Capsaicin alleviates the imbalance in xenobiotic metabolizingenzymes and tumor markers during experimental lungtumorigenesis

P. Anandakumar Æ S. Kamaraj Æ S. Jagan ÆG. Ramakrishnan Æ C. Naveenkumar Æ S. Asokkumar ÆT. Devaki

Received: 14 January 2009 / Accepted: 28 April 2009 / Published online: 18 May 2009

� Springer Science+Business Media, LLC. 2009

Abstract Lung cancer is currently a leading cause of

death all over the world. Environmental risk factors, par-

ticularly genotoxic chemicals such as polycyclic aromatic

hydrocarbons (PAH), are likely to account for a much

higher mortality. Xenobiotic metabolizing enzymes are

potentially chief determinants in both the susceptibility to

the mutagenic effects of chemical carcinogens and in the

response of tumors to chemotherapy. The well-known

carcinogen benzo(a)pyrene (B(a)P) of PAH family was

given orally (50 mg/kg body weight) to induce lung cancer

in Swiss albino mice. B(a)P induction altered the levels of

cytochromes (P450, b5), activities of phase I biotransfor-

mation enzymes (NADPH-cytochrome P450 reductase,

NADH-cytochrome b5 reductase and epoxide hydrolase),

phase II enzymes (glutathione-S-transferase, UDP-glucu-

ronyl transferase and DT-diaphorase), and the levels of

serum tumor markers. Treatment with capsaicin (CAP)

(10 mg/kg body weight) to the lung carcinoma mice

restored back the activities of phase I and II biotransfor-

mation enzymes and the levels of tumor markers to near

normalcy. The above findings were substantiated by

immunoblotting and immunohistochemical analysis of

cytochrome P450 1A1 (CYP1A1) in the lung tissues. Our

present study unravels that CAP can effectively detoxify

the carcinogens which discloses its anti-carcinogenic effect

during experimental lung cancer.

Keywords Benzo(a)pyrene � Lung cancer � Capsaicin �Phase I and II detoxification enzymes � Tumor markers

Introduction

The most important organs of xenobiotic metabolism are

the liver, which is strategically located to deal with

chemicals entering the body through the alimentary tract,

and the lungs, which have to take care of air-borne pollu-

tants. Most of the chemicals entering the body are not

reactive themselves and require metabolic activation by a

variety of enzymes responsible for drug metabolism to

exert their genotoxicity. Cytochrome P450 (CYP), a phase

I drug-metabolizing enzyme, is one of the major enzymes

mainly involved in the activation of carcinogens [1]. Many

of the CYP genes are known to exist in variant forms that

have different activities. Since many carcinogens require

metabolic activation before binding to DNA, individuals

with an elevated metabolic capacity to activate specific

carcinogens may be at an increased risk of cancer [2].

Phase II reactions are carried out by a class of widely

distributed enzymes that detoxify carcinogens either by

destroying their reactive centers or by conjugating them to

endogenous ligands facilitating their excretion [3]. Thus,

the activation of the carcinogens by phase I enzymes and

its detoxification, and elimination by phase II enzymes are

in absolute balance that exist in cells, and is an important

determinant of whether exposure to such carcinogens will

result in toxicity and neoplasia [2].

Benzo(a)pyrene (B(a)P) is one among the chief con-

stituents of smoke components, and in vivo metabolic

activation of B(a)P by xenobiotic enzymes leads to a highly

reactive metabolite 7,8-diol-9,10-epoxide-benzo(a)pyrene

(BPDE) [4]. BPDE is highly carcinogenic and mutagenic

and is capable of forming DNA adducts as well as chro-

mosomal aberrations. If DNA adducts are not efficiently

removed and repaired prior to DNA replication, mutations,

DNA strand breaks, or other genetic alterations may result,

P. Anandakumar � S. Kamaraj � S. Jagan � G. Ramakrishnan �C. Naveenkumar � S. Asokkumar � T. Devaki (&)

Department of Biochemistry, University of Madras,

Guindy Campus, Chennai 600-025, Tamil Nadu, India

e-mail: devakit@yahoo.co.uk

123

Mol Cell Biochem (2009) 331:135–143

DOI 10.1007/s11010-009-0151-0

which contribute to the process of carcinogenesis [5]. The

levels of BPDE-induced DNA damage vary widely and

reflect individual variation in susceptibility to a tobacco-

related carcinogen challenge [6]. Therefore, BPDE is a

relevant challenge mutagen to study the susceptibility to

lung carcinogenesis.

Recent epidemiological and experimental studies have

demonstrated the chemopreventive effects of capsaicin

(CAP) against chemical-induced carcinogenesis. These

studies have provided evidence of CAP’s protection against

variety of cancers [7, 8]. CAP is widely consumed

throughout the world and is also known to possess other

beneficial properties including anti-inflammatory, anti-

fungal and analgesic effects [9]. Mechanisms proposed

to explain CAP’s modification of cancer susceptibility

include effects on carcinogen metabolism, formation of

DNA adducts, and free radical scavenging [10–12]. Studies

from our laboratory have also proved the chemoprotective

role of CAP in different dimensions [13–17].

Hence, this study was motivated to prove that CAP may

extend its chemopreventive potential through modulating

phase I and II enzymes and decrease the levels of tumor

markers during B(a)P-induced lung cancer.

Materials and methods

Materials

Benzo(a)pyrene and CAP were purchased from M/s. Sigma

chemicals, St. Louis, USA. All other chemicals were of

analytical grade procured from M/s. SRL Chemicals Pvt.

Ltd., Mumbai.

Animals

Healthy male Swiss albino mice weighing 20–25 g

(8–10 weeks old) obtained from Veterinary College,

Chennai were used throughout the experiment. This study

was ethically approved by the Ministry of Social Justices

and Empowerment, Government of India, and by the Animal

Ethics Committee guidelines of our Institution (IAEC No.

01/024/08). The animals were housed under conditions of

controlled temperature (26 ± 2�C) with 12-h day/night

cycle. They were fed with standard rat/mice pellet diet (M/s.

Hindustan Lever Ltd., Mumbai) under the trade name Amrut

rat/mice feed and were given access to water ad libitum.

Experimental design

Experimental animals were divided into four groups of six

mice each as follows. Group I (control) received olive oil

throughout the course of the experiment. Group II (B(a)P)

was treated with benzo(a)pyrene [50 mg/kg body weight

(b.wt.) dissolved in olive oil] orally twice in a week (1st

day and 4th day) for four successive weeks. Group III

(CAP) received capsaicin (10 mg/kg b.wt. dissolved in

olive oil) intraperitoneally once in a week for 14 weeks to

assess the cytotoxicity (if any) induced by CAP. Group IV

(B(a)P ? CAP) received B(a)P (as in Group II) along with

CAP (10 mg/kg b.wt. dissolved in olive oil) intraperito-

neally. CAP treatment was started 1 week prior to the first

dose of B(a)P administration and continued for 14 weeks.

Dosing regime for the experimental animals was fixed

based on our previous studies [13–17].

All the procedures were performed at temperature

ranging between 0 and 4�C. At the end of the experimental

period, the animals were sacrificed. Liver and lung tissues

were isolated, washed in ice cold 1.15% KCl, and

homogenized. The homogenate was centrifuged at

9,0009g for 20 min, and the resulting supernatant was

further centrifuged at 105,0009g for 1 h at 4�C to obtain

the microsomal fraction. Blood was also collected, and the

serum was separated for other estimations. The following

biochemical estimations were carried out in the supernatant

and in the serum.

Biochemical analysis

Isolation of microsomes: Microsomes were isolated by the

method of Hanioka et al. [18]. The lung and liver were

homogenized with four volumes of medium containing

Tris–KCl buffer in a glass homogenizer. The homogenate

was centrifuged at 9,0009g for 20 min in a refrigerated

centrifuge, and the supernatant was decanted and recen-

trifuged at 105,0009g for 60 min. The microsomal pellet

was recentrifuged in Tris–KCl buffer and resedimented

twice as above. The washed microsomal pellet was finally

resuspended in phosphate buffer (4 mg of protein/ml) and

used for the analysis. The microsomal protein content was

estimated by the method of Lowry et al. [19].

Microsomal biotransformation enzymes

Phase I drug metabolizing enzymes

Estimation of cytochrome P450

Cytochrome P450 was estimated by the method of Omura

and Sato [20]. Microsomes suspended in phosphate buffer

(4 mg/ml) were reduced by a few milligrams of solid

sodium dithionate. Then, 1 ml of water saturated with

carbon monoxide was added. The absorbance of the sam-

ples was scanned at 400–500 nm. The level of cytochrome

P450 was expressed as nmoles/mg protein.

136 Mol Cell Biochem (2009) 331:135–143

123

Estimation of cytochrome b5

The amount of cytochrome b5 was measured by the

method of Omura and Sato [20]. To the microsomal sus-

pension, containing 4 mg of protein/ml in phosphate buf-

fer, 1 ml of NADH was added. The absorbance spectrum

between 400 and 500 nm was read against the blank con-

taining microsomal suspension alone. The level of cyto-

chrome b5 was calculated using the molar extinction

coefficient of 185 mM/cm between 424 and 409 nm and

was expressed as nmoles/mg protein.

Assay of NADPH-cytochrome P450 reductase

The activity of NADPH-cytochrome P450 reductase was

assayed by the method of Phillips and Langdon [21]. The

assay mixture containing 2.5 ml of buffer, 0.2 ml of

potassium cyanide, and 0.1 ml of cytochrome c was mixed

gently. After 3 min, 0.1 ml of NADPH was added and the

change in optical density was recorded at 30-s intervals for

3 min at 550 nm. The activity of NADPH-cytochrome

P450 reductase was expressed as nanomoles of cytochrome

c oxidized/min/mg protein.

Assay of NADH-cytochrome b5 reductase

NADH-cytochrome b5 reductase activity was determined

according to the method of Strittmater and Velick [22]. To

2 ml of buffer mixture, 0.1 ml of potassium cyanide and

0.1 ml of microsomal suspension, and 0.1 ml of NADH

were added, and the change in optical density at 430 nm

for 3 min at 15-s interval was taken. The specific activity

was expressed as nanomoles of ferricyanide reduced/min/

mg protein.

Phase II enzymes

Assay of glutathione-S-transferase

The activity of glutathione-S-transferase (GST) was

assayed by the method of Habig et al. [23]. To 1 ml of

tissue homogenate, 1 ml of phosphate buffer, 1.7 ml of

water and 0.1 ml of CDNB was added. After incubation at

37�C for 15 min, 0.1 ml of GSH was added, and the

change in optical density was read at 340 nm. The GST

activity was expressed as micromoles of CDNB conju-

gated/min/mg of protein.

Assay of UDP glucuronyl transferase

The UDP glucuronyl transferase (UDP-GT) was assayed

by the method of Bock et al. [24] with slight modification.

To 0.5 ml of Tris–HCl added 0.2 ml of TritonX-100,

0.05 ml of MgCl2, 0.05 ml of p-nitrophenol, and 0.18 ml

of water was added and then finally 0.1 ml of sample was

added and incubated at 37�C for 20 min. A quantity of

0.1 ml of UDP-glucuronic acid was added and the reaction

was arrested at different time intervals of 0, 10, and 20 min

by using TCA. The precipitate was centrifuged, and the

supernatant of 1 ml was taken. A quantity of 0.25 ml of

NaOH was added and read at 450 nm. The activity of

UDP-GT was expressed as nanomoles of p-nitrophenol

liberated/min/mg protein.

Assay of DT-diaphorase

Activity of DT-diaphorase (DTD) was assayed according to

the method of Ernest [25, 26]. To 2.5 ml of Tris–HCl buffer

(0.05 M), 0.1 ml of NADH (0.3 mM), 0.5 ml of dichloro-

phenol indophenol (0.04 mM), and 0.5 ml of BSA (0.07%)

were added and mixed well. A quantity of 0.1 ml of the

sample was added just before reading at 600 nm. Readings

were taken at 30-s interval for 2 min. Activity of DTD is

expressed as cytochrome C reduced/min/mg of protein.

Analysis of carcinoembryonic antigen and neuron

specific enolase

Carcinoembryonic antigen (CEA) and neuron specific

enolase (NSE) were measured in blood serum by chemi-

luminescent immunoassay (fully automated ADVIA cen-

taur, Bayer USA Chemiluminescense system).

Western blot analysis

Immunoblotting was performed according to the method of

Ramakrishnan et al. [27]. Approximately, 50 lg of the total

cell lysate was mixed with equal volume of 29 sample

buffer, boiled for 5 min at 95�C, cooled, loaded on each

lane of 10% polyacrylamide gel, and separated by sodium

dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–

PAGE) at room temperature. The resolved proteins were

electrophoretically transferred to Nitrocellulose mem-

branes. The membranes were then blocked in 5% non-fat

milk in Tris-buffered saline with 0.1% Tween 20 for 1 h at

room temperature and probed with the following primary

antibodies: Cytochrome P450 1A1 (CYP1A1) mouse

monoclonal antibody (Dr. John J. Stegeman, WHOI,

Massachusetts and Dr. Harry. V. Gelboin, NCI, Maryland)

at a concentration of 3 lg/ml and b-actin (Sigma) mouse

monoclonal antibody at a dilution of 1:2,000 overnight at

4�C. The blots were then extensively washed with Tris-

buffered saline with 0.1% Tween 20, and then incubated

with anti-mouse and anti-mouse HRP-labeled secondary

antibody (Genei, Bangalore, India) at a dilution of 1:2,000

for 1 h at room temperature. After extensive washes in

Mol Cell Biochem (2009) 331:135–143 137

123

TBS-T, the bands were visualized by treating the mem-

branes with 3,30-diaminobenzidine tetrahydrochloride

(SRL, Mumbai, India). The membranes were photographed

and quantitated with image analysis software, imagej, NIH,

USA. Densitometry data presented in bar graphs are ‘‘fold

change’’ as compared with control.

Immunohistochemistry of cytochrome P450 1A1

The method of Ramakrishnan et al. [27] was followed for

immunohistochemistry. Tissue sections were deparaffi-

nized in two changes of xylene at 60�C for 20 min each

and hydrated through a graded series of alcohol; the slides

were incubated in a citrate buffer (pH 6.0) for three cycles

of 5 min each in a microwave overnight for antigen

retrieval. The sections were then allowed to cool to room

temperature and then rinsed with 19 Tris buffered saline

(TBS), and treated with 0.3% H2O2 in methanol for 10 min

to block endogeneous peroxidase activity. Non-specific

binding was blocked with 3% BSA in room temperature

for 1 h. The sections were then incubated with CYP1A1

(Dr. John J. Stegeman, WHOI, Massachusetts and

Dr. Harry V. Gelboin, NCI, Maryland) mouse monoclonal

antibody at a concentration of 0.3 lg/ml in 1% BSA in

TBS for two 1-h periods with a total of 0.3 ml per slide

added as 0.15 ml at time 0 and 60 min at 4�C overnight.

The slides were washed with TBS and then incubated

with anti-mouse HRP labeled secondary antibody (Genei,

Bangalore, India), at a dilution 1:500 for 1 h in room

temperature. The peroxidase activity was visualized by

treating the slides with 3,30-diaminobenzidine tetrahydro-

chloride (SRL, Mumbai, India); the slides were counter-

stained with Meyer’s hamatoxylin. Negative controls were

incubated with TBS instead of primary antibodies. Quan-

titative analysis was made in a blinded manner under a

light microscope. Each section was examined at high

magnification (409), and the ratio of positive area to lung

tissue area was calculated. The result was regarded as the

mean of five different fields on each section.

Data analysis

All data were expressed as mean ± SD for six mice. The

results were computed statistically (SPSS Software Pack-

age) using one-way ANOVA. Post-hoc testing was per-

formed for inter comparisons using the LSD. P \ 0.05 was

considered significant.

Results

Effect of CAP on cytochrome P450 and cytochrome b5

Table 1 represents the level of lung and liver microsomal

Cyt P450 and Cyt b5 in control and experimental group of

animals. There was a significant (P \ 0.05) increase in

lung and liver Cyt P450 and Cyt b5 levels in cancer-

bearing animals (Group II) when compared to control

animals of Group I. Treatment with CAP markedly

(P \ 0.05) decreased Cyt P450 and Cyt b5 levels in Group

IV animals when compared to lung cancer animals of

Group II.

Effect of capsaicin on phase I enzymes

Activities of lung and liver microsomal phase I drug-

metabolizing enzymes are represented in Table 2. Highly

significant (P \ 0.05) increase in the activities of NADPH

Cyt P450 reductase, NADH Cyt b5 reductase, and epoxide

hydrolase was noted in lung cancer-induced animals

(Group II) when compared to control animals (Group I).

The enzyme activities were significantly (P \ 0.05)

reversed to near normalcy on treatment with CAP in Group

IV animals when compared to lung cancer-bearing animals

(Group II).

Effect of capsaicin on phase II enzymes

Table 3 depicts the activities of phase II drug-metabolizing

enzymes in the lung and liver of control and experimental

Table 1 Levels of Cyt P450 and Cyt b5 in lung and liver microsomes of control and experimental animals

Parameters Group I (control) Group II (B(a)P) Group III (CAP) Group IV (B(a)P ? CAP)

Lung

Cyt P450 0.62 ± 0.06 0.99 ± 0.09a 0.63 ± 0.06 0.75 ± 0.07b

Cyt b5 0.47 ± 0.04 0.87 ± 0.07a 0.48 ± 0.04 0.55 ± 0.05b

Liver

Cyt P450 0.79 ± 0.07 1.21 ± 0.11a 0.78 ± 0.07 0. 88 ± 0.08b

Cyt b5 0.59 ± 0.05 0.94 ± 0.08a 0.58 ± 0.05 0.67 ± 0.06b

Values represent the mean ± SD for six mice. Statistical significance at P \ 0.05, as compared with a Group I, b Group II. Units: Cyt b5 and Cyt

P450—nmol/mg protein

138 Mol Cell Biochem (2009) 331:135–143

123

of group of animals. Significant (P \ 0.05) decrease in the

activities of GST, UDP-GT, and DTD was noticed in lung

cancer-bearing animals (Group II) when compared to

Group I control animals. Supplementation with CAP sig-

nificantly (P \ 0.05) restored the activities of these

enzymes to normalcy in Group IV animals when compared

to Group II lung cancer animals.

Effect of capsaicin on tumor markers

Figure 1 presents the level of CEA in the serum of control

and experimental animals. The level of CEA was found to

be markedly (P \ 0.05) elevated in the tumor-bearing

animals (Group II) when compared to normal control

animals of Group I. On CAP administration, CEA levels

were significantly (P \ 0.05) retained to near normalcy in

Group IV animals when compared to Group II lung cancer-

bearing animals.

The influence of CAP on the level of serum NSE is

shown in Fig. 2. The level of NSE was found to be sig-

nificantly (P \ 0.05) increased in the tumor-bearing ani-

mals (Group II) when compared with Group I control

animals. On CAP treatment, the NSE level was markedly

(P \ 0.05) sustained to near normalcy in Group IV animals

when compared to tumor-bearing animals of Group II.

Table 3 Activities of lung and liver microsomal phase II xenobiotic metabolizing enzymes in control and experimental group of animals

Parameters Group I (control) Group II (B(a)P) Group III (CAP) Group IV (B(a)P ? CAP)

Lung

GST 1.67 ± 0.17 1.18 ± 0.11a 1.68 ± 0.17 1.56 ± 0.14b

UDP-GT 3.85 ± 0.40 2.62 ± 0.25a 3.87 ± 0.40 3.20 ± 0.31b

DTD 1.97 ± 0.20 1.17 ± 0.10a 1.96 ± 0.20 1.76 ± 0.18b

Liver

GST 6.84 ± 0.67 1.28 ± 0.13a 6.85 ± 0.68 5.14 ± 0.50b

UDP-GT 10.22 ± 1.10 4.56 ± 0.50a 10.20 ± 1.10 8.37 ± 0.84b

DTD 5.11 ± 0.50 1.14 ± 0.13a 5.10 ± 0.50 4.13 ± 0.42b

Values represent the mean ± SD for six mice. Statistical significance at P \ 0.05, as compared with a Group I, b Group II. Units: GST—lmol of

CDNB conjugated/min/mg protein; UDP-GT—units/min/mg protein; DTD—cytochrome c reduced/min/mg protein

Table 2 Activities of lung and liver microsomal phase I xenobiotic metabolizing enzymes in control and experimental group of animals

Parameters Group I (control) Group II (B(a)P) Group III (CAP) Group IV (B(a)P ? CAP)

Lung

NADPH Cyt P450 reductase 0.21 ± 0.02 0.61 ± 0.06a 0.20 ± 0.02 0.37 ± 0.03b

NADH Cyt b5 reductase 0.42 ± 0.04 0.79 ± 0.07a 0.41 ± 0.04 0.53 ± 0.05b

Epoxide hydrolase 1.32 ± 0.12 5.28 ± 0.53a 1.31 ± 0.12 1.96 ± 0.20b

Liver

NADPH Cyt P450 reductase 6.38 ± 0.64 10.11 ± 1.01a 6.36 ± 0.63 7.24 ± 0.73b

NADH Cyt b5 reductase 6.12 ± 0.62 9.81 ± 0.99a 6.13 ± 0.62 7.05 ± 0.70b

Epoxide hydrolase 5.22 ± 0.53 8.82 ± 0.89a 5.22 ± 0.53 6.01 ± 0.60b

Values represent the mean ± SD for six mice. Statistical significance at P \ 0.05, as compared with a Group I, b Group II. Units: NADPH Cyt

P450 reductase—nanomoles of Cyt c oxidized/min/mg protein; NADH Cyt b5 reductase—nanomoles of ferricyanide reduced/min/mg protein;

Epoxide hydrolase—lmol of resourfin/min/mg protein

0

0.5

1

1.5

2

2.5

3

3.5

CONTROL B(a)P CAP B(a)P+CAP

ng

/mL

CEA a

b

Fig. 1 Figure represents the levels of serum CEA in the control and

experimental group of animals. Values represent the mean ± SD for

six mice. Statistical significance at P \ 0.05, as compared witha Group I, b Group II

Mol Cell Biochem (2009) 331:135–143 139

123

Effect of capsaicin on the protein expression

of cytochrome P450 1A1

Figure 3 represents the immunoblot analysis to confirm the

expression of CYP1A1 in all the defined experimental

animals. Their expression under tightly controlled physio-

logical situation is very optimal (lane 1) in control tissues.

CYP1A1 expression was very high in B(a)P-induced lung

cancer animals (lane 2). B(a)P along with CAP treatment

group caused a significant reduction in CYP1A1 expression

when compared to cancerous mice (lane 4), whereas CAP-

alone-treated mice showed expression similar to control

(lane 3).

Figure 4 shows the immunoreactive indices for

CYP1A1, which were found to be significantly upregulated

in lung cancer-bearing Group II animals (Fig. 4b) com-

pared to normal control animals (Fig. 4a) and CAP-alone-

treated animals (Fig. 4c). B(a)P along with CAP treatment

group markedly downregulated CYP1A1 expression in

Group IV animals (Fig. 4d).

Discussion

It is widely accepted that metabolic activation of xenobi-

otics by phase I enzymes is required for their cytotoxic,

mutagenic, and carcinogenic activities [28]. Studies dem-

onstrated that B(a)P after sequential metabolic activation

principally by phase I enzyme, CYP1A1 generates BPDE

[29]. BPDE is believed to be the ultimate carcinogenic

metabolite of B(a)P that leads to the formation of DNA

adducts that results in cancer. These findings were in

concordance with our present study, as we have noticed an

increase in the expression of CYP1A1 together with ele-

vations in the levels of Cyt P450, Cyt b5, and in the

activities of phase I xenobiotic metabolizing enzymes,

namely, NADPH Cyt P450 reductase, NADH Cyt b5

reductase, and epoxide hydrolase in B(a)P administered

lung cancer-bearing animals.

Substantial evidence implicates that CAP modulates the

microsomal cytochrome P450 enzymes 3A1, 2A2, 2B1,

2B2, 2C6, and 2C11 and thereby affects the metabolism of

carcinogens [10]. Moreover, CAP was found to suppress

the activity of rat epidermal aryl hydrocarbon hydroxylase

that is linked to the cytochrome P450 1A isoform respon-

sible for the metabolism of B(a)P and other PAH [30].

Earlier studies on CAP established that this alkaloid com-

pound was found to suppress the metabolism and covalent

DNA binding of B(a)P in human and mouse keratinocytes

[31]. In addition, in this study, CAP treatment effectively

reduced the activities of phase I xenobiotic metabolizing

enzymes suggesting its anti-mutagenic/anti-carcinogenic

activity against PAH, which was analogous with the pre-

vious findings.

Phase II enzymes such as GST, UDP-GT and DTD are

considered to be a major mechanism of protection against

chemical stress and initiation of carcinogenesis [3].

Mounting evidence has suggested a relevant mechanism

between the induction of phase II detoxification enzymes

and cancer chemoprevention [32]. GSTs are a family

of enzymes that catalyzes the conjugation of reactive

0

2

4

6

8

10

12

14

16

18

20

CONTROL B(a)P CAP B(a)P+CAP

ng

/mL

NSE a

b

Fig. 2 Figure depicts the serum NSE levels in the control and

experimental group of animals. Values represent the mean ± SD for

six mice. Statistical significance at P \ 0.05, as compared witha Group I, b Group II

Fig. 3 Figure presents the immunoblot analysis of CYP1A1 and

b-actin (internal control) in the lung of control and experimental

group of animals. a Lanes 1–4 represent groups 1–4 of control and

experimental animals, respectively. b Densitometric analysis of

CYP1A1 immuno blotting. Densitometry data was presented as ‘‘fold

change’’ as compared with control. Values represent the mean ± SD

for six mice. Statistical significance at P \ 0.05, as compared witha Group I, b Group II

140 Mol Cell Biochem (2009) 331:135–143

123

chemicals with GSH (reduced glutathione) and plays a

major role in protecting cells. After generating conjugated

GSH, these are subsequently eliminated via a GSH con-

jugate-recognizing transport [33]. Many naturally occur-

ring chemopreventive agents, including CAP, have been

reported to convert the DNA-damaging entities into

excretable metabolites through induction of GST [34].

Glucuronidation, catalyzed by UDP-GT family of

enzymes, is a major metabolic pathway of endogenous

steroids, bile acids, drugs, and carcinogens [35]. Several

phytochemicals are known to cause elevation in the

activities of GST through the induction of the microsomal

detoxification enzyme UDP-GT gene complex [36]. DTD

is a flavoprotein that catalyses two-electron reduction of

quinones, quinone imines, and nitrogen oxides. This reac-

tion prevents the formation of semiquinones by one elec-

tron reduction and, in turn, the generation of free radicals

from the autooxidation of semiquinones. Reduction of

quinines and nitrogen oxide might also make them avail-

able for conjugation with UDP-glucuronic acid, facilitating

their excretion. Hence, DTD acts as an early cellular

defense against tumorigenesis [37]. In this study, CAP

supplementation augmented the activities of all these three

important phase II enzymes in the lung and liver from its

basal constituent levels divulging its chemopreventive

action.

Tumor markers are substances usually identifiable in the

blood that indicates the presence or extent of tumor in the

body. Cancer chemoprevention and therapy depends on

the investigation of these tumor markers. CEA is an

oncofetal glycoprotein that is used clinically as a tumor

marker to detect the recurrence of many types of tumors

[38]. CEA is one of the most popular tumor markers

measured in lung cancer, and its various clinical applica-

tions have been reported [39]. CEA is detected in abnor-

mally high levels in metastatic carcinomas of non-digestive

organs, such as breast, lung, prostate, and ovary [40]. The

other protein that is most widely used as neuroendocrine

serum marker in clinical practice is the cc-isomer of the

ubiquitous enzyme enolase referred to as NSE [41]. This

serum marker has been extensively evaluated regarding its

clinical applicability. The use of NSE as an indicator of

disease status during chemotherapy and follow-up has been

evaluated in independent studies; decrease and increase of

NSE level are well correlated with tumor response during

chemotherapy [42]. Moreover, serum NSE measurements

may be of value in assessment of tumor burden in lung

cancer at presentation and a helpful parameter during

therapy [43]. The observed rise in serum CEA and NSE

levels in B(a)P administered animals could be associated

with production rates of tumor, its location and stage, size,

differentiation, and vascularity. CAP treatment lowered the

levels of CEA and NSE, which is a good prognosis for

tumor regression, and inhibition of metastasis.

In conclusion, CAP inhibits phase I enzymes that bio-

activates the carcinogen B(a)P and elevates the activity of

phase II enzymes which consecutively enhances the

detoxification process thereby aids in the elimination of

carcinogen. Furthermore, reduced levels of serum tumor

markers in the CAP-treated group reflected the anti-cancer

property of CAP. Hence, this study demonstrates that the

biotransformation enzyme-modifying capability of CAP

Fig. 4 Figure shows the

immunohistochemical staining

of CYP1A1 in the lung of

control and experimental group

of animals (409). Plates a–drepresent the lung sections of

groups 1–4 of experimental

animals, respectively. Arrowsindicate immunostained areas

Mol Cell Biochem (2009) 331:135–143 141

123

might play an important role in its anti-carcinogenic

potency which strongly suggests a possible cancer che-

mopreventive potential for CAP against B(a)P-induced

experimental lung cancer.

Acknowledgments First author P. Anandakumar wishes to thank

University Grants Commission, New Delhi, India for the financial

assistance in the form of Junior Research fellowship. The authors

S. Kamaraj and S. Jagan wishes to thank ICMR, CSIR, New Delhi,

India, respectively, for the financial assistance in the form of Senior

Research fellowship. The authors are thankful to Dr. John J. Steg-

eman, Woods Hole Oceanographic Institution, Massachusetts, and Dr.

Harry V. Gelboin, National Cancer Institute, Maryland for their kind

gift of CYP1A1 primary mouse monoclonal antibody.

References

1. Ding X, Kaminsky LS (2003) Human extrahepatic cytochromes

P450: function in xenobiotic metabolism and tissue-selective

chemical toxicity in the respiratory and gastrointestinal tracts.

Annu Rev Pharmacol Toxicol 43:149–173. doi:10.1146/annurev.

pharmtox.43.100901.140251

2. Srinivasan P, Suchalatha S, Babu PV, Devi RS, Narayan S, Sabitha

KE, Shyamala Devi CS (2008) Chemopreventive and therapeutic

modulation of green tea polyphenols on drug metabolizing

enzymes in 4-nitroquinoline 1-oxide induced oral cancer. Chem

Biol Interact 172:224–234. doi:10.1016/j.cbi.2008.01.010

3. Selvendiran K, Banu SM, Sakthisekaran D (2005) Oral supple-

mentation of piperine leads to altered phase II enzymes and

reduced DNA damage and DNA–protein cross links in ben-

zo(a)pyrene induced experimental lung carcinogenesis. Mol Cell

Biochem 268:141–147. doi:10.1007/s11010-005-3702-z

4. Graslund A, Jernstrom B (1989) DNA–carcinogen interaction:

covalent DNA-adducts of benzo(a)pyrene 7,8-dihydrodiol 9,10-

epoxides studied by biochemical and biophysical techniques. Q

Rev Biophys 22:1–37

5. Shinozaki R, Inoue S, Choi KS (1998) Flow cytometric mea-

surement of benzo[a]pyrene-diol-epoxide-DNA adducts in nor-

mal human peripheral lymphocytes and cultured human lung

cancer cells. Cytometry 31:300–306. doi:10.1002/(SICI)1097-

0320(19980401)31:4\300::AID-CYTO10[3.0.CO;2-U

6. Zhang H, Spitz MR, Tomlinson GE, Schabath MB, Mina JD, Wu

X (2002) Modification of lung cancer susceptibility by green tea

extract as measured by the comet assay. Cancer Detect Prev

26:411–418. doi:10.1016/S0361-090X(02)00127-7

7. Miller CH, Zhang Z, Hamilton SM, Teel RW (1993) Effects of

capsaicin on liver microsomal metabolism of the tobacco-specific

nitrosamine NNK. Cancer Lett 75:45–52. doi:10.1016/0304-3835

(93)90206-O

8. Surh YJ, Lee SS (1995) Capsaicin, a double-edged sword: tox-

icity, metabolism, and chemopreventive potential. Life Sci

56:1845–1855. doi:10.1016/0024-3205(95)00159-4

9. Chowdhury B, Mukhopadhyay S, Bhattacharayay D, De AK

(1996) Capsaicin, a unique anti-oxidant, anti-inflammatory,

analgesic compound with antifungal activity against dermato-

phytes. Med Sci Res 24:669–670

10. Teel RW (1997) Effects of capsaicin on rat liver S9-mediated

metabolism and DNA binding of aflatoxin. Nutr Cancer 15:27–32

11. Tanaka T, Kohno H, Sakata K, Yamada Y, Hirose Y, Sugie S, Mori

H (2002) Modifying effects of dietary capsaicin and rotenone on 4-

nitroquinoline 1-oxide-induced rat tongue carcinogenesis. Carci-

nogenesis 23:1361–1367. doi:10.1093/carcin/23.8.1361

12. Kogure K, Goto S, Nishimura M, Yasumoto M, Abe K, Ohiwa C,

Sassa H, Kusumi T, Terada H (2002) Mechanism of potent an-

tiperoxidative effect of capsaicin. Biochim Biophys Acta 1573:

84–92

13. Anandakumar P, Jagan S, Kamaraj S, Ramakrishnan G, Clara JB,

Pathitha D, Kavitha T, Devaki T (2009) Ameliorating effect of

capsaicin on alternations in lipid metabolism during mice lung

carcinoma. Arch Pharm Res 32:229–234. doi:10.1007/s12272-

009-1140-2

14. Anandakumar P, Kamaraj S, Jagan S, Ramakrishnan G, Devaki T

(2009) Lysosomal abnormalities during experimental lung car-

cinogenesis—defensive role of capsaicin. Fundam Clin Pharma-

col 23:97–103. doi:10.1111/j.1472-8206.2008.00637.x

15. Anandakumar P, Jagan S, Kamaraj S, Ramakrishnan G,

Vinodhkumar R, Devaki T (2008) Beneficial influence of cap-

saicin on lipid peroxidation, membrane-bound enzymes and

glycoprotein profile during experimental lung carcinogenesis.

J Pharm Pharmacol 60:803–808. doi:10.1211/jpp.60.6.0017

16. Anandakumar P, Kamaraj S, Jagan S, Ramakrishnan G, Vin-

odhkumar R, Devaki T (2008) Capsaicin modulates pulmonary

antioxidant defense system during benzo(a)pyrene-induced lung

cancer in Swiss albino mice. Phytother Res 22:529–533. doi:

10.1002/ptr.2393

17. Anandakumar P, Kamaraj S, Jagan S, Ramakrishnan G, Vin-

odhkumar R, Devaki T (2008) Stabilization of pulmonary mito-

chondrial enzyme system by capsaicin during benzo(a)pyrene

induced experimental lung cancer. Biomed Pharmacother

62:390–394. doi:10.1016/j.biopha.2007.09.005

18. Hanioka N, Jinno H, Nishimura T, Ando M (1997) Changes in

cytochrome P450 enzymes by 1,1-dichloroethylene in rat liver

and kidney. Arch Toxicol 72:9–16. doi:10.1007/s002040050462

19. Lowry OH, Rosebrough NH, Farr AL, Randall RI (1951) Protein

measurement with the folin-phenol reagent. J Biol Chem 193:

265–275

20. Omura T, Sato R (1964) The carbon monoxide-binding pigment

of liver microsomes. II. Solubilization, purification, and proper-

ties. J Biol Chem 239:2379–2385

21. Phillips AH, Langdon RG (1962) Hepatic triphosphopyridine

nucleotide-cytochrome c reductase: isolation, characterization,

and kinetic studies. J Biol Chem 237:2652–2660

22. Strittmatter P, Velick SF (1956) A microsomal cytochrome

reductase specific for diphosphopyridine nucleotide. J Biol Chem

221:277–286

23. Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione-S-trans-

ferase. The first enzymatic step in mercapturic acid formation.

J Biol Chem 249:7130–7139

24. Bock KW, Burchell B, Ditton GJ et al (1983) UDP-glucuronosyl

transferase activities: guidelines for consistent interim terminol-

ogy and assay condition. Biochem Pharmacol 32:953–955. doi:

10.1016/0006-2952(83)90610-X

25. Ernest L, Danielson L, Ljunggren M (1982) DT-diaphorase-

purification from the soluble fraction of rat liver cytoplasm.

Biochem Biophys Acta 58:171–188

26. Talalay P, Benson AM (1982) Elevation of quinone reductase

activity by anticarcinogenic antioxidants. Adv Enzyme Regul

20:287–300. doi:10.1016/0065-2571(82)90021-8

27. Ramakrishnan G, Augustine TA, Jagan S, Vinodhkumar R,

Devaki T (2007) Effect of silymarin on N-nitrosodiethylamine

induced hepatocarcinogenesis in rats. Exp Oncol 29:39–44

28. Selvendiran K, Thirunavukkarasu C, Singh JPC, Padmavathi R,

Sakthisekaran D (2005) Chemopreventive effect of piperine on

mitochondrial TCA cycle and phase-I and glutathione-metabo-

lizing enzymes in benzo(a)pyrene induced lung carcinogenesis in

Swiss albino mice. Mol Cell Biochem 271:101–106. doi:10.1007/

s11010-005-5615-2

142 Mol Cell Biochem (2009) 331:135–143

123

29. Szeliga J, Dipple A (1998) DNA adduct formation by polycyclic

aromatic hydrocarbon dihydrodiol epoxides. Chem Res Toxicol

11:1–11. doi:10.1021/tx970142f

30. De AK, Agarwal K, Mukherjee A, Sengupta D (1995) Inhibition

by capsaicin against cyclophosphamide-induced clastogenicity

and DNA damage in mice. Mutat Res 335:253–258

31. Modly CE, Das M, Don PS, Marcelo CL, Mukhtar H, Bickers DR

(1986) Capsaicin as an in vitro inhibitor of benzo(a)pyrene

metabolism and its DNA binding in human and murine kerati-

nocytes. Drug Metab Dispos 14:413–416

32. Premalatha B, Sachdanandam P (2000) Modulating role of

Semecarpus anacardium L. nut milk extract on Aflatoxin B1

biotransformation. Pharmacol Res 41:19–24. doi:10.1006/phrs.

1999.0544

33. Song LL, Kosmeder JW, Lee SK, Gerhauser C, Lantvit D, Moon

RC, Moriarty RM, Pezzuto JM (1999) Cancer chemopreventive

activity mediated by 40-bromoflavone, a potent inducer of phase

II detoxification enzymes. Cancer Res 59(3):578–585

34. Manson MM, Ball HW, Barrett MC, Clark HL, Judah DJ, Wil-

liamson G, Neal GE (1997) Mechanism of action of dietary

chemoprotective agents in rat liver: induction of phase I and II

drug metabolizing enzymes and aflatoxin B1 metabolism. Car-

cinogenesis 18:1729–1738. doi:10.1093/carcin/18.9.1729

35. King C, Tang W, Ngui J, Tephly T, Braun M (2001) Charac-

terization of rat and human UDP-glucuronosyltransferases

responsible for the in vitro glucuronidation of diclofenac. Toxicol

Sci 61(1):49–53

36. Moon YJ, Wang X, Morris ME (2006) Dietary flavonoids: effects

on xenobiotic and carcinogen metabolism. Toxicol In Vitro

20:187–210. doi:10.1016/j.tiv.2005.06.048

37. Begleiter A, Leith MK, Curphey TJ, Doherty GP (1997) Induc-

tion of DT diaphorase in cancer chemoprevention and chemo-

therapy. Oncol Res 9:371–382

38. Sivaramakrishnan V, Shilpa PNM, Praveen Kumar VR, Niranjali

Devaraj S (2008) Attenuation of N-nitrosodiethylamine-induced

hepatocellular carcinogenesis by a novel flavonol-Morin. Chem

Biol Interact 171:79–88. doi:10.1016/j.cbi.2007.09.003

39. Sakao Y, Tomimitsu S, Takeda Y, Natsuaki M, Itoh T (2004)

Carcinoembryonic antigen as a predictive factor for postoperative

tumor relapse in early-stage lung adenocarcinoma. Eur J Car-

diothorac Surg 25:520–522. doi:10.1016/j.ejcts.2004.01.029

40. Devipriya S, Ganapathy V, Shyamaladevi CS (2006) Suppression

of tumor growth and invasion in 9,10 dimethyl benz(a)anthracene

induced mammary carcinoma by the plant bioflavonoid quercetin.

Chem Biol Interact 162:106–113. doi:10.1016/j.cbi.2006.04.002

41. Pujol JL, Boher JM, Grenier J, Quantin X (2001) Cyfra 21-1,

neuron specific enolase and prognosis of non-small cell lung

cancer: prospective study in 621 patients. Lung Cancer 31:221–

231. doi:10.1016/S0169-5002(00)00186-0

42. Quoix E, Purohit A, Faller-Beau M, Moreau L, Oster JP, Pauli G

(2000) Comparative prognostic value of lactate dehydrogenase

and neuron-specific enolase in small-cell lung cancer patients

treated with platinum-based chemotherapy. Lung Cancer 30:127–

134. doi:10.1016/S0169-5002(00)00131-8

43. Lamy P, Grenier J, Kramar A, Pujol JL (2000) Pro-gastrin-

releasing peptide, neuron specific enolase and chromogranin A as

serum markers of small cell lung cancer. Lung Cancer 29:197–

203. doi:10.1016/S0169-5002(00)00113-6

Mol Cell Biochem (2009) 331:135–143 143

123