statistical
analysis revealed no significant effect in the activity of EROD or MROD in
lung microsomes isolated from rats treated with the lowest dose (3.75 mg/kg)
of DEP while the increase observed in the activity of EROD and MROD was
found to be statistically significant in lungs isolated from rats treated with
higher doses (7.5- or 15- or 30 mg/kg) of DEP (Table 3.1). Though the
increase observed in the activity of pulmonary EROD or MROD in rats treated
with the highest dose of DEP (30 mg/kg) was found to be statistically
70
significant when compared to the controls, the magnitude of increase in
enzyme activity subsequently declined when the dose of DEP was increased
to 30 mg/kg than in the lungs isolated from rats exposed to relatively lower
doses (15 mg/ kg and 7.5 mg/kg) of DEP (Table 3.1).
In contrast, CYP2B1 dependent PROD activity showed a dose dependent
decrease in the enzyme activity. This decrease in the activity of PROD in lung
was found to be statistically significant at 7.5, 15 and 30 mg/kg doses of DEP.
However, the decrease observed in the activity of CYP2B1 dependent PROD
at lowest dose (3.75mg/kg) of DEP was not statistically significant (Table 3.1).
As observed with EROD and MROD, CYP2E1 dependent NDMA-d activity
also showed an increase in enzyme activity in lung microsomes isolated from
rats exposed to different doses of DEP. The increase in the activity of NDMA-
d was found to be dose-dependent upto 15 mg/kg of DEP. The magnitude of
increase in the enzyme activity then declined in the lung microsomes isolated
from rats treated transtracheally with the highest dose of 30 mg/kg when
compared to rats receiving 7.5- or 15 mg/ kg of DEP. Though the increase in
the activity of NDMA-d at highest dose was relatively less when compared to
the lower dose of 7.5- or 15 mg/kg, the increase observed was statistically
significant when compared to the controls. However the increase observed at
lowest dose (3.75 mg/kg) was not found to be statistically significant (Table
3.1).
Transtracheal instillation of different doses of DEP was found to increase the
activity of cytosolic GSTs 24 hours after the treatment. Dose dependent
increase was observed upto 15 mg/kg DEP dose and the increase in enzyme
activity subsequently declined in the lungs isolated from the rats treated with
the highest dose (30 mg/kg) of DEP. Though this increase in the activity of
GST was relatively less when compared to rats receiving relatively lower
doses (7.5- or 15 mg/kg) of DEP, the increase in the activity of GST in the rats
receiving the highest dose (30 mg/kg) was statistically significant as observed
in the animals receiving relatively lower doses (7.5- or 15 mg/kg) of DEP.
Statistical analysis revealed that the increase observed in the activity of
cytosolic GST in the lungs isolated from the rats treated with the lowest dose
(3.75 mg/kg) of DEP was not found to be statistically significant (Table 3.1).
71
Table 3.1: Effects of transtracheal instillation of DEP on the activity of
drug metabolizing enzymes in lungs
Category ERODa MROD
a PROD
a NDMA-d
b GST
c
Control 6.1± 0.45 2.7± 0.14 8.2± 0.51 1.31±0.06 94.2± 4.70
DEP(3.75mg/kg) 8.3± 0.41* 3.2±0.20* 8.10± 0.51 1.41±0.07 104.3± 5.70
DEP(7.5mg/kg) 13.4±0.75* 4.2± 0.23* 6.82±0.35* 1.83±0.10* 141.8± 12.0*
DEP(15mg/kg) 20.4± 1.83* 5.8±0.43* 3.82±0.34* 2.10±0.21* 187.4±12.12*
DEP(30mg/kg) 11.3± 0.71* 3.9± 0.24* 1.62±0.15* 1.68±0.09* 123.8±11.3*
All the values are mean+ S.E. of 6 animals.
a: pmoles resorufin/min/mg protein.
b:nmoles HCHO formed/min/mg protein
c: nmoles conjugate formed/min/mg protein
* p<0.05 when compared to the controls
3.4.2 Effect of DEP on drug metabolizing enzymes in rat PBL
The effect of DEP on the activity of drug metabolizing enzymes in
peripheral blood lymphocytes is shown in Table3. 2. As evident from the table,
freshly prepared PBL isolated from rats treated transtracheally with 3.75- or
7.5- or 15- or 30 mg/kg of DEP showed similar pattern of alterations in CYP
dependent enzymes at all the doses of DEP. The activity of EROD and
MROD showed dose dependent increase in EROD and MROD activity at
relatively lower doses of DEP (3.75, 7.5, 15 mg/kg), while the magnitude of
increase observed at the highest dose was lesser when compared to the
lower dose of DEP (15 mg/kg). Similar to that seen with lungs, the increase
observed at 3.75 mg/kg was not found to be statistically significant when
compared to the controls while the increase observed with relatively higher
doses (7.5- or 15- or 30 mg/kg) was found to be statistically significant (Table
3.2). In contrast, CYP2B1 dependent PROD activity showed dose dependent
decrease in the enzyme activity in PBL isolated from rats treated
transtracheally with different doses of DEP. However the decrease observed
in PBL was found to be statistically significant in the rats receiving relatively
higher doses (7.5- or 15- or 30 mg/kg) of DEP. Likewise, as observed in
lungs, no significant effect was seen in the activity of PROD in PBL isolated
from rats treated transtracheally to the lowest dose (3.75 mg/kg) of DEP
(Table 3.2).
72
CYP2E1 dependent NDMA-d activity also showed dose dependent
increase in the enzyme activity in PBL isolated from rats treated with 3.75- or
7.5- or 15 mg/kg of DEP. The magnitude of induction subsequently declined in
the PBL isolated from rats treated with the highest dose (30 mg/kg) of DEP.
Though the magnitude of increase in NDMA-d activity in PBL isolated from the
rats receiving the at highest dose of DEP was less compared to the rats which
were treated with lower dose (7.5- or 15 mg/kg) of DEP, the increase
observed was found to be statistically significant as observed with the rats
receiving 7.5- or 15 mg/kg of DEP. No statistically significant effect was
observed in the activity of NDMA-d in the rats receiving the lowest dose (3.75
mg/kg) of DEP (Table 3.2).
As seen in lung cytosol, lymphocyte GST also showed a similar pattern
of increase in enzyme activity all the doses of DEP. The magnitude of
induction in lymphocyte GST was found to be lesser when compared to the
lung enzyme. As observed with the lung enzymes, the magnitude of induction
at highest dose (30 mg/kg) of DEP was lesser when compared to the
induction in the enzyme activity seen in rats treated with relatively lower doses
(7.5- or 15 mg/kg) of DEP. Statistical analysis revealed that the increase
observed in the activity of GST in PBL isolated from rats exposed to 7.5- or
15- or 30 mg/kg DEP dose was found to be statistically significant. The
increase observed in the activity of blood lymphocyte GST in the rats treated
with lowest dose (3.75 mg/kg) of DEP was not found to be statistically
significant (Table 3.2).
Table 3.2: Effects of transtracheal instillation of DEP on the activity of
drug metabolizing enzymes in peripheral blood lymphocytes.
Category ERODa MROD
a PROD
a NDMA-d
b GST
c
Control 0.8± 0.07 1.0± 0.10 0.47±0.02 0.67± 0.03 26.2± 1.45
DEP(3.75mg/kg) 1.0± 0.09 1.1± 0.17 0.45±0.02 0.68± 0.03 27.4± 1.53
DEP(7.5mg/kg) 1.4± 0.24* 1.4± 0.11* 0.40±0.02* 0.85±0.07* 33.8±1.90*
DEP(15mg/kg) 2.0± 0.26* 1.7± 0.20* 0.26±0.03* 0.98± 0.09* 37.7± 2.50*
DEP(30mg/kg) 1.3± 0.14* 1.3 ± 0.17 0.17 ± 0.02* 0.77 ± 0.04 31.2± 1.70*
All the values are mean+ S.E. of 6 animals.
a: pmoles resorufin/min/mg protein.
b:nmoles HCHO formed/min/mg protein
c: nmoles conjugate formed/min/mg protein.
* p<0.05 when compared to the controls.
73
3.4.3 Effect of DEP on lipid peroxidation and total glutathione (GSH)
content
Transtracheal administration of different doses of DEP produced a
significant increase in the GSH content in lung homogenates and PBL at all
the doses except in the rats receiving the lowest dose (3.75 mg/kg) of DEP.
Glutathione content increased in dose dependent manner upto 15 mg/kg DEP
dose and subsequently declined at highest dose (30 mg/kg) of DEP.
Statistical analysis revealed that the increase observed in the lungs isolated
from rats treated with the highest dose (30 mg/kg) of DEP and at other lower
doses (7.5- or 15 mg/kg) of DEP was found to be statistically significant. The
increase in glutathione content observed at lowest dose (3.75 mg/kg was not
found to be statistically significant. Similar to that seen with the tissue
homogenates, blood GSH also showed similar pattern of increase in all the
four doses (3.75- or 7.5- or 15- or 30 mg/kg) of DEP. As observed with the
lung homogenates, the increase observed at the highest dose (30 mg/kg) was
of lesser magnitude than in the animals exposed to relatively lower doses
(7.5- or 15 mg/kg) of DEP. Statistical analysis revealed that the increase
observed was found to be statistically significant at all the doses except in the
animals exposed to the lowest dose (3.75 mg/kg) of DEP (Table 3.3).
Transtracheal administration of different doses (3.75- or 7.5- or 15- or
30 mg/kg) of DEP leads to dose dependent increase in LPO (both enzymatic
and nonenzymatic) in all the doses of DEP in lung tissue homogenate.
Statistically significant increase in LPO was found in all the doses of DEP in
rats, however in the lowest (3.75 mg/kg) dose, the increase observed was not
found to be statistically significant (Table 3.3). Similar to lung homogenate,
lymphocytes also showed dose dependent increase in LPO (both enzymatic
and nonenzymatic) in all the doses (3.75- or 7.5- or 15- or 30 mg/kg) of DEP
(Table 3.3). Similar to lung homogenate, lymphocyte also showed statistically
significant dose dependent in all the three doses (7.5- or 15- or 30 mg/kg) of
DEP. However, the increase observed at lowest dose (3.75 mg/kg) was not
found to be statistically significant. The magnitude of induction in lymphocytes
was several fold lower compared to lung homogenate (Table 3.3)
74
Table 3.3: Effects of transtracheal instillation of diesel exhaust particles
on GSH content and lipid peroxidation in lungs and peripheral blood
lymphocytes
Lungs
Category GSHa LPO
c(non -enzymic) LPO
d (enzymic)
Control 1.80± 0.16 7.01±0.7 43.2± 2.0
DEP(3.75mg/kg) 2.09±0.14 8.92±1.0 49.4±2.0
DEP(7.5mg/kg) 3.63± 0.34* 15.1± 1.37* 72.3± 4.0*
DEP(15mg/kg) 7.64± 0.4* 20.1± 1.7* 113.4± 5.0*
DEP(30mg/kg) 2.54± 0.1* 29.9± 2.4* 163.3± 10.0*
Lymphocytes
Category GSHa LPO
c(non -enzymic) LPO
d (enzymic)
Control 1.50± 0.11 0.13± 0.01 24.2± 1.0
DEP(3.75mg/kg) 1.62± 0.11 0.14± 0.02 26.4± 1.0
DEP(7.5mg/kg) 2.54± 0.3* 0.17± 0.02* 31.3± 2.0*
DEP(15mg/kg) 3.58± 0.12* 0.22± 0.02* 35.3± 2.0*
DEP(30mg/kg) 1.93 ± 0.02* 0.33± 0.02* 42.4± 3.0*
All the values are mean+ S.E. of 6 animals.
a: µmoles GSHformed formed/g tissue
b: µmoles GSHformed formed/ml
c: nmoles MDAformed /hour/g tissue
d: pmoles MDA formed/min/mg protein
*p<0.05 when compared to the controls.
3.4.4 Effect of DEP on protein expression of CYP1A1/1A2 isoenzymes
Western blot analysis of microsomal proteins isolated from lungs of control
rats revealed vey faint immunoreactivity, comigrating with CYP1A1/1A2, with
polyclonal antibody raised against rat liver CYP1A1/1A2 (Fig.3.1).
Transtracheal instillation of DEP resulted in a significant increase in the
immunoreactivity comigrating with hepatic CYP1A1/1A2 in the lanes
containing microsomal proteins isolated from lungs of rats treated with
different doses of DEP.
Densitometric analysis revealed that the increase in the immunoreactivity
observed in lungs isolated from DEP treated rats revealed a dose-dependent
response in the lanes containing microsomal proteins isolated from rats
75
treated with 3.75- or 7.5- or 15 mg/kg of DEP. The immunoreactivity declined
in the microsomal proteins isolated from rats treated with the highest dose of
DEP (30 mg/kg), even though marked increase in immunoreactivity was
observed when compared to the controls (Figure- 3.1). Due to the extremely
low levels of expression of CYP1A1/1A2 in PBL and low level of induction
observed after DEP treatment, identification of CYP1A1/1A2 induction in PBL
was beyond the limits of the detection used in the present study (data not
shown).
3.4.5 Effect of DEP on protein expression of CYP2E1
Western blotting studies with polyclonal antibody raised against rat lung
CYP2E1 revealed considerable immunoreactivity in lung microsomal proteins
obtained from control or DEP treated rats. Transtracheal instillation of DEP
resulted in a significant increase in the immunoreactivity comigrating with
hepatic CYP2E1 in the lanes containing microsomal proteins isolated from
lungs of rats treated with different doses of DEP. A dose-dependent increase
was observed in immunoreactivity corresponding to CYP2E1 in the lanes
containing microsomal proteins isolated from rats treated with 3.75- or 7.5- or
15 mg/kg of DEP. The immunoreactivity declined in the microsomal proteins
isolated from rats treated with the highest dose of DEP (30 mg/kg), even
though marked increase in immunoreactivity was observed when compared to
the controls (Figure 3.2). As observed with CYP1A1/1A2 in PBL, extremely
low levels of expression of CYP2E1 in PBL and low level of induction
observed after DEP treatment were beyond the limits of the detection of our
assay system (data not shown).
76
3.4.6 Immunocytochemical localization of CYP 1A1/1A2 in PBLs after
DEP treatment
Blood lymphocytes isolated from control rats when incubated with
polyclonal antibody (primary antibody) raised against rat liver CYP1A1/1A2
and the secondary antibody labeled with FITC showed positive staining for
CYP1A1/1A2 as observed by fluorescence microscopy. Superimposition of
fluorescence exhibited by FITC with DAPI, the nuclear stain revealed that the
CYP1A1/1A2 mediated fluorescence was localized in the cytosol (Figure 3.3).
Further, the lymphocytes isolated from DEP treated rats (15 mg/kg) showed
marked increase in the expression of CYP1A1/1A2 as characterized by
increase in the intensity of fluorescence in DEP treated lymphocytes
compared to control lymphocytes (Figure 3.3).
77
3.4.7 Immunocytochemical localization of CYP2E1 in PBLs after DEP
treatment
Blood lymphocytes isolated from control rats when incubated with
polyclonal antibody (primary antibody) raised against rat liver CYP2E1 and
the secondary antibody labeled with FITC showed positive staining for
CYP2E1 as observed by fluorescence microscopy. Superimposition of
fluorescence exhibited by FITC with DAPI, the nuclear stain revealed that the
CYP2E1 mediated fluorescence was localized in the cytosol (Figure 3.4).
Further, the lymphocytes isolated from DEP treated rats (15 mg/kg) showed
marked increase in the expression of CYP2E1 as characterized by increase in
the intensity of fluorescence in DEP treated lymphocytes compared to control
lymphocytes (Figure 3.4).
3.4.8 Semi-quantitative Reverse Transcriptase (RT)-PCR analysis
Prior to PCR amplification of cDNA obtained after reverse transcription
of the RNA, extracted from lungs or PBL isolated from control or DEP
exposed rats, with primers of CYPs, normalization was carried out with
primers of β-actin, the house keeping gene. As shown in Fig. 5, DEP
treatment did not produced any significant effect on the mRNA expression of
β- actin in RNA extracted from lungs or blood lymphocytes of control or DEP
treated rats.
78
3.4.8.1 mRNA expression of CYP1A1
PCR amplification utilizing primers specific for rat lung CYP1A1 produced
band of correct size (341bp) in the RNA samples isolated from lungs of control
or DEP treated rats. Similar to that seen in lung, PCR amplification of the RT
product obtained from blood lymphocytes of control or DEP (30mg/kg body
weight) treated rats revealed the formation of band of correct size (341 bp)
(Figure 3.5.1). The mRNA expression profile of CYP1A1 was almost similar in
both, lungs and blood lymphocyte except that the intensity of the band formed
was several fold higher in the PCR product obtained from the lungs of DEP
treated rats indicating that the CYP1A1 transcript is expressed to much higher
extent in the lungs. Densitometric analysis revealed that CYP1A1 was
expressed at very low levels in the lungs isolated from control rats (Fig.3.5.1).
79
DEP exposure was found to increase the expression of CYP1A1 both in the
lungs and PBL. A dose dependent increase in the expression of of CYP1A1 was
observed, both in lungs and PBL in the rats receiving different doses (3.75- or
7.5- or 15 mg/kg) of DEP. However, when the dose of DEP was increased to 30
mg/kg, a decline in the magnitude of induction was observed in both, lungs and
PBL than in the lungs or PBL isolated from rats treated with relatively lower
doses (7.5- or 15 mg/kg) of DEP (Fig. 3.5.1).
80
3.4.8.2 mRNA expression of CYP1A2
PCR amplification, utilizing primers specific for rat lung CYP1A2, of the
RT product generated from the RNA isolated from lungs or blood lymphocytes
of control or DEP treated rats demonstrated that distinct bands of correct size
(795bp) were formed, both in the lungs and blood lymphocytes (Fig 3.5.2). As
observed with CYP1A1, mRNA expression of CYP1A2 was observed in
control lungs and blood lymphocytes isolated from control rats which was
indicative of constitutive expression of CY1A2 in both lung and blood
lymphocytes. DEP treatment resulted in a significant dose dependent
increase in the mRNA expression of CYP1A2 in both the lungs and blood
lymphocytes at relatively lower doses (3.75- or 7.5- or 15mg/kg) (Fig. 3.5.2).
An increase in mRNA expression of CYP1A2 was also observed, both in
lungs and PBL isolated from the rats treated with the highest dose of DEP (30
mg/kg). However, the magnitude of increase observed at this dose was less
when compared in PBL or lungs isolated from rats treated with relatively lower
doses of 7.5- or 15.0 mg/kg of DEP (Fig. 3.5.2).
81
3.4.8.3 mRNA expression of CYP1B1
PCR amplification of the RT product, synthesized from RNA extracted
from blood lymphocytes or lungs of control or DEP exposed rats, resulted in
the formation of PCR product of correct size (312 bp), both in the lungs and
blood lymphocytes (Fig. 3.5.3). Transtracheal instillation of DEP was found to
significantly increase the mRNA expression of CYP1B1 in whole lungs at the
lower doses (3.75- or 7.5- or 15 mg/kg). Densitometric analysis revealed that
this increase in mRNA expression of CYP1B1 in lungs was dose dependent
(Fig. 3.5.3). Though the increase in mRNA expression of CYP1B1 in rat lungs
persisted even in the rats receiving the highest dose (30 mg/kg) of DEP, the
magnitude of increase was much lesser when compared to the lower doses
(7.5- or 15 mg/kg) (Fig. 3.5.3). Similar to that observed in lungs, transtracheal
instillation of DEP was found to produce a dose dependent increase in the
mRNA expression of CYP1B1 in the blood lymphocytes at the lower doses
(Fig. 3.5.3) while a decline in the magnitude of induction was observed at the
highest dose (30 mg/kg) of DEP when compared to the rats receiving 7.5- or
15 mg/kg of DEP (Fig. 3.5.3).
82
3.4.8.4 mRNA expression of transcription factors (AhR and Arnt)
RT-PCR studies utilizing primers specific for rat liver AhR produced
bands of correct size of 340 bp (AhR) or 413 bp (Arnt) in the RNA samples
extracted from both, lungs and blood lymphocytes isolated from either, control
or DEP treated rats (Fig 3.5.4 & 3.5.5). Consistent with the increase in the
expression of CYP1A (1A1 and 1A2) and CYP1B1, DEP treatment was found
to significantly increase the mRNA expression of AhR and Arnt, in rat lungs
following exposure of DEP (Fig. 3.5.4 & 3.5.5). As observed with the
expression of CYPs, the increase in the expression of AhR observed after the
exposure of relatively lower doses of DEP (3.75- or 7.5- or 15 mg/kg) was
found to be dose-dependent. The treatment of highest dose of DEP (30
mg/kg) also resulted in a increase in the mRNA expression of AhR (Fig.
3.5.4), though this increase in mRNA expression of AhR was found to be of
much lesser magnitude when compared to the lower doses (7.5- or 15.0
mg/kg). Similar to that observed in rat lungs, transtracheal instillation of DEP
resulted in a dose-dependent increase in the mRNA expression of AhR in
blood lymphocytes isolated from rats treated with the lower doses (3.75- or
7.5- or 15.0 mg/kg) of DEP. As observed in the lungs, transtracheal instillation
of the highest dose (30 mg/kg) of DEP also resulted in an increase in mRNA
expression of AhR in blood lymphocytes, though this increase was of a much
lesser magnitude when compared to that observed after treatment of lower
doses of DEP (Fig. 3.5.4). As observed with AhR, transtracheal instillation of
DEP was found to increase the mRNA expression of ARNT in lungs in a dose
dependent manner at the lower doses (3.75- or- 7.5- or 15mg/kg) while the
levels were found to decline after the treatment of the highest dose (30 mg/kg)
of DEP, though they remained increased when compared to the controls (Fig.
3.5.5). Similar expression profile of ARNT was also seen in the blood
lymphocytes with significantly increased expression of ARNT being observed
at the lower doses of DEP (Fig.3.5.5). As observed with lungs, the increase in
the mRNA expression in blood lymphocytes declined after intra-tracheal
instillation of the highest dose (30 mg/kg) of DEP (Fig. 3.5.5).
83
84
3.4.8.5 mRNA expression of CYP2E1
PCR amplification of the RT product obtained from RNA extracted from
blood lymphocytes or lung of control or DEP(30mg/kg body weight) treated rats
using primers specific for rat lung CYP2E1 produced band of correct size (473
bp) in both, lungs and blood lymphocytes . As evident from the figure and
densitometeric analysis, a dose dependent increase in the mRNA expression of
CYP2E1 was observed, both in blood lymphocytes and lungs isolated from rats
treated with 3.75- or 7.5- or 15 mg/kg of DEP. An increase in the mRNA
expression of CYP2E1 was also observed after the treatment of highest dose
(30 mg/kg) of DEP, though the magnitude of increase was found to be less than
seen after the treatment of lower doses (7.5- or 15 mg/kg) of DEP (Fig. 3.5.6).
3.4.8.6 mRNA expression of GST isoforms (GSTPi,GSTM1 and GSTM2)
RT-PCR studies utilizing primers specific for rat liver GST produced
bands of correct size of 573 bp (GSTPi) or 501 bp (GSTM1) or GSTM2
(383bp) in the RNA samples extracted from both, lungs and blood
lymphocytes isolated from either, control or DEP treated rats (Fig
85
3.5.7,3.5.8,3.5.9 ). As observed with the expression of CYPs, the increase in
the expression of GST isoforms (GSTPi,GSTM1,GSTM2) observed after the
exposure of relatively lower doses of DEP (3.75- or 7.5- or 15 mg/kg) was
found to be dose-dependent. The treatment of highest dose of DEP (30
mg/kg) also resulted in a increase in the mRNA expression of GST isoforms
(GSTpi, GSTM1, GSTM2) (Fig. 3.5.7, 3.5.8, 3.5.9), though this increase in
mRNA expression of GST isoforms was found to be of much lesser
magnitude when compared to the lower doses (7.5- or 15.0 mg/kg). Similar to
that observed in rat lungs, transtracheal instillation of DEP resulted in a dose-
dependent increase in the mRNA expression of GSTPi, GSTM1, GSTM2 in
blood lymphocytes isolated from rats treated with the lower doses (3.75- or
7.5- or 15.0 mg/kg) of DEP. As observed in the lungs, transtracheal instillation
of the highest dose (30 mg/kg) of DEP also resulted in an increase in mRNA
expression of GST isoforms in blood lymphocytes, though this increase was
of a much lesser magnitude when compared to that observed after treatment
of lower doses of DEP (Fig. 3.5.7, 3.5.8, 3.5.9).
86
3.4.9 Quantitative Real-time PCR (qRT-PCR) studies in rat lung
The mRNA expression of β-actin, a housekeeping gene was used as
an endogenous control and was normalized by the software itself. The
expression of β-actin was found to be uniform throughout all the samples
87
(control or DEP treated) analyzed confirming the integrity of RNA used in
assays. DEP treatment increased the mRNA expression of PAH-inducible
CYP1A1, CYP1A2 and 1B1 isoenzymes in lungs of DEP treated rats (Table
3.4). CYP1A1 was found to be maximally induced followed by CYP1B1 and
1A2. Likewise, DEP was found to significantly increase the mRNA expression
of CYP2E1, the ethanol inducible CYP isoenzyme in lungs (Table 3.4).
Further, the mRNA expression of CYP1A1, 1A2, 1B1 and 2E1 increased in a
dose- dependent manner when the dose of DEP was increased up to 15
mg/kg dose in both lungs and PBL. Thereafter, when the dose of DEP was
increased to 30 mg/kg, a decline in the magnitude of induction in CYPs was
observed, though the increase at the highest dose remained statistically
significant when compared to the controls (Table 3.4). A trend towards the
increase was also observed in the mRNA expression of Ahr & Arnt in lungs
and PBL, though the increase was not found to be statistically significant
(Table 3.4).
Table 3.4: Effects of transtracheal instillation of DEP on the relative
mRNA expression of CYP isoenzymes, AhR and ARNT in rat lungs
Each reaction was performed in triplicate on cDNA samples in 96 well optical plates The
threshold cycle value (Ct value) of each sample was normalized with Ct value of endogenous
control ( - actin) ( Ct). Fold change is calculated from Ct value of each sample Ct= Ct of
treated- Ct of control. p<0.05 when compared with the controls. All the values are mean+
S.E. of 6 animals. *p<0.05 when compared to the controls.
3.5 Quantitative Real-time PCR (qRT-PCR) studies in PBL
The mRNA expression of β-actin, a housekeeping gene was used as
an endogenous control and was normalized by the software itself. The
expression of β-actin was found to be uniform throughout all the samples
(control or DEP treated) analyzed confirming the integrity of RNA used in
assays. The expression of CYP1A1, 1A2, 1B1 and 2E1 isoenzymes was
Category CYP1A1 CYP1A2 CYP1B1 CYP2E1 AhR ARNT
Control 1.00± 0.07 1.00± 0.09 1± 0.06 1.00±0.09 1.00±0.06 1.00±0.07
DEP(3.75mg/kg 1.7±0.06* 1.4±0.11 1.6±0.09* 1.3± 0.1 1.09±0.1 1.2±0.11
DEP(7.5mg/kg) 2.5±0.14* 1.6±0.09* 2.2±0.15* 2.4±0.16* 1.1±0.06 1.25±0.06
DEP(15mg/kg) 3.2±0.31* 2.0±0.20* 2.7± 0.22* 2.8±0.25* 1.3±0.07 1.3±0.07
DEP(30mg/kg) 2.0±0.22* 1.48±0.11* 1.4±0.07* 2.2±0.19* 1.1±0.05 1.18±0.06
88
found to be lower in blood lymphocytes when compared to the lungs (Table
3.4 & 3.5). DEP treatment increased the mRNA expression of PAH-inducible
CYP1A1, CYP1A2 and 1B1 isoenzymes in PBL (Table 3.5). CYP1A1 was
found to be maximally induced followed by CYP1B1 and 1A2. Similar pattern
of induction was observed in PBL after exposure to DEP. Likewise, DEP was
found to significantly increase the mRNA expression of CYP2E1, the ethanol
inducible CYP isoenzyme in freshly prepared PBLs. As evident from the
tables, similar pattern of induction of CYP2E1 mRNA was observed in PBLs
and lungs (Table 3.4 & 3.5). Further, the mRNA expression of CYP1A1, 1A2,
1B1 and 2E1 increased in a dose- dependent manner when the dose of DEP
was increased up to 15 mg/kg dose in both lungs and PBL. Thereafter, when
the dose of DEP was increased to 30 mg/kg, a decline in the magnitude of
induction in CYPs was observed, though the increase at the highest dose
remained statistically significant when compared to the controls (Table 3.5). A
trend towards the increase was also observed in the mRNA expression of
AhR & Arnt in PBL, though the increase was not found to be statistically
significant (Table 3.5).
Table 3.5: Effects of transtracheal instillation of DEP on the relative mRNA
expression of CYP isoenzymes, AhR and ARNT in rat PBL
Each reaction was performed in triplicate on cDNA samples in 96 well optical plates The
threshold cycle value (Ct value) of each sample was normalized with Ct value of endogenous
control ( - actin) ( Ct). Fold change is calculated from Ct value of each sample Ct= Ct of
treated- Ct of control. p<0.05 when compared with the controls. All the values are mean+
S.E. of 6 animals. *p<0.05 when compared to the controls.
3.5.1 Quantitative mRNA expression of GST Isoforms in rat lung
RT-PCR studies further demonstrated that mRNA expression of phase
II enzymes, GSTPi, GSTM1 and GSTM2 was induced in lungs of DEP
treated rats at at all doses of DEP. As observed with enzyme data, the
Category CYP1A1 CYP1A2 CYP1B1 CYP2E1 AhR ARNT
Control 1.00± 0.08 1.0± 0.06 1±0.07 1.0± 0.09 1.00±0.05 1.00±0.09
DEP(3.75mg/kg 1.28± 0.12 1.1± 0.11 1.1±0.11 1.1±0.13 1.00±0.07 1.00±0.05
DEP(7.5mg/kg) 1.68±0.07* 1.4± 0.07* 1.53±0.08* 1.5±0.07* 1.1±0.05 1.11±0.05
DEP(15mg/kg) 1.83±0.17* 1.7± 0.14* 1.73±0.15* 1.9±0.16* 1.2±0.07 1.13±0.07
DEP(30mg/kg) 1.5 ± 0.08* 1.2 ±0.05 1.31±0.1 1.3 ± 0.1 1.00±0.05 1.11±0.06
89
magnitude of increase observed at the highest dose (30 mg/kg) was of lesser
magnitude when compared to the relative lower doses of DEP (3.75- or 7.5-
or 15 mg/kg) in lungs of DEP treated animals (Table 3.6). GSTPi, which is
primarily involved in the detoxification of free radicals, exhibited maximum
increase in its expression in lungs. Significant increase in the mRNA
expression of GSTM1 and M2, that specifically detoxify the reactive
intermediates generated from PAHs, was also lungs isolated from DEP
treated animals (Table 3.6).
Table 3.6: Effects of transtracheal instillation of DEP on the relative
mRNA expression of GST isoenzymes in rat lungs
Category GSTPi GSTM1 GSTM2
Control 1.00± 0.09 1.00± 0.09 1.00± 0.09
DEP(3.75mg/kg) 1.42±0.1 1.28±0.1 1.12±0.11
DEP(7.5mg/kg) 2.74±0.17* 2.03±0.15* 1.63±0.11*
DEP(15mg/kg) 3.63±0.20* 2.84±0.23* 2.42±0.25*
DEP(30mg/kg) 1.90±0.11* 1.53±0.11* 1.34±0.11
Each reaction was performed in triplicate on cDNA samples in 96 well optical plates The
threshold cycle value (Ct value) of each sample was normalized with Ct value of endogenous
control ( - actin) ( Ct). Fold change is calculated from Ct value of each sample Ct= Ct of
treated- Ct of control. p<0.05 when compared with the controls. All the values are mean+
S.E. of 6 animals. *p<0.05 when compared to the controls
3.5.2 Quantitative mRNA expression of GST Isoforms in rat PBL
RT-PCR studies further demonstrated that mRNA expression of phase
II enzymes, GSTPi, GSTM1 and GSTM2 was induced in freshly isolated
PBLs at all doses of DEP, though the increase observed in blood
lymphocytes was of lesser magnitude when compared to those in the lungs.
As observed with enzyme data, the magnitude of increase observed at the
highest dose (30 mg/kg) was of lesser magnitude when compared to the
relative lower doses of DEP (3.75- or 7.5- or 15 mg/kg), both in freshly
prepared PBLs and lungs (Table 3.6 & 3.7). Interestingly, GSTPi, which is
primarily involved in the detoxification of free radicals, exhibited maximum
increase in its expression, both in freshly prepared PBLs and lungs.
Significant increase in the mRNA expression of GSTM1 and M2, that
90
specifically detoxify the reactive intermediates generated from PAHs, was
also observed in blood lymphocytes and lungs isolated from DEP treated
animals (Table 3.6& 3.7).
Table 3.7: Effects of transtracheal instillation of DEP on the relative
mRNA expression of GST isoenzymes in rat PBL
Category GSTPi GSTM1 GSTM2
Control 1.00± 0.07 1.00± 0.15 1.00± 0.16
DEP(3.75mg/kg) 1.20±0.15 1.13±0.15 1.13±0.11
DEP(7.5mg/kg) 1.81±0.08* 1.74±0.18* 1.43±0.08*
DEP(15mg/kg) 2.13±0.19* 2.06±0.24* 1.84±0.14*
DEP(30mg/kg) 1.53±0.11* 1.23±0.1 1.23±0.09
Each reaction was performed in triplicate on cDNA samples in 96 well optical plates The
threshold cycle value (Ct value) of each sample was normalized with Ct value of endogenous
control ( - actin) ( Ct). Fold change is calculated from Ct value of each sample Ct= Ct of
treated- Ct of control. p<0.05 when compared with the controls. All the values are mean+
S.E. of 6 animals. *p<0.05 when compared to the controls.
3.6 Discussion
The present study has demonstrated that transtracheal instillation of
DEP produces an increase in the activity of CYP1A-dependent EROD and
MROD, CYP2E1- mediated NDMA-d and a decrease in CYP2B1-regulated
PROD activity in freshly prepared PBLs isolated 24 hours after exposure to
DEP. However, in contrast to the previous reports indicating dose-dependent
increase in the activity of CYP1A1 enzymes after intratracheal (transoral)
instillation of similar doses of DEP (Rengasamy et al., 2003), relatively lesser
magnitude of induction of pulmonary CYPs was observed at the highest dose
(30 mg/kg) than at relatively lower doses (7.5- or 15 mg/kg) of DEP in the
present study. This could possibly be attributed to the direct effects of DEP
after transtracheal instillation when compared to transoral instillation where
DEP could be metabolized by tracheal CYPs prior to reaching the lungs (Lee
et al., 1998; Gerde et al., 1997). Lee et al., 1998 have earlier reported that
trachea is equipped with CYPs such as CYP1A1 and CYP2B1,the levels of
which were reported to be induced by treatment with CYP inducers in rats.
Likewise Gerde et al., (1997) suggested that instillation of benzo(a)pyrene into
91
the lungs results in extensive metabolism and approximately 28% of the
instilled dose was bound to tracheal tissues.
RT-PCR and immunocytochemical studies have shown that induction
in the activity of CYP enzymes in PBL is associated with an increase in the
mRNA and protein expression of CYP1A1, 1A2, 1B1 and 2E1 isoenzymes. As
PBL isolated from 3-methylcholanthrene (MC) or ethanol pre-treated rats have
been reported to mimic the induction of CYP1A-, 1B1 or CYP2E1 isoenzymes
observed in rat tissues (Dey et al 2001, 2005; Saurabh et al., 2010), the
induction of CYP1A-, 1B1 or CYP2E1 in freshly isolated PBL could be
attributed to the PAHs and heterocyclic amines present in DEP (Yamasaki et
al., 2000; Kuljukka-Rabb et al., 2001). Studies have demonstrated that
CYP1A1& 2E1 expressed in PBL are induced by PAHs and ethanol
respectively & show similarities in their responsiveness with the tissue
enzyme.The bioactivation of DEP extracts and their major nitrated PAH
components, 1-nitropyrene and dinitropyrenes, by human cytochromes P450
1A1, 1A2, and 1B1 using Escherichia coli membranes (Yamasaki et al.,
2000). Kuljukka-Rabb et al., (2001) investigated time- and dose-dependent
DNA adduct formation by PAHs derived from three diesel particulate extracts,
diesel particulate matter Standard Reference Material 1650 (SRM),
benzo[a]pyrene (B[a]P) and 5-methylchrysene (5-MeCHR) in a human
mammary carcinoma cell line (MCF-7). Based on the DNA adduct formation
PAH derived from DEP extracts such as fluorene, phenanthrene, anthracene,
fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[e]pyrene,
benzo[b]fluoranthene, benzo[k]fluoranthene, B[a]P, dibenz[a,h]anthracene,
benzo[ghi]perylene and indeno[1,2,3-cd]pyrene were classified into non-
carcinogenic, weak and strong carcinogenic PAHs.
Similar isoenzyme specific alterations in the expression of pulmonary
CYPs were reported in rat lungs after DEP exposure (Hatanaka et al., 2001;
Rengasamy et al., 2003; Zhao et al., 2004). Hatanaka et al., (2001)
demonstrated that the CYP isoenzymes including CYP1A1, CYP1A2, and
CYP1B1 were induced by inhalation of DEP. In rats exposed to DEP, elevated
mRNA levels of CYP1A1 and CYP1B1 and increased 7-ethoxyresorufin O-
deethylase (EROD) activity were found not only in the lung, but also in the
liver. Intratracheally instilled DEP (5,15,35 mg/kg) were reported to result in a
92
dose dependent increase of CYP1A1 protein and EROD activity at 1 day post-
exposure, and the enzyme level declined with time and returned to control
level at five days post-exposure in rat lungs (Rengasamy et al.,2003). Studies
carried out by Zhao et al., (2004) also showed that following instillation of
DEP, maximal induction of CYP1A1 was observed after 1 day of exposure
with basal levels reaching at seven day post exposure in rats.
An increasing trend was observed in the expression of Ahr and Arnt in
lungs and lymphocytes following DEP exposure have suggested similarities in
the regulatory mechanisms responsible for CYP1A1, 1B1 and 1A2 induction
in rats. PAHs present in DEP acts as ligands and binds to AhR in the cytosol
which translocates into the nucleus and heterodimerises with Arnt and then
binds to promoter region of xenobiotic response elements (XRE) to induce
transcription of CYP genes. However, no significant increase observed in the
expression of AhR and Arnt in rat lungs and lymphocytes could be attributed
to the presence of high concentrations of fluoranthrene and other ligands
present in our DEP sample which exhibit low affinity for Ah receptor
(Piskorska-Pliszczynska et al., 1986).
In contrast, increase in the activity of EROD and NDMA-d the marker
enzymes of CYP1A- and CYP2E1, a decrease in the the activity PROD, a
marker enzyme of CYP2B1 was observed in present study. Studies have
shown that CYP2B1 the major constitutive CYP isoenzyme expressed in
lungs was decreased in lungs isolated from DEP treated rats. Further the
activity remained decreased over a 5-7-day post exposure period
(Rengasamy et al., 2003; Zhao et al., 2004). The observed decrease in
CYP2B1 activity could be explained by the direct effect of compounds
(toluene and xylene) present in DEP which downregulate the expression of
certain CYPs in lungs (Furman et al., 1998; Verschoyle, 2001). A significant
inhibition of the activity of cytochrome P-4502B1 (CYP2B1), and CYP4B1
enzymeswas observed in rats treated with toluene (Furman etal.,1998).
Verchoyle et al., (1993) also reported significant inhibition in lung CYP2B1
activity in p-xylene treated rats, while no change was observed in the activity
of CYP1A1. The mechanism by which these agents inhibit CYP2B1 appears
to involve the formation of an aldehyde–heme adduct (Raner et al., 1997).
Another mechanism of DEP downregulation of CYP2B1 may involve nitric
93
oxide (NO). NO avidly binds to reactive heme–iron centers of several
enzymes and thereby regulates their activities. Bacterial lipopolysaccharide
(LPS) has been shown to downregulate CYP2B1 in rat hepatocytes by an
NO- dependent mechanism (Ferrari et al., 2001), which was completely
prevented by NO synthase inhibitors. Yang et al., (2001) have shown that
DEP exposure resulted in a moderate increase in macrophage production of
NO (Yang et al., 2001). This suggests that NO may be partly responsible for
the downregulation of CYP2B1observed in DEP exposed rats.
As observed with CYPs, a similar pattern of increase in the activity of
GSTs and mRNA expression of specific GSTs (GSTPi, GSTM1 & GSTM2)
was observed in PBLs and lungs isolated from rats treated with different
doses of DEP. Similar to that seen with phase 1 enzymes the magnitude of
induction observed in the GSTs was lesser at highest dose (30 mg/kg) when
compared to relatively lower doses (7.5 &15 mg/kg) of DEP in both
lymphocytes and lungs isolated from DEP treated rats. Though, contrasting
effects of DEP have been reported on pulmonary GSTs, Ueng et al., (1998)
reported increase in the activity of GST after inhalation and intratracheal
instillation of motorcycle exhausts in lungs and liver of rats. This increase in
the activity of GSTs was attributed to the presence of PAHs and
polychlorinated biphenyls (PCBs) present in the exhaust that acts as
bifunctional inducers to induce CYPs and GSTs (Irigaray & Belpomme, 2010).
It has been reported that components of DEP activate antioxidant response
element (AREs) by interaction with transcription factor NRf2 that may lead to
activated expression of various antioxidant enzymes such as GSTs and heme
oxygenase 1(Li et al., 2004). A decrease in the activity of GSTs in rat and
mouse lung has also been reported after intratracheal instillation of DEP
(Rengasamy et al., 2003; Sagai et al., 1993). Rengasamy et al., (2003)
showed sustained decrease in the activity of GST in both dose dependent and
time dependent manner in DEP treated rats. Further studies have shown that
DEP inhibited GST activity in mouse lung after DEP exposure (Sagai et al.,
1993). The discrepancies has been attributed to the differences in the dose,
particle distribution or particle clearance produced by various methods of
exposure (Osier and Oberdoster, 1997). Likewise, increase in the mRNA
expression of GSTpi, which constitute 90% of total lung GSTs, is consistent
94
with the earlier studies indicating involvement of GSTPi in the detoxification of
lipid peroxides and DNA oxidation products formed in large amounts following
exposure to DEP (Koike etal., 2004; Li et al., 2004). Koike et al., (2004), using
cDNA microarray analysis demonstrated changes in gene expression of
antioxidative genes including GST using rat epithelial cells following exposure
to organic extracts of DEP. Li et al., (2004) demonstrated that aromatic and
polar DEP fractions, which are enriched in polycyclic aromatic hydrocarbons
and quinones, respectively, induce the expression of HO-1, GST, and other
phase II enzymes in human macrophages and epithelial cells and postulated
occurrence of cytoprotective mechanism which protects cells against the
proinflammatory and oxidising effects of diesel exhaust particles. Similarly,
increase in the expression of GSTmu suggests its role in the detoxification of
reactive intermediates generated during oxidative metabolism of PAHs.
That the increase in the expression of drug metabolizing enzymes may
lead to formation of reactive intermediates was provided by our data indicating
a dose-dependent increase in the lipid peroxidation (both enzymatic and non-
enzymatic) as well as GSH contents in blood lymphocyte and lungs.
Inhalation or intratracheal instillation of DEP in mice has been reported to
produce several folds increase in lipid peroxide formation in lungs which may
lead to the induction of lipid peroxidation in lungs (Martin et al., 1998; Nel,
1998; Sagai, 1993; Whitekus et al., 2002; Donaldson, 2003). Generation of
ROS is a multidimensional phenomenon and several enzymes including
CYP2E1 and CYP1A1 are known to be involved in their generation (Wan &
Diaz-Sanchez, 2007; Bonvallot et al., 2001). Likewise, coordinated increase of
GSH content and GST activity observed in our study suggest the increased
cellular GSH demand due to its rapid utilization for conjugation by GST.
Earlier studies have shown that exposure of rats to DEP induced time
dependent increase in GSH in rat lungs and a moderate increase in
lymphocyte GSH along with increase in glutathione reductase activity (Al-
Humadi et al., 2002). Studies on human volunteers exposed to DEP and other
air pollutants also showed increase in cellular GSH in bronchoalveolar lavage
and peripheral lungs (Behndig, 2006). Elevated concentrations of airway GSH
has been reported in various air pollutant studies. The observed increase in
95
the glutathione content reflects increased rate of glutathione synthesis, allied
to increased export to the cell surface (Blomberg, 1999; Mudway, 2004).
In summary, transtracheal instillation of DEP in rat was found to
increase the catalytic activity and expression of CYP 1A1, 1A2 and 2E1 and
decrease in the expression of CYP2B1 in lungs and lymphocytes at all doses
of DEP. The induction pattern observed in lungs of DEP treated rats
correlated with the induction pattern of CYPs in blood lymphocytes, though
the magnitude of induction was lesser in the blood lymphocytes of DEP
treated rats. Transtracheal instillation of DEP was also found to increase the
catalytic activity of antioxidant enzymes and content of antioxidants such as
GST& GSH in rat lungs and PBL at all the doses of DEP. As observed with
CYPs the induction pattern observed in lungs of DEP treated rats correlated
with the induction pattern of GSTs in blood lymphocytes. Further significant
increase in the basal lipid peroxidation in rat blood lymphocytes in DEP
treated rats has indicated similarities in lipid peroxidation in rat blood
lymphocytes with the lung. Similarities in the increase of lipid peroxidation in
lymphocytes with that of lungs could be of immense significance in identifying
oxidative damage which can lead to pulmonary toxicity after exposure of DEP.
To conclude, similarities found in the alterations of blood lymphocyte CYPs,
particularly CYP1A1, 1B1 and CYP2B1 with the lung enzymes after
transtracheal instillation of DEP of significance as these CYPs play an
important role in mutagen activation in lungs and thus could be used as a
surrogate for monitor tissue expression of toxication and detoxication
enzymes to predict the toxicity of vehicular emissions.
Chapter 4
Similarities in DEP induced alterations in Xenobiotic
metabolizing enzymes and DNA damage in blood derived
and lung derived cell lines
96
Chapter 4
Similarities in DEP induced alterations in Xenobiotic metabolizing
enzymes and DNA damage in blood derived and lung derived cell lines
4.1 Introduction
In the preceding chapters we have shown that mRNA expression
profiles of genes involved in drug metabolism and toxicity are expressed in
freshly prepared PBL and show similarities in their responsiveness to the lung
enzymes in rats transtracheally instilled with DEP. Our data in the chapter 2
have further shown that transtracheal instillation of different doses of DEP to
adult wistar rats resulted in significant alterations in the catalytic activity of
cytochrome P450 (CYP) dependent enzymes and glutathione S-transferases
(GSTs) in lungs and freshly prepared PBL isolated form rats 24 hrs after the
exposure. Consistent with the enzymatic analysis, results from western blot
and real-time PCR (RT-PCR) have indicated that the increase in the activity of
EROD, MROD and NDMA-d is associated with an increase in the protein
expression of CYP1A1/CYP1A2 and CYP2E1 protein after DEP treatment in
both freshly prepared PBL and lungs 24 hrs after DEP exposure. The data
suggested similarities in the responsiveness and regulation of these drug
metabolizing enzymes (DMEs) in PBL with the lung enzymes and that PBL
expression profiles of DMEs could be used as a surrogate to monitor tissue
expression.
To further demonstrate the suitability of using PBL as a surrogate,
studies have also been carried out in established human blood cell lines to
assess basal expression and chemical induction responsiveness of these
xenobiotic metabolizing genes. Krovat et al., (2000) reported that constitutive
CYP expression profiles were conserved across established human blood cell
lines. The basal expression of the xenobiotic metabolizing CYPs in blood
derived cell lines was found to be highly similar to that observed in freshly
prepared PBL, however the inductive response was not manifested in these
blood cell lines, which could be attributed to various factors including the
inducers used and the induction protocol used, given the low levels of the
expression of CYPs and other drug metabolizing enzymes in PBL. However,
as observed with other established cell lines, the in vivo patterns of
97
expression are well maintained in blood derived cell lines indicating the
suitability of these cell lines as a model to study tissue specific toxic events.
Attempts were therefore made to study the effect of DEP on the
expression of PAH responsive CYPs in IM9, the blood derived cell line and
compared with lung derived cell lines, the well established cell model used for
monitoring the toxicity of diesel exhausts. Studies were also carried out to
investigate the role of CYP mediated metabolism in the toxicity including
genotoxicity of DEP.
4.2 Material and methods
4.2.1 Particle preparation and characterization
DEP (National Institute of Standards and Technology, Standard
Reference material 2975, Gaithersburg, MD) was suspended in complete
DMEM-F12 medium at a concentration of 1mg/ml (stock) followed by
sonication at 100 mega hertz. Size distribution and zeta potential of DEP were
determined using dynamic light scattering and phase analysis light scattering
(PALS) in a Zetasizer Nano-ZS, Model ZEN3600 equipped with 4.0mW,
633nm laser (Malvern instruments Ltd., UK).
4.2.2 Cell culture and treatment
Human lung carcinoma epithelial cell line - A549 (ATCC no. CCL-
185TM) used in the study was originally procured from National Centre for
Cell Sciences, Pune, (India) and human B lymphoblastic cell line (IM9)(ATCC
no.CCL-159 TM) were obtained from American Type Culture Collection
(Rockville, MD). A549 and IM9 cells were cultivated in DMEM F-12 and RPMI
1640 culture media respectively. Both the culture media was supplemented
with 10% fetal bovine serum (FBS), 0.2% sodium bicarbonate (NaHCO3),
1ml/100ml medium 1% antibiotic-antimycotic at 370C in 5% CO2-95%
atmospheric air under high humid conditions. After 24h attachment of A549
cells, suspension of DEP (1mg/ml) in DMEM-F12 medium, diluted to
concentration range of 1-100µg/ml was added to the cells for 6-48h as per the
demand of endpoint.
98
4.2.3 Exposure
For cytotoxicity studies, cells were seeded in 96-well plates (10,000
cells per well). The cells were then exposed for 96 hr to different
concentrations diesel exhaust particles (DEP), For genotoxicity testing, cells
were seeded in six-well plates (Corning, International Medical, Brussels; A549
: 20,000 cells per well ). After the exposure the tissue culture medium was
removed, the cells were washed with PBS and trypsinized. The cells were
centrifuged for 5 min at 1500 rpm (350g) and resuspended in 200 ml PBS
(final cell suspension).
4.2.4 MTT assay
MTT assay provides an indication of mitochondrial integrity & activity,
which is interpreted as a measure of percent cell viability. The assay was
carried out following the protocol described earlier by Pandey et al., (2006). In
brief, cells (1x104) were allowed to adhere for 24 h under high humid
environment in 5% CO2- 95% atmospheric air at 370C in 96-well culture
plates. The medium was aspirated and cells were subjected to expose for 6-
48 h with selected dosages of MWCNTs (0.5-100 µg/ml) in fresh medium.
Tetrazolium bromide salt (5mg/ml of stock in PBS) was added 10µl/well in
100µl of cell suspension and plate were incubated for 4 h. At the end of
incubation period, the reaction mixture was carefully taken out and 200 ml of
DMSO was added to each well. The plates were kept on rocker shaker for 10
min at room temperature and then analyzed at 550 nm using Multiwell
microplate reader (Synergy HT, Bio-Tek, USA). Untreated sets were also run
under identical conditions and served as basal control.
For identifying the role of CYP1A1 in DEP mediated genotoxicity, a
flask containing A549 or IM9 cells were pre-incubated with 3-
methylcholanthrene (MC; 4µm for A549 and 15 µm for IM9), an inducer of
CYP1A1 catalysed reactions for 12 hrs and then exposed to DEP for 6 hr (for
A549) or 12 hr (for IM9) along with MC. The cells after incubation were
processed accordingly for comet assay, ROS generation and mRNA
expression of CYP1A1. For studying the protein expression of CYP1A1, a
flask of cells after preincubation, were incubated with DEP for 48 hours and
then processed for western blotting. Another batch of cells were also treated
99
with CYP1A1 inhibitor, α naphthoflavone, (α-NF; 20µm for A549 and 10 µm
for IM9) together with DEP to visualise synergestic effect of DEP along with
MC. The concentrations of inducer (MC) and inhibitor (α-NF) were based on
the previous published reports (Wang et al., 2001; Ferrecatu et al., 2010).
4.2.5 Comet assay
Slides were prepared in duplicate according to the method of
Bajpayee et al., (2002) with some modifications. In brief, slides were
immersed in freshly prepared chilled lysing solution (2.5 M NaCl, 100 mM
EDTA, 10 mM Tris pH 10.0 and 1% Triton X‐100, pH 10) for 2h. After lysis,
the slides were placed in a horizontal gel electrophoresis tank (Life
Technologies, Gaithersburg, MD) filled with fresh, chilled electrophoresis
solution (1 mM Na2EDTA and 300 mM NaOH, pH > 13). The slides were left
in this solution for 10 min to allow DNA unwinding. Electrophoresis was
conducted for 15 min at 0.7 V/cm and 300 mA at 4°C. All the steps were
performed under dimmed light to avoid additional DNA damage. Following
electrophoresis, Tris buffer (0.4 M Tris pH 7.5) was added drop wise to
neutralize excess alkali and this was repeated three times. Slides were then
stained with ethidium bromide (20 µg/ml, 75 µl/ slide) for 10 min in the dark.
They were dipped once in chilled distilled water to remove excess ethidium
bromide and subsequently coverslips were placed over them. The slides were
stored in a dark, humidified chamber and analysed within 3–4 h.
Slides were analysed using an image analysis system (Kinetic Imaging,
Liverpool, UK) attached to a fluorescent microscope (Leica, Germany). The
images were captured by CCD camera and transferred to a computer and
analysed using Komet 5.0 software. The parameters studied were tail DNA
(%), tail length (estimated leading edge from the nucleus; µm) and tail
moment (arbitrary units) (Olive et al., 1990, 1992). The tail moment is defined
as the distance between the centre of mass of the tail and the centre of mass
of the head, in micrometres, multiplied by the percentage of DNA in the tail.
This number was then compared with the total DNA content. Images from 50
cells (25 from each slide) were analysed.
100
4.2.6 Quantitative Real Time-PCR (qRT-PCR) Analysis
Total RNA was isolated from control and treated A549 and IM9 cells
using TRIzol reagent as described previously in section 2.2.3 of chapter 2.
Further cDNA was synthesized by High-Capacity cDNA Reverse Transcription
Kit by the method described in section 2.2.4 of chapter 2. The PCR reaction
mixture for human CYP1A1, 1B1 and β-Actin and PCR conditions were same
as that described in section 3.3.4 of chapter 3.
4.2.7 Enzymatic analysis
The activity of 7-ethoxyresorufin-O-deethylase (EROD) a catalytic
marker of CYP1A1 catalysed reactions were determined in A549 or IM9 cells
by the method of Parmar et al., 1998 with slight modification. Briefly, A549 or
IM9 cells were grown to its almost full confluency at 37°C and then were
treated with DEP, 3-MC and α-NF for desired period and concentration as
described above. After treatment, cells were washed with PBS (pH 7.4) and
cells were collected in tube using a cell scraper and lysed using a sonicator at
frequency of 60MHz/10s and the process continued for seven times to
completely lyse the cells. The lysate was then used for EROD assay as
described in section 3.2.6 of chapter 3. Levels of resorufin in the supernatant
was measured using a Cary Eclipse Fluorescence spectrophotometer at
excitation wavelength of 550nm and emission wavelength of 585nm with a slit
width on 10nm each and integration time of one second. All experiments were
performed three times with different preparations.
4.2.8 Immunocytochemical analysis
Immunocytochemical analysis for Control and treated A549 and IM9
cells was done according to protocol as described in section 3.3.6 of chapter
3.
4.2.9 Determination of ROS
ROS production was determined by the method as described by
Halliwell and Whiteman, (2004) using dichloro- dihydrofluorescein-diacetate
(DCFDA), a non fluorescent probe which is converted into highly fluorescent
(2’, 7’ dichlorofluorescein) molecule in presence of ROS. Briefly cells were
placed on six well cell culture plates at 3X105 and allowed to adhere for 24
hours in CO2 incubator at 37ºC. The medium was then replaced with the
complete medium containing desired concentration of DEP, inducer MC and
inhibitor α-NF for the given time period. After treatment cells were detached
from wells and spun for 5 minutes at 1,000 rpm and again resuspended in
phosphate buffer saline containing 10µm DCFDA in the dark at 37°C for 30
minutes. After 30 minutes the cells were pelleted and resuspended in PBS to
be analyzed by multiplate reader with an
of 525 nm
4.3 Statistical Analysis
Students `t’ test was employed to calculate the statistical significance
between control and treated groups. P<0.05 was considered to be significant
when compared with the controls.
4.4 Results
4.4.1 Particle Characterstistics
The mean particulate diameter and zeta potential in tissue culture DMEM
medium was approximately 184nm and
shown in fig 4.1.
4.4.2 Cytotoxicity
The average cytotoxicity
A549 and IM9 cells shows that viability of cells are decreased as the time and
concentration is increased in all the three compounds mentioned above in
both A549 and IM9 cells (Table 4.1
101
hours in CO2 incubator at 37ºC. The medium was then replaced with the
complete medium containing desired concentration of DEP, inducer MC and
NF for the given time period. After treatment cells were detached
ells and spun for 5 minutes at 1,000 rpm and again resuspended in
phosphate buffer saline containing 10µm DCFDA in the dark at 37°C for 30
minutes. After 30 minutes the cells were pelleted and resuspended in PBS to
multiplate reader with an absorbance of 488 nm and emission
Statistical Analysis
Students `t’ test was employed to calculate the statistical significance
between control and treated groups. P<0.05 was considered to be significant
when compared with the controls.
Particle Characterstistics
The mean particulate diameter and zeta potential in tissue culture DMEM
medium was approximately 184nm and -30.2mV respectively for DEP as
he average cytotoxicity of DEP at different concentrations and time in
A549 and IM9 cells shows that viability of cells are decreased as the time and
concentration is increased in all the three compounds mentioned above in
IM9 cells (Table 4.1-4.4).
hours in CO2 incubator at 37ºC. The medium was then replaced with the
complete medium containing desired concentration of DEP, inducer MC and
NF for the given time period. After treatment cells were detached
ells and spun for 5 minutes at 1,000 rpm and again resuspended in
phosphate buffer saline containing 10µm DCFDA in the dark at 37°C for 30
minutes. After 30 minutes the cells were pelleted and resuspended in PBS to
absorbance of 488 nm and emission
Students `t’ test was employed to calculate the statistical significance
between control and treated groups. P<0.05 was considered to be significant
The mean particulate diameter and zeta potential in tissue culture DMEM
ctively for DEP as
at different concentrations and time in
A549 and IM9 cells shows that viability of cells are decreased as the time and
concentration is increased in all the three compounds mentioned above in
102
Table 4.1: Cytotoxicity, as assessed by MTT assay in various
concentrations of DEP in A549 cells
Concentration 12 hour 24 hour 48 hour 96 hour
Con 0.70+ 0.05 0.81+ 0.1 0.71+ 0.05 0.79+ 0.05
DEP(1ng/ml) 0.66+ 0.07 0.76+ 0.07 0.72+ 0.05 0.72+0.07
DEP(100 ng/ml) 0.68+ 0.05 0.70+ 0.07 0.68+ 0.08 0.73+ 0.05
DEP(10 µg/ml) 0.67+ 0.04 0.72+0.05 0.58+ 0.03* 0.42+ 0.03*
DEP(100 µg/ml) 0.64+ 0.06 0.73+ 0.08 0.41+ 0.03* 0.28+ 0.01*
Data are mean +SEM of three independent experiments, with six replicates per sample.
Table 4.2: Cytotoxicity, as assessed by MTT assay in various
concentrations of DEP in IM9 cells
Concentration 12 hour 24 hour 48 hour 96 hour
Con 0.50+ 0.05 0.54+ 0.1 0.61+ 0.05 0.59+ 0.05
DEP(1ng/ml) 0.56+ 0.07 0.62+ 0.07 0.52+ 0.05 0.58+0.07
DEP(100 ng/ml) 0.58+ 0.05 0.65+ 0.07 0.68+ 0.08 0.63+ 0.05
DEP(10 µg/ml) 0.61+ 0.04 0.52+0.05 0.58+ 0.03 0.52+ 0.03
DEP(50 µg/ml) 0.64+ 0.06 0.31+ 0.08* 0.21+ 0.03* 0.11+ 0.01*
Data are mean +SEM of three independent experiments, with six replicates per sample.
Table 4.3: Cytotoxicity, as assessed by MTT assay in various
concentrations of MC in A549 and IM9 cells
Lung (A549)
Conc 12 hour 24 hour 48 hour 96 hour
Con 0.54+0.05 0.61+0.1 0.55+ 0.05 0.59+ 0.05
ΜC (2 µm) 0.56+0.07 0.66+0.07 0.62+ 0.05 0.52+0.07
ΜC (4 µm) 0.58+0.05 0.60+ 0.07 0.68+ 0.08 0.63+ 0.05
ΜC (10 µm) 0.57+0.04 0.62+0.05 0.58+ 0.03 0.49+ 0.03
ΜC (20 µm) 0.54+0.06 0.63+ 0.08 0.51+ 0.03 0.58+ 0.01
ΜC (50 µm) 0.55+0.05 0.66+ 0.08 0.57+ 0.07 0.38+0.01*
ΜC (100 µm) 0.51+0.07 0.67+ 0.09 0.65+ 0.05 0.21+0.02*
Lymphocyte (IM9)
Con 0.49+ 0.03 0.51+ 0.04 0.48+ 0.05 0.49+ 0.05
ΜC (2 µm) 0.46+0.04 0.46+ 0.05 0.52+ 0.05 0.55+0.05
ΜC (4 µm) 0.53+0.05 0.50+ 0.06 0.59+ 0.07 0.53+ 0.06
ΜC (10 µm) 0.51+0.04 0.52+0.05 0.58+ 0.05 0.44+ 0.05
ΜC (20 µm) 0.54+0.06 0.53+ 0.08 0.51+ 0.04 0.48+ 0.01
ΜC (50 µm) 0.55+0.05 0.56+ 0.08 0.57+ 0.07 0.28+ 0.01*
ΜC (100 µm) 0.31+0.03* 0.24+ 0.02* 0.11+ 0.01* 0.09+ 0.01*
Data are mean +SEM of three independent experiments, with six replicates per sample. *
p<0.05 compared to control.
103
Table 4.4.Cytotoxicity, as assessed by MTT assay in various concentrations of α-NF in A549 and IM9 cells
Lung (A549) Conc 12 hour 24 hour 48 hour 96 hour
Con 0.55+ 0.05 0.54+ 0.1 0.53+ 0.05 0.57+ 0.05
α-NF (5µm) 0.50+ 0.05 0.54+ 0.1 0.51+ 0.07 0.59+ 0.06
α-NF (10µm) 0.56+ 0.05 0.52+ 0.07 0.38+0.02* 0.29+0.001*
α-NF (15 µm) 0.58+ 0.05 0.38+ 0.03 0.14+0.02* 0.01+ 0.005*
α-NF (20 µm) 0.51+ 0.04 0.19+0.01* 0.11+0.01* 0.01+ 0.003*
α-NF (50 µm 0.42+ 0.04 0.19+0.02* 0.05+0.01* 0.01+ 0.001*
Lymphocyte (IM9
Con 0.44+ 0.05 0.48+ 0.1 0.41+ 0.05 0.45+ 0.05
α-NF (5µm) 0.40+ 0.05 0.44+ 0.1 0.41+ 0.05 0.49+ 0.05
α-NF (10µm) 0.46+ 0.05 0.42+ 0.07 0.20+ 0.02* 0.09+0.001*
α-NF (15 µm) 0.48+ 0.05 0.33+ 0.03 0.18+ 0.02* 0.04+ 0.005*
α-NF (20 µm) 0.41+ 0.04 0.12+0.01* 0.17+ 0.01* 0.03+ 0.003*
α-NF (50 µm) 0.22+ 0.04 0.17+ 0.02* 0.09+ 0.01* 0.01+ 0.001*
Data are mean +SEM of three independent experiments, with six replicates per sample. * p<0.05
compared to control.
4.4.3 DEP induced DNA damage in A549 and IM9 cells
Exposure of DEP causes significant concentration dependent DNA
damage in A549 and IM9 cell line as assessed by olive tail moment (OTM), a
parameter of Comet assay, when added at different concentration to the cells
(Table 4.5). Maximum DNA damage (117.5%) was observed when DEP at the
concentration of 100µg/ml was added to the cells. However, at this
concentration, mortality of the cells was also observed. Based on the ability of
DEP to induce DNA damage as well as its cytotoxic effect, 12.5 µg/ml of DEP
for A549 and 25 µg/ml of DEP for IM9 (concentration in the medium) was
used as a potential genotoxic dose to assess DEP induced damage as well as
to identify the role of CYP1A1 in DEP induced DNA damage.
104
Table 4.5: Effect of diesel exhaust particles on DNA damage as
assessed by comet assay in A549 and IM9
Concentration of DEP OTM % increase OTM % increase
Lung (A549) Lymphocyte (IM9)
Control 0.87 +0.11 0.81+0.10
3.12 µg/ml 0.92 +0.5 5.7% 0.85+0.11 4.93
6.25 µg/ml 1.4+0.19 60.9% 0.92+0.14 13.5
12.5 µg/ml 1.9+0.2* 118.4% 1.01+0.13 24.69
25 µg/ml 3.34+0.5* 283% 1.52+0.16* 87.6
50 µg/ml 8.1+ 2.0* 831% 1.78+0.19* 119.7
100 µg/ml 11.1+ 3.3* 1175% 2.06+0.21* 154.3
Data are mean + SEM of three independent experiments. For each experiment the olive tail
moment of 50 cells was calculated. * p<0.05 compared to control.
4.4.4 Effect of pretreatment of CYP1- modifiers on DEP mediated
induction of CYP mRNA, protein expression and associated enzyme
activity
In order to investigate the role of PAH-metabolizing CYP1A1 and
CYP1B1 isoenzymes in DEP-induced DNA damage, the mRNA expression of
CYP1A1 and CYP1B1 was determined by RT-PCR in control and treated
A549 and IM9 cells. Pretreatment of MC (4µM) to the A549 cells for 18h
resulted in several fold higher induction in the mRNA expression of CYP1A1
(159-fold) or CYP1B1 (17.4-fold) in the treated cells (Table 4.6 ). The mRNA
expression of CYP1A1 and CYP1B1 was increased by 25 and 4.9 folds
respectively in cells exposed to DEP for 6h. When the MC pretreated cells
were exposed to DEP, still higher increase in the mRNA expression of
CYP1A1 (200-fold) and CYP1B1 (13.9 fold) was observed. This synergistic
effect of MC and DEP in the expression of CYP1A1 and 1B1 was lowered to
about 105 and 5.6 fold respectively when α-NF, an inhibitor of CYP1A1 and
1B1 catalyzed reaction was added to these cells (Table 4.6). For IM9 cells,
Pre-treatment of MC (15µM) to the IM9 cells for 12h resulted in induction of
CYP1A1 (2.84 fold) & CYP1B1 (1.62-fold) in the treated cells (Table 4.6).
Further mRNA expression of CYP1A1 and CYP1B1 was increased by 1.54
fold and 1.31 fold respectively in cells exposed to DEP for 6h. When the MC
pre-treated cells were exposed to DEP, still higher increase in the mRNA
105
expression of CYP1A1 (2.91 fold) and CYP1B1 (1.66 fold) was observed. This
synergistic effect of MC and DEP in the expression of CYP1A1 and 1B1 was
lowered to about 1.57 fold and 1.36 fold respectively when CYP1A1 and
CYP1B1 inhibitor α-NF was added to these cells (Table 4.6).
Table 4.6: Effects induced by 3-MC, DEP and α-NF on the relative mRNA
expression of CYP 1A1 and 1B1 in A549 and IM9
Category CYP1A1 CYP1B1 Category CYP1A1 CYP1B1
Lung (A549) Lymphocyte (IM9)
Control A549 1+0.25 1+ 0.18 Control IM9 1+0.13 1+ 0.2
α-NF (20 µm) 1.2+0.15 1.1+0.1 α-NF (10 µm) 1.09+ 0.17 1.1+ 0.11
DEP(12.5 µg/ml) 20+ 4.5* 4.9+ 1.3* DEP(25µg/ml) 1.64+0.14* 1.31+ 0.2
MC (4 µm) 129+ 20.0* 7.4+ 2.5* MC (15 µm) 2.84+ 0.22* 1.62+ 0.18*
MC+DEP 170+ 32.0* 11.9+ 4.3* MC+DEP 2.91+ 0.21* 1.66+ 0.2*
MC+ DEP+ α-NF 75+ 10.2* 5.6+ 1.5* MC+ DEP+ α-NF 1.77+ 0.18* 1.36+ 0.17
Each reaction was performed in triplicate on cDNA samples in 96 well optical plates The
threshold cycle value (Ct value) of each sample was normalized with Ct value of endogenous
control (β- actin) (∆ Ct). Fold change is calculated from∆∆Ct value of each sample∆∆Ct=∆Ct of
treated-∆Ct of control. p<0.05 when compared with the controls. All the values are mean+ S.E.
of 6 animals. *p<0.05 when compared to the controls.
As shown in Table 4.7 , activity of EROD, a marker for CYP1A1 and 1B1
isoenzymes, was significantly increased (380%) in A549 cells treated with
DEP. Exposure of DEP to the cells also resulted in increase (100%) in the
activity of EROD (Table 4.7). In-vitro addition of DEP to the cells preincubated
with MC led to still higher increase (500%) in the enzyme activity when
compared to the cells treated with DEP or MC alone. Treatment of α-NF to the
cells treated with MC and DEP reduced the extent of induction in the EROD
activity in A549 cells. About 150% increase in the activity of these cells was
observed when compared to the cells treated with MC+DEP (500%) or MC
(380%) or DEP (100%) alone (Table 4.7).
When IM9 cells were exposed to DEP or DEP modulator of CYPs,
activity of EROD, was significantly increased (162%) in cells treated with MC.
Exposure of DEP to the cells resulted in increase (75%) in the activity of
106
EROD (Table 4.7). In vitro addition of DEP to the cells, preincubated with MC,
lead to a still higher increase (250%) in the enzyme activity when compared to
the cells treated with DEP or MC alone. Treatment of α-NF to the cells treated
with MC and DEP showed about 112.5% increase in the EROD activity when
compared to the cells treated with MC+DEP (250%) or MC (162.5%) or DEP
(75%) alone (Table 4.7).
Table 4.7: Effects induced by 3-MC, DEP and α-NF on the EROD activity
in human A549 and IM9 cell line
Category ERODa % increase Category EROD
a % increase
Lung(A549) Lymphocyte(IM9)
Control A549 0.1+ 0.01 Control IM9 0.08+0.01
α-NF (20 µm) 0.11+ 0.01 10 α-NF (10 µm) 0.082+0.07 2.4
DEP(12.5 µg/ml) 0.2+ 0.02* 100 DEP(25µg/ml) 0.14+0.01* 75
MC (4 µm) 0.48+0.05* 380 MC (15 µm) 0.21+0.02* 162.5
MC+DEP 0.6+0.07* 500 MC+DEP 0.28+0.04* 250
MC+ DEP+ α-NF 0.25+ 0.01* 50 MC+ DEP+ α-NF 0.17+0.02 25
Data are mean + SEM of three independent experiments. * p<0.05 compared to control
Immunocytochemical studies have further demonstrated the involvement of
CYP1A1 induction in DEP mediated toxicity. A549 or IM9 cells when
incubated with monoclonal antibody raised against human CYP1A1 (primary
antibody) and the secondary antibody labelled with FITC showed positive
staining (green fluorescence) for CYP1A1 as observed by fluorescence
microscopy. As evident from the Figure, intensity of the staining was greater
in A549 cells indicating higher basal expression of CYP1A1 in these cells
when compared to IM9 cells. Superimposition of fluorescence exhibited by
FITC with DAPI, the nuclear stain revealed that the CYP1A1 mediated
fluorescence was localized in the cytoplasm of the cell (Fig. 4.2, 4.3). Cells
(A549 or IM9) isolated after pretreatment with MC revealed intensity of much
higher magnitude in A549 cells when compared to IM9 cells. As observed with
control cells, superimposition of fluorescence exhibited by FITC with DAPI,
the nuclear stain revealed that the CYP1A1 mediated fluorescence was
localized in the cytoplasm of the cell. Exposure of DEP to A549 or IM9 cells
also increased the intensity of positive staining for CYP1A1, though the
magnitude of increase was less in these cells (A549 or IM9) when compared
to the cells exposed to MC.
DEP showed marked increase in the expression of CYP1A1 as characterized
by increase in the intensity of fluorescence treated A549 cells
The increase in the intensity of positive staining for CYP1A1 was higher in the
cells exposed to the combination of MC and DEP when compared to MC
alone or DEP alone (Fig. 4.2, 4.3). Pretreatment of cells with
reduced positive staining in both cells when the cells exposed to MC or DEP
alone.
107
magnitude of increase was less in these cells (A549 or IM9) when compared
to the cells exposed to MC. Further, cells isolated after exposure
DEP showed marked increase in the expression of CYP1A1 as characterized
by increase in the intensity of fluorescence treated A549 cells
The increase in the intensity of positive staining for CYP1A1 was higher in the
osed to the combination of MC and DEP when compared to MC
alone or DEP alone (Fig. 4.2, 4.3). Pretreatment of cells with α
reduced positive staining in both cells when the cells exposed to MC or DEP
magnitude of increase was less in these cells (A549 or IM9) when compared
posure of MC and
DEP showed marked increase in the expression of CYP1A1 as characterized
by increase in the intensity of fluorescence treated A549 cells or IM9 cells.
The increase in the intensity of positive staining for CYP1A1 was higher in the
osed to the combination of MC and DEP when compared to MC
alone or DEP alone (Fig. 4.2, 4.3). Pretreatment of cells with α-NF resulted in
reduced positive staining in both cells when the cells exposed to MC or DEP
4.4.5 Involvement of reactive o
To further identify if ROS are generated during CYP mediated
metabolic activation and toxicity of chemical ingredients present in DEP, in
vitro studies were initiated with DEP in the presence of CYP modifiers.
Treatment of A549 cells with DCFDA show
in ROS formation in cells incubated with DEP compared to control A549 cells
as measured through increased bright fluorescence of DCFDA suggesting
that ROS are generated during the met
To further identify the involvement of CYP1A1 in this generation of ROS, DEP
was added to the cells preincubated with MC. Significant increase (209.5%) in
the generation of ROS in the cells exposed to MC alone while a s
magnitude of increase (384.4%) in the formation of ROS was evident when
the MC preincubated ce
when α-NF was preincubated along with MC and the cells were then exposed
to DEP, the formation of
108
Involvement of reactive oxygen species in DEP induced toxicity
To further identify if ROS are generated during CYP mediated
metabolic activation and toxicity of chemical ingredients present in DEP, in
vitro studies were initiated with DEP in the presence of CYP modifiers.
Treatment of A549 cells with DCFDA showed considerable increase (136.2
in ROS formation in cells incubated with DEP compared to control A549 cells
as measured through increased bright fluorescence of DCFDA suggesting
that ROS are generated during the metabolism of chemicals present in DEP.
To further identify the involvement of CYP1A1 in this generation of ROS, DEP
was added to the cells preincubated with MC. Significant increase (209.5%) in
the generation of ROS in the cells exposed to MC alone while a s
magnitude of increase (384.4%) in the formation of ROS was evident when
the MC preincubated cells were exposed to DEP (Table 4.8
NF was preincubated along with MC and the cells were then exposed
to DEP, the formation of ROS was significantly reduced (142.2%)
xygen species in DEP induced toxicity
To further identify if ROS are generated during CYP mediated
metabolic activation and toxicity of chemical ingredients present in DEP, in
vitro studies were initiated with DEP in the presence of CYP modifiers.
ed considerable increase (136.2%)
in ROS formation in cells incubated with DEP compared to control A549 cells
as measured through increased bright fluorescence of DCFDA suggesting
abolism of chemicals present in DEP.
To further identify the involvement of CYP1A1 in this generation of ROS, DEP
was added to the cells preincubated with MC. Significant increase (209.5%) in
the generation of ROS in the cells exposed to MC alone while a still higher
magnitude of increase (384.4%) in the formation of ROS was evident when
s were exposed to DEP (Table 4.8). Interestingly,
NF was preincubated along with MC and the cells were then exposed
ROS was significantly reduced (142.2%)
109
demonstrating that PAH-inducible CYPs (CYP1A1) is involved in the
formation of ROS during the metabolic activation of chemical ingredients
present in DEP (Table 4.8).
Similarly as seen with lung A549 cells, lymphocyte IM9 cells also
showed statistically significant increase in the formation of ROS in all set of
treated IM9 cells. IM9 cells incubated with DEP showed increase of upto
66.9% compared to control cells. Incubation of cells with MC or as combined
with DEP showed increase in the fluorescence of upto 115.9% and 159.4%
respectively. Interestingly, when α-NF was preincubated along with MC and
the cells were then exposed to DEP, the formation of ROS was significantly
reduced (86.95%) (Table 4.8).
Table 4.8: Reactive oxygen species production (ROS) by 3-MC, DEP,
MC+DEP and MC+DEP+ α-NF in cultured A549 and IM9 cell line.
Data are mean + SEM of three independent experiments, with three replicates per sample.
* p<0.05 compared to control
4.4.6 Role of CYP1A1 in DEP mediated DNA damage
To identify the involvement of CYP1A1 as well as to assess similarity
in the DEP induced DNA damage in both A549 and IM9 cell line, effect of
DEP in the presence of CYP1A1 inducer (MC) and inhibitor (α-NF) of DEP
was studied in lung and lymphocyte cell line. Exposure of DEP (12.5µg/ml for
6h) or MC (4µM for 18 h) showed increase in tail moment value by about
174% and 394% respectively when added to the control A549 cells (Table
4.9). However, addition of DEP to the cells, preincubated with MC, further
increased DNA damage as assessed by measuring the olive tail moment
significantly to 394% when compared to the effect seen in control cells alone.
Category DCFfluorescence % Inc. Category DCFfluorescence % Inc.
Lung (A549) Lymphocyte (IM9)
Control A549 116+ 9.8 Control IM9 41.4+ 5.7
α -NF (20 µm) 122.8+ 10.2 5.8 α-NF (10 µm) 43.2+ 5.4 4.3
DEP(12.5µg/ml) 274+ 33.6* 136.2 DEP(25µg/ml) 69.1+ 6.2* 66.9
MC(4µm) 359+ 37.0* 209.5 MC (15 µm) 89.4+ 8.1* 115.9
MC+DEP 562+ 45* 384.4 MC+DEP 107.4+ 10.4* 159.4
MC+DEP+ α-NF 281+ 14.8* 142.2 MC+ DEP+ α-NF 77.4+ 5.5* 86.95
110
Further, as evident from Table 4.9, addition of α-NF to the cells incubated with
DEP and MC significantly reduced the DNA (191%) when the data was
compared to the cells containing DEP alone (174%) or cells exposed to
DEP+MC (394%). Addition of α-NF to the cells alone did not produced any
significant DNA damage as evident by the alterations in the olive tail moment
assessed by Comet assay (Table 4.9).
As compared to A549 cells, exposure of DEP (25 /ml for 12h) or MC
(15µM for 12 h) showed increase in tail moment value by about 71.26% and
96.5% respectively when added to the control IM9 cells (Table 4.9). However,
addition of DEP to the cells preincubated with MC further increased DNA
damage, as assessed by measuring the olive tail moment, significantly to
137.9% when compared to the effect seen in control cells alone. Further, as
evident from Table 4.9, addition of α-NF to the cells incubated with DEP and
MC significantly reduced the DNA (74.71%) when the data was compared to
the cells containing DEP alone (71.26%) or cells exposed to DEP+MC
(394%). Addition of α-NF to the IM9 cells alone did not produced any
significant DNA damage as evident by the alterations in the olive tail moment
assessed by Comet assay (Table 4.9).
Table 4.9: Genotoxic effect induced by CYP1A1 inducer 3-MC and diesel
exhaust particles in A549 cells with and without CYP1A1 inhibitor alpha
naphthoflavone.
Category OTM % increase Category OTM % increase
Lung (A549) Lymphocyte (IM9)
Control 0.91+ 0.2 Control 0.87+ 0.1
α-NF(20 µm) 1.1+ 0.1 20.8% α-NF (10 µm) 0.89+ 0.2 2.29
DEP(12.5 µg/ml) 2.5+ 0.3* 174% DEP(25µg/ml) 1.49+ 0.14* 71.26
MC (4 µm) 3.5+0.5* 284% MC (15 µm) 1.71+ 0.2* 96.5
MC+DEP 4.5+ 0.9* 394% MC+DEP 2.07+ 0.22* 137.9
MC+ DEP+ α-NF 2.71+ 0.2* 191% MC+ DEP+ α-NF 1.52+ 0.11* 74.71
Data are mean + SEM of three independent experiments. For each experiment the olive tail
moment of 50 cells was calculated. * p<0.05 compared to control.
111
4.5 Discussion
In the preceding chapters we have shown similarities in the expression
profiles of drug metabolizing enzymes involved in the toxicity of DEP in freshly
prepared PBLs and lung. This, along with the previous reports on the
similarities in the regulation of blood lymphocyte CYP1A, 2B & 2E1 with tissue
enzymes has suggested that freshly isolated PBL could be used as a
surrogate to monitor toxicity of DEP and other toxic chemicals (Vanden
Heuvel et al., 1993; Raucy et al., 1999; Dey et al., 2001, 2006; Saurabh et al.,
2010, 2011). Further, significant increase in the expression of CYP1A1 or
CYP2E1 in freshly prepared blood lymphocytes isolated from patients
suffering from lung cancer or alcoholic cirrhosis have suggested that CYP1A1
or CYP2E1 mRNA profiles of freshly blood lymphocytes could be used as a
biomarker to predict these diseases (Shah et al.,2009; Khan et al., 2011).
Krovat et al., (2000) using blood derived cell lines (HEL,IM9,HL60, THP)
observed poor responsiveness of some of the CYPs, though the constitutive
pattern of expression of xenobiotic metabolizing CYPs was maintained in
blood cell lines indicating the suitability of using blood derived cell lines for
understanding cell specific and tissue specific toxic events.
The data of the present dissertation showed that as observed with
A549, the lung derived cell line, the expression of PAH metabolizing CYP1A1
& 1B1 were expressed in IM9, the lymphocyte derived cell line. Comparison of
∆Ct values revealed that as reported with the tissues, the basal expression of
CYP1B1 was greater than 1A1 in IM9 cell line. Similar constitutive mRNA
expression of CYP1A1 & 1B1 has been reported in freshly prepared PBL
isolated from rats (Sharma et al., 2013). Exposure of MC to IM9 cells was
found to increase the mRNA expression of CYP1A1 and 1B1 in IM9 cells. As
observed with A549 cells, a greater magnitude of increase was observed in
the expression of CYP1A1 when compared to CYP1B1 after exposure of MC
to the IM9 cells. Similar pattern of increase in the mRNA expression of
CYP1A1 and 1B1 was reported in freshly prepared PBL isolated from MC
pretreated rats when compared to the controls (Saurabh et al., 2010). As
reported with the freshly prepared PBL and tissues, the mRNA expression of
CYP1A1 and 1B1 and their induction following exposure of MC was less in
112
IM9 when compared to A549 cells. Further, the increase in the protein
expression of CYP 1A1, as demonstrated by immunocytochemical analysis
have shown that like in the tissues and PBL, the induction of CYP1A1 is
transcriptionally regulated in blood derived cell line. The increase in the EROD
activity in IM9 cells following exposure of MC have shown that as observed in
the tissues as well as PBL, CYP1 family of isoenzymes expressed in blood
cells is catalytically active. Similar response of MC though of a higher
magnitude was observed in A549 cells, the lung derived cell line.
Exposure of DEP to IM9 cells was found to significantly increase the
expression of CYP1A1 and 1B1. Similar increase though of a greater
magnitude was observed in A549 cells. In the preceding chapters, similar
increase in the expression of these CYP enzymes was observed in PBL and
the lungs isolated from rats treated with DEP. Though there are no reports
available on the effects of DEP on blood derived cell lines, DEP or DEP
extracts have been reported to increase the activity of CYP1A1 in A549 cells
or other lung derived cell line (Bonvallot et al., 2001; Iwanari et al., 2002;
Baulig et al., 2003; Mahadevan et al., 2004; Vogel et al., 2005; Zhao et al.,
2006; Iba et al., 2010; Totlandsdal et al., 2010) As suggested earlier in in vivo
studies, the induction of CYP1A1 or 1B1 in lymphocyte or lung derived cell
have demonstrated that the increase in CYP1A1 or 1B1 activity could be
attributed to the PAHs present in DEP (Yamasaki et al., 2000; Kuljukka Rab et
al., 2001; Iba et al., 2010; Srivastava et al., 2012). Immunocytochemical
studies and enzymatic assays have further indicated that the DEP induced
increase in the mRNA expression of CYP1A & 1B1 is associated with an
increase in the protein expression of CYP1A1, CYP1B1 and EROD activity in
both lymphocyte and lung derived cell lines further providing evidence for
similarities in the regulation of blood lymphocyte CYPs with the tissue
enzymes after DEP exposure.
Further, greater magnitude of increase in the expression of CYP1A and
1B1 isoenzymes following exposure of DEP in A549 or IM9 cells pretreated
with MC has shown that CYP1A1 and 1B1 catalyse the metabolic activation of
chemicals present in DEP. As pretreatment of MC is known to enrich the
levels of CYP1A1 and 1B1 isoenzymes in tissues or specific cell ines,
113
enrichment of these CYP isoenzymes in IM9 or A549 cells may lead to the
greater metabolic activation of DEP as evident by greater magnitude of
increase in the expression of CYP1A1 or 1B1 in these cell lines, when
compared to those exposed to MC alone or to DEP alone. Greater magnitude
of induction of CYP1A1 and CYP1B1 in the lung cells is further consistent with
the in vivo studies indicating the greater magnitude of alterations in lungs
when compared to PBL. That CYP1A1&1B1 catalyze the bioactivation of DEP
was further evident when α-naphthoflavone (α-NF), an inhibitor of CYP1A1,
1B1 catalysed reactions, was added to the cells pretreated with MC. Similar to
that seen with A549 cells, pretreatment of α-NF and MC to IM9 cells prior to
the exposure of DEP showed much greater decrease in the expression of
CYP1A1 and 1B1 isoenzymes suggesting occurrence of similar regulatory
mechanisms for DEP and PAHs for CYP induction in both, lung or lymphocyte
derived cell lines.
In addition to the binding of PAH to AhR that increase the expression of
CYP1A isoenzymes (Whitlock, 1996; Denison, 1998), PAHs are reported to
enhance the generation of ROS such as superoxide anion, hydroxyl radical,
and hydrogen peroxide by inducing the levels of CYP1A1 (Guenrich, 1988; Xu
et al., 2005). Potent CYP1A inducer such as MC or TCDD also leads to
increased ROS generation by inducing the levels of CYP1A (Park et al., 1996;
Liu et al., 2001). Consistent with the previous reports that exposure to PAH
increase the production of ROS, our study also showed that treatment of IM9
or A549 cells with MC leads to the increased production of ROS in IM9 or
A549 cells. Higher magnitude of ROS production in A549 cell line can be
related to greater magnitude of induction of CYP1A1 in A549 cells. Similar to
that observed with MC, exposure of DEP also leads to the increased
production of ROS in IM9 or A549 cells thus demonstrating that PAHs present
in DEP account for increase in the production of reactive oxygen species
(ROS) either directly or through metabolic activation of PAHs, halogenated
aromatic hydrocarbons (HAH), and redox-active quinines present in DEP
(Ichinose et al., 1997; Kumagai et al., 1997; Hiura et al., 1999; Wan and Diaz
Sanchez, 2007). Studies have provided evidence of DEP induced ROS
generation via metabolic activation of organic compounds such as PAHs
adsorbed on DEP through CYP1A1, microsomal P450 reductase and quinone
114
reductases (Bonvallot et al., 2001; Kumagai et al., 1997). Further, exposure of
DEP in A549 or IM9 cells pretreated with MC potentiated the generation of
ROS in either IM9 or A549 cells indicating that the increase in the ROS
production in either IM9 or A549 cells is due to the increase in the expression
of CYP1A or 1B1 enzymes. Decreased ROS formation in both IM9 and A549
cells pretreated with MC and α-NF demonstrate that depletion of the levels of
CYP1A- or 1B1 by α-NF may account for the decrease in the generation of
ROS from DEP. That the increase in the generation of ROS, due to the
increased expression of CYP1A and 1B1 isoenzymes, may lead to toxic
manifestations was demonstrated in the present study indicating oxidative
DNA damage in the blood cells exposed to DEP. Studies have shown that
reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), Hydroxyl
radicals (.OH), Superoxide (O2.-) and singlet oxygen(1O2) as well as reactive
nitrogen species (RNS) including nitric oxide and peroxynitrite generated by
DEPs have a impact on DNA damage and are considered as a major
determinant of genotoxic properties (Marnett, 2000; Knaapen, 2004). Further
treatment of DEP also induced considerable DNA damage in both IM9 and
A549 cells with higher magnitude of damage in A549 cells evident from comet
assay. This is in agreement with previous studies showing that DEP induces
DNA damage in various types of cell lines (Don porto Carero et al., 2001;
Danielson et al., 2008; Jantzen et al., 2012). As observed with CYP1A1
induction, pretreatment of MC with DEP was found to increase the DNA
damage suggesting the increased generation of ROS in the pretreated IM9 or
A549 cells. Previous in vivo and in vitro studies have shown that bioactivation
of PAHs present in DEPs by CYP1A isoenzymes is partially responsible in
mediating genotoxic response as seen through DNA strand breaks or through
formation of DNA adduct (8-OHdG) formation in exposed cells (Ichinose et
al.,1997; Danielsen et al., 2008; Iba et al., 2013; Vattanasit et al., 2014). The
reduction in the extent of DNA damage following preincubation of A549 or IM9
cells with α-NF, has further provided evidence that DNA damage induced by
DEP was mediated through ROS generated during the CYP catalysed
metabolic activation.
In conclusion, present study has shown that responsiveness of CYPs
expressed in blood cells is retained under in vitro conditions. As observed
115
with in vivo studies and A549 cells, DEP was found to induce the expression
of PAH-responsive CYP1A1 and 1B1 in IM9 cells. These studies also showed
that PAH responsive CYPs catalyse the metabolic activation of chemicals
present in DEP. Our data also provided evidence that PAHs in DEP leads to
ROS generation that caused DNA damage in lymphocytes and lung derived
cells. The present data exhibiting similarities in the responsiveness of DEP in
blood derived cell line with lungs cells has further provided evidence that
blood lymphocytes can be used not only for studying cell specific toxic but
more importantly as a surrogate to monitor tissue expression and as a reliable
and less invasive tool for monitoring toxicity of environmental chemicals and
adverse drug effects in clinical settings.
Summary
116
SUMMARY
The mixed function oxidase system (MFO), localized in the
endoplasmic reticulum of many mammalian cells, contain many enzymes
including cytochrome P450s (CYPs), cytochrome b5, and NADPH-
cytochrome P450 reductase. CYPs act as a terminal oxidase for the electron
transport system of mixed-function oxidase, which is implicated in the
biotransformation of many xenobiotics including drugs, toxins, and
carcinogens and endogenous substrates, such as steroids and fatty acids.
Although liver is considered to be a repository of CYPs and other MFOs,
freshly prepared peripheral blood lymphocytes (PBLs) are being increasingly
used as a surrogate to monitor tissue effects caused by exposure to toxic
agents. Lymphocytes have advantages for use in the development of least
invasive assays to screen human population for toxicant exposure. The use of
PBL in identifying genotoxicity of environmental toxicants is well established.
Cytogenetic alterations in PBL with known or suspected genotoxic
carcinogens have been used as biomarkers in genotoxicity studies. Smoking
related PAH-DNA adducts in human lymphocytes have proved to be a good
dosimetric exposure markers.
There has been an interest to develop expression profiles of CYPs in
peripheral blood samples as a tool for predicting adverse drug effects &
exposure of environmental chemicals. DNA array studies have provided
evidence for expression of majority of CYPs and their respective transcription
factors in freshly prepared peripheral blood lymphocytes (PBL), suggesting
that CYP expression profiles in PBL could be used as a biomarker predicting
the exposure of drugs & environmental chemicals. Recent studies from our
laboratory have shown similarities in the expression of xenobiotic
metabolizing CYPs in freshly prepared PBL with the tissue enzymes.
Polycyclic aromatic hydrocarbon (PAH)-metabolizing CYP1A-, 1B1
isoenzymes and ethanol metabolizing CYP2E1 were found to be expressed in
rat and human PBL and showed similarities in the mechanism of regulation of
these CYPs in PBL with the tissue enzymes. Further, clinical studies
indicating that CYP1A1 and CYP2E1 mRNA could be used as a biomarker for
identifying tobacco induced lung cancer and alcoholic liver cirrhosis
117
respectively have prompted studies to investigate PPBL expression profiles of
candidate genes as a surrogate to monitor toxicity of environmental chemicals
Vehicular emissions continue to be the major source of air pollution in
developing as well as developed nations. Diesel exhaust particles (DEPs) are
the major contributors of vehicular emissions and the risk to these exhaust
emissions is relatively high in the developing countries where because of poor
regulatory compliance, the exposure is often higher than the permissible limits
laid down by the regulatory agencies. The majority of DEPs are classified as
fine (2.5-0.1 mm) or ultrafine (< 0.1 mm) particles, but these primary DEPs
can coalesce to form aggregates of varying sizes. It has been postulated that
because smaller particles have a greater relative surface area, they should
carry proportionally more chemicals and have greater biologic effects.
Growing epidemiological and experimental evidence have demonstrated
correlation between exposure to DEPs and adverse health outcomes. DEPs
consist primarily of an elemental carbon core with a large surface area to
which organic compounds are adsorbed, many of which are known to alter the
microsomal xenobiotic metabolizing enzymes resulting in altered metabolism
as well as increased generation of reactive oxygen species (ROS) which
leads to oxidative stress resulting in a variety of toxic manifestations. Most of
the mechanistic studies have attributed the proinflammatory and adjuvant
effects of DEPs to these chemical constituents. DNA microarrays have
provided a mechanistic insight of the adverse effects of DEP in lungs. The
role of drug metabolizing enzymes, oncogenes, inflammatory response genes
and other stress related genes have been identified in DEP induced
pulmonary toxicity. A good correlation has been reported between the
increase in DNA mutation frequency and the extent of DNA adducts formation
in the cells of rat lungs after exposure to DEP.
To validate expression profiles of target genes as a tool for monitoring
tissue expression and toxicity of DEP, the present study attempted to
investigate i) Taqman based low density array (TLDA) to identify similarities in
the mRNA expression of target genes altered by exposure to diesel exhaust
particles (DEPs) in freshly prepared PBLs and lungs; ii) to identify similarities
in the alterations in the mRNA and protein expression and activity of CYPs
118
and glutathione S-transferases (GSTs), involved in the metabolic activation
and detoxification of PAHs present in DEPs, in PBL and lungs of rats exposed
to DEP; and iii) Using in vitro approaches, identify the role of CYP mediated
metabolic pathways in DEP induced genotoxicity in IM9, the blood derived cell
line after exposure to DEP.
For TLDA based studies, adult wistar rats were treated transtracheally
with single dose of 7.5- or 15- or 30 mg/kg of DEP. The rats were sacrificed
24 hrs after the exposure and blood and lungs were processed for Real time-
PCR (RT-PCR). TLDA data revealed that DEP treatment simultaneously
increased the expression of CYP1A1, 1A2, 1B1 and 2E1 isoenzymes,
involved in the metabolism of PAHs present in DEP in both rat lungs and PBL.
Though the expression of these PAH metabolizing CYPs was found to be of
relatively higher magnitude than PBL, the pattern of induction was found to be
similar in both, lungs and PBL. This increase in CYPs was associated with a
simultaneous increase in the expression of transcription factors such as AhR,
Arnt, c-fos, fosl1 and jun as well as MAPK 8, 9, 10 in both, lungs and PBL
after DEP exposure reports indicating participation of various regulatory
components in the regulation of CYPs and reflection of these regulatory
pathways in PBL. Likewise as observed with CYPs, various GST isoforms
(GSTA5, GSTM1, GSTM2, GSTM3, GSTM5, GSTP, GSTO1, GSTO2, GSTK,
mGST) and antioxidant enzymes such as superoxide dismutase (SOD),
metallothionein, peroxiredoxin and transcription factors also showed
similarities in the alteration of expression in PBL and lungs isolated from rats
treated with DEP indicating that like phase I enzymes, similarities were
observed in the responsiveness of the phase II enzymes to DEP in PBL with
the tissue enzymes. As observed with the lungs, an similar increase in the
expression of several chemokines (CCL2, CCL5), cytokines (IL-1β, IL-6, TGF-
β and IL-12) and adhesion molecules (ICAM1, VCAM) was observed in PBL
isolated from DEP exposed rats. Our data further revealed almost similar
pattern of increase in the mRNA expression of DNA repair genes such as
DNA glycosylase (OGG1), DNA topoisomerase, poly ADP ribose polymerase
and PCNA and apoptotic genes such as BbC3, PDCD8, BID,BAD, BCL2
family and caspase 3 in both, lungs and PBL isolated from DEP treated rats.
The data thus indicating similarities in the responsiveness of candidate genes,
119
involved in the toxicity of DEP, in PBL with the lungs after exposure to DEP
demonstrates that expression profiles of genes in PBL could be used as a
surrogate for monitoring the alterations in tissue expression induced by
exposure of fine and ultrafine particulate matter present in vehicular
emissions.
Studies were further carried out to validate the alterations in the
expression of blood lymphocyte CYPs and GSTs, involved in the metabolic
activation and detoxification of DEP, by investigating similarities or
differences, if any, in the activity of these enzymes in PBL and lungs isolated
from rats exposed to DEP. Transtracheal instillation of different doses (3.75-
or 7.5- or 15- or 30 mg/kg) of DEP resulted in significant alterations in the
catalytic activity of CYP dependent enzymes and GSTs in lungs and freshly
prepared PBL isolated form rats 24 hrs after the exposure. Statistically
significant increase in the enzymatic activity of CYP1A1 and CYP1A2
dependent 7-ethoxyresorufin-O-deethylase (EROD) & 7-methoxyresorufin-O-
deethylase (MROD) was observed in rat lungs. Similar to that seen in lungs,
lymphocytes also showed similar pattern of induction of EROD & MROD
activity at all doses of DEP with magnitude of induction being relatively less
than the tissue enzymes. Statistically significant and similar change was
observed in both lymphocytes and lungs with maximum increase being
observed in the rats receiving 15 mg/kg dose of DEP. The activity of CYP2E1
dependent N-nitrosodimethylamine demethylase (NDMA-d) activity also
showed similar increase at all the doses in both, lymphocytes and lungs. In
contrast, a dose dependent decrease in the activity of CYP2B1 dependent 7-
pentoxyresorufin-O-deethylase (PROD) was obsered in both, lymphocytes and
lungs. Similar to that seen with CYPs, a similar pattern of increase in the
activity of GSTs was observed in PBL and lungs isolated from rats exposed to
different doses of DEP. Consistent with the enzymatic analysis and RT-PCR
studies, immunocytochemical studies revealed that the increase in the activity
of EROD, MROD and NDMA-d is associated with an increase in the protein
expression of CYP1A1/ CYP1A2 and CYP2E1 protein after exposure of DEP
both, freshly prepared PBL and lungs 24 hrs after DEP exposure. Thus our
data indicating similarities in the alterations in the expression of PAH-
metabolizing CYPs and GSTs in PBL with the lung enzymes suggests that
120
expression profiles of blood lymphocyte CYPs and GSTs could be used as
biomarkers for predicting exposure of DEP in monitoring studies.
Mechanistic studies were carried out in established human blood cell
lines to further demonstrate the suitability of using PBL as a surrogate for
monitoring tissue expression as well as toxicity of DEP. Both, in A549, the
lung derived cell line and IM9, the lymphocyte derived cell line were found to
constitutively express the PAH- metabolizing CYP1A1, 1B1 isoenzymes. As
observed in PBL or lungs isolated from rats treated with DEP, a concentration
dependent increase in the expression and activity of CYP1A1 and 1B1
isoenzymes was observed in A549 or IM9 cells exposed to DEP. DEP
exposure also induced concentration dependent DNA damage as
demonstrated by Comet assay in A549 or IM9 cells. This DNA damage was
attributed to increased generation of ROS following DEP exposure in A549 or
IM9 cells. Induction studies carried out to identify the role of PAH-metabolizing
CYPs in DEP induced DNA damage revealed that similar to that seen in PBL
or lungs isolated from rats pretreated with CYP1A- isoenzyme inducer (MC)
or A549 cells exposed to MC, the mRNA, protein expression and associated
catalytic activity was significantly increased in IM9 cells exposed to MC. The
greater magnitude of increase in the expression and activity of CYP1A and
1B1 following exposure of DEP in A549 or IM9 cells pretreated with MC has
shown that CYP1A1 and 1B1 isoenzymes, which specifically catalyse the
metabolic activation of PAHs present in DEP, are further induced after DEP
exposure resulting in synergistic/ additive effect on CYP1A1/1B1 in both,
A549 or IM9 cells. That the increase in expression of CYP1A1/ 1B1 is of
toxicological significance was shown by increased generation of ROS and
DNA damage as evident by Comet assay in IM9 or A549 cells pretreated with
MC and subsequently exposed to DEP. Further evidence for the role of PAH-
metabolizing CYPs in the toxicity of DEP was provided when α -NF, a specific
inhibitor of CYP1A1 was added to the cells exposed to DEP along with MC.
While addition of a-NF alone to the control cells (IM9 or A549 cells) did not
produce any significant effect on the expression of CYP1A1/1B1 or ROS
generation or DNA damage, addition of α -NF cells to the cells exposed to MC
resulted in significant inhibition of the enzyme activity or ROS generation or
DNA damage when compared to the cells (A549 or IM9) exposed to MC
121
alone. That the CYP1A1/1B1 isoenzymes enriched in A549 or IM9 cells
following exposure of MC and DEP together catalyse the DNA damage and
ROS generation was evident by the lesser magnitude of DNA damage, ROS
generation and CYP1A1 mRNA and protein expression in cells pretreated
with a-NF along DEP and MC. The present study thus exhibiting similarities in
the toxicity of DEP in blood derived cell lines with lungs cells has further
provided support to our data indicating that freshly prepared peripheral blood
lymphocytes can be used to monitor toxicity of DEP.
In conclusion, the data of present study have provided evidence for
similarities in the responsiveness of candidate genes, involved in the toxicity
of DEP, in freshly prepared PBL with the lungs after exposure to DEP.
Similarities in the regulation of of PAH- metabolizing CYPs and GSTs in PBL
with the tissue enzymes after exposure to DEP has led us to suggest that
activity of blood lymphocyte CYPs and GSTs could be used as a surrogate to
monitor tissue damage. Mechanistic studies further demonstrated similarities
in ROS generation and resulting DNA damage in blood derived cell lines and
lung cell lines exposed to DEP. The present dissertation has thus
demonstrated the suitability of using expression profiles of mixed function
oxidases in freshly prepared peripheral lymphocyte as a surrogate for
monitoring tissue expression and in evaluating toxicity of DEP and other fine
and ultrafine particulate matter present in vehicular emissions.
References
122
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List of Publications
154
List of Publications and Abstracts originating from thesis
1. Srivastava A, Yadav S, Sharma A, Dwivedi UN, Flora SJ, Parmar D:
Similarities in diesel exhaust particles induced alterations in
expression of cytochrome P-450 and glutathione S-transferases in
rat lymphocytes and lungs. Xenobiotica 2012, 42(7):624-32.
2. Srivastava A, Sharma A, Yadav S, Flora SJ, Dwivedi UN, Parmar D:
Gene expression profiling of candidate genes in peripheral blood
mononuclear cells for predicting toxicity of diesel exhaust
particles. Free Radic Biol Med 2013, 67C:188-194.
3. Ankita Srivastava, Sanjay Yadav, Kumar Saurabh, Devendra Parmar:
Similarities in the alteration of cytochrome P450s in blood
lymphocytes and lungs following exposure to diesel exhaust
particles (DEPs) presented at 28 annual conference Society of
Toxicology(STOX Ludhiana). October 16-18,2008.
