INVESTIGATION OF THE INTERINDIVIDUAL VARIABILITY IN HEPATIC CYTOCHROME P450 CYP3A4: ASSOCIATION WTH CYP3A4*IB

Antonio Ciaccia

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Pharmacology University of Toronto

0 Copyright by Antonio Ciaccia 1999

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Abstract

Investigation of the interindividual variability in hepatic Cytochrome P450 CYP3A4:

Association with CYP3A4 * I B. M.Sc. 1999. Antonio Ciaccia Department of

Pharmacology. University of Toronto

In this study, we determined the association of CYP3A4VB (CYP3A4-V) to the

large interindividual variability in hepatic CYP3A4, We observed a non-normal

distribution in the amount of immunoreactive CYP3A4 present in 48 human liver

samples. Probit and NTV analyses reveaied that 38% of these samples had CYP3A4

protein levels beiow an apparent antimode of 21 pmol CYP3A4 / mg microsomal protein.

Interestingly, probit and NTV plots of testosterone 6B-hydroxylation activity, a marker

for CYP3A4 activity, of two liver banks also revealed approximately 40% of the

population below an analogous antimode. This data suggests that approximately 40% of

the Caucasian population foms a distinct subpopulation with low CYP3A4 activity. To

determine whether CYP3AI*IB associates with this low activity, we characterized our

liver banks via a novel CY;P3A4*IB genotype assay. We found a CYP3AJ*IB allele

f?equency of 6% in the Caucasian population. Furthemore, we report no association

between C W 3 A l * l B and testosterone 6B-hydroxylation. This study suggests

CYP3A4*IB allele has no association with CYP3A4 activity.

I would like to thank:

Dr. ?K Kulow and Dr. B.K Tang for their support and guidance.

Our collaborators, Dr. E.A. Roberts, Dr. A.B. Okey, Dr. W. Li, Dr- D. Grant, Dr. R. Voorman and Dr. T. Rebbeck. for their assistance with this project.

John Giannone, Dr. Judy Wong, Ewa Hamann, Pefer Kurd and Dr. Vural &demir for their technical expertise and invaluuble suggestions. Withouf these individuals this work would not have been possible.

My colieagues andpends throughout the Department of Phmmacology, for their fiiendrhip and encouragement.

My family, for their understanding and uncondilional love.

Siacey, for her patience and support.

List of Abbreviations

ARMS

ASA

ASP

BTE

CAT

CSGE

CYP

ER6 PXRE

HFLaSE

NFSE

P450s

PAMSA

PASA

rcc

RXR

SrNE

SSCP

UTR

Amplification Rehctory Mutation System

Allele Specific A m p ~ c a t i o n

Ailele-Specific PCR

Basic Transcription Element

Chloramphenicol Acetyltransferase

Conformation Sensitive Gel Electmphoresis

Cytochrome P450

Everted Repeat 6 PXR Response Element

p4SOHFLa-Specific Element

P4SONF-Spe~ific Element

Cytochromes P450s

PCR Amplification of Multiple Specific Alleles

PCR Amplification of Specinc Aileles

Genetic Component Value

9-cis Retinoic Acid Receptor

Short Interspersed Repetitive Element

Single Strmded Conformation Polymorphism

Untranslated Region

List of Figures

Figure 1 .

Figure 2 .

Figure 3 .

Figure 4-

Figure 5-

Figure 6-

Figure 7-

Figure 8-

Figure 9-

Figure IO .

Figure 1 1 .

Figure 12 .

Figure 13 .

Figure 14 .

5' Regdatory region of CYP3A4 .............................................................. 11

........................ Sequence alignment between hPXR, SXR, hPAR and mPXR 13

A h family tree ................................................................................................... 21

....................................................... Schematic of allele specific amplification -36

...................................................... Imrnunoblotted CYP3 A4 Standard Curve -40

Probit and NTV piots of immunoreactive CYP3A4 ............. ... .................... 42

Probit plots of testosterone 6f3-hydroxyiation and Vmm .................................... 43

......... Correlation analysis between immunoreactive CYP3A4.. ............... .... 46 and testosterone 6a-hydroxylation .

Allele specific amplification of CYP3A4*IB ................ .... ......................... 48

Sequence of CYP3A4 *I l? .............................................................................. 49

............................................... Phenotype-genotype cornparison -53

Probit and NTV plots of testosterone 6phydroxylation ................................. 54 fiom V-Series livers .

CYP3A4 mRNA .............................................................................................. 57

............................... RT-PCR amplification of P-Actin and 3.0kb transcript - 3 9 of CYP3A4 .

List of Tables

Table 1 . Members o f the human Cytochromes P450 Superfamily ..................................... 4

.............................. Table 2 . Reported nucleotide and amino acid variations in CYP3A4 15

Table 3 . Immunoreactive CYP3A4 levels of L-series Iivers ............................................ 41

Table 4 . Multiple correlation analysis .............................................................................. 45

Table 5 . CYP3APIB allele fkquencies ............ .... .................................................... 51

List of Appendices

Appendix 1 . Correlation analysis between testosterone 6B-hydroxylation ............... ..... 78 and aryl hydrocarbon hydroxyIase .

Appendk 2 . Characteristics of L-series livers ...................... .. ...................................... 79

Appendix 3 . Characteristics and CYP3A4 * l B genotype of V-senes livers ..................... 80

Appendix 4 . Characteristics of Human DNA samples and CYP3A4 *IB genotype ......... 81

Appendix 5 . Kinetic parameten of L-senes livers ............. ... ....................................... 84

Table of Contents

. . Abstract ............................ ... .......................................................................................... 11

... ....................................................................................................... Acknowledgements ui

.......................................................... ..................... .. List of Abbreviations .... ...-.. iv

List of Figures .............................................................................................................. v

List of Tables ................................................................................................................ vi

.. List of Appendices ............................ ... ................................................................... mi

... .............................. ..................*.............................................. Table of Contents ..... wii

......................................................................................................... Introduction ....... ..,. 1 1 . Cytochromes P450 Superfamily ........................................................................ 1

1.1 Oxidative Cycle ..................................................................................... 1 ............................................ 1 -2 Cytochromes P450 Nomenclature and Families 2

2 . Pharmacogenetics of Human Cytochromes P450 ............................................... 3 2.1 CYPlA ........................................ .. 3 .......................

. ..................................................................*....................... 3 CYP3A Family ..... ...... 6 3.1 CYP3A4 ......................................................................................................... 8

.................................................................... 3.2 Variability in CYP3A4 Activity 8 .......................................................................... 3.3 Gene Structure of CYP3A4 10

.......................................................................................... 3.4 CYP3A4 mRNA 14 ............................................................................ 3 -5 CYP3AI *Il3 (CYP3A4- 16 ............................................................................. . 4 Allele Specinc Amplification 17

. 5 Alu Family ........................................................................................................... 19

........................................................................................ ................... Rationale ...... 22

............................................................................................... Hypothesis .......... .... 23

.................................................................................................................. Objectives 23

Materials and Methods .............................. .. ................... 24 ............................ Caucasian Human Liver Tissue .. ....................................... 2 4

.............................................................................................. h u n o b i o t Analysis 24 ............................................................................................ RNA/DNA Extraction 2 6

Alternate Method of DNA Extraction .............................................. 28 ............................................................................ Reverse Transcription of mRNA 29

................................................................. PCR Amplincation of p-Actin cDNA 30 PCR Amplincation of CYP3A4 3'UTR (cDNA and genomic DNA) .................... 3 1

.......................... PCR Amplification of Spromoter region of CYP3A4 .... .... 3 1

......................... Extraction and Purification of PCR Products From Agarose Gels 32 .............................................................................. Subcloning and Transformation 33

Preparation of Plasmid DNA .................................................................................. 34 ................................................................................................ Restriction Analysis 35

................................................................................. Allele Specific Amplification 35 ............................................................................. Data Analysis .................... ..... 38

Results ......................................................................................................... 39 .............................................................................................. Irnmunoblot Analysis 39

Correlation Analyses ............................................................................................ 39 .............................................................................................. S equence of C P 3 A 4 44

Allele Specific Amplification Assay ................... ... ......................................... 44 Genotype Frequency Estimation ............................................................................. 50

.................................. CYP3A4 *Il3 Genotype and Testosterone 6fbHydroxylation 52 ..................................................................... Sequence of the S'region of CP3A4 55

Sequence of the 3'UTR of CYP3A4 ....................................................................... 56 Expression of CYP3A4 mRNA transcripts ........................................................... 56

Discussion ................... ., ....... ............................................................................... 60 Immunoblot Analysis ............................................................................................ 60

................................................................................. Allele Specific Amplification 62 ............................................................................. Genotype Frequency Estimation 63

................ ............. CYP3A4 *IB Genotype and Testosterone 6P-Hydroxylation ... 64 .......................................................................... Sequence of Non-Coding Regions 65

..................... Future Studies -... ............................................................................ 66

.................................................................................................................... References 68

Appendices ................................................................................................................. 77

Introduction

1. Cytochromes P450 Superfamily

Cytochromes P450 (P450s) are a superfamily of dnig metabolizing enzymes.

P450s are hemoproteins that contain a single heme prosthetic group linked to a

polypeptide composed of 400 - 500 amino acids. Located primarily in the smooth

endoplasmic reticulum of the Iiver, these membrane bound proteins are involved in the

metabolism of a wide variety of endogenous compounds and foreign chernicals

(Guengerich, 1995; Guengerich and Shimada, 1991).

1.1 Oxidative Cycie

P450s catalyze many types of reactions including O-dealkylations, deaminations

and dehydrogenations, however, the role these enzymes play in the mixed function

oxidase system is by far the most important. NADPH-cytochrome P450 reductase

catalyzes the traasfer of an electron fiom NADPH to the oxidized fonn of a cytochrome

P450-substrate complex. After this rate-lirniting step, P450s use one atom of molecular

oxygen for substrate oxidation. Following the donation of a second electron via either

NADPH or NADH, the second atom of molecular oxygen is consurned for the formation

of water and the oxidized substrate-cytochrorne P450 complex dissociates (Lewis, 1996).

1.2 Cytochromes P450 Nomenclature and Families

At the early period of P450 research, the classification of novel P450s was

entirely at the discretion of the individuai investigator. Unfortunately, this system of

nomenclature leads to overlap in the naming of individual P450s. For example, PB-B

and PB-4 were both used to describe CYP2Bl (Nelson et al-, 1993). The current

classification of P450s is based on amino acid similarity and uses CYP, for Qtocbrome

P450, followed by an Arabic number designating the family, a letter for the subfamily -

and an Arabic number for the individual gene within the subfamily (Nebert et al., 199 1 ;

Nelson et al., 1993). When describing the gene of a P450 protein, Cn) is italicized. The

mouse gene, however, is designated Cyp. As a general guideline, P450 proteins with

greater than 40% amino acid sequence similarity belong to the same family and those

with greater than 55% similarity are assigned to the same gene subfamily.

With the advancement of molecular biology, the number of identified

C ytochrome P450 sequences has swollen to approximately 1 O00 (Nelson, 1999).

Humans have 48 sequenced CIiP genes and 14 pseudogenes. Interestingly, CW2CI O,

CYP3A3 and CYPlA9 have been rernoved fiom the Cytochromes P450 Superfamily, as

they are most likely sequencing artefacts (Nelson, 1999). Of the 16 Human Cytochrome

P450 Families, CYP 1 -CYP3 metabolize drugs and other xenobiotics while CYP4-CYP5 1

are responsible endogenous processes such as steroid and bile acid biosynthesis.

There are several key features of P450s that allow them to effectively metabolize

xenobiotics. First of d l , P450s have a broad substrate specificity. CYP3A4, for

example, is able to catalyze stmcturally diverse cornpoumis such as cocaine and

cyclosporin (LeDuc et al-, 1993). Secondly, multiple isoforms of P450s may metabolize

the same substratte. For example, CYP2D6, CYP2C9 and CYP3A4 are ail responsible for

the 4-hydroxylation of tamoxifen (Crewe et al., 1997). Furthemore, a substrate may be

metabolized by P450s at multipie locations. Debrisoquine, for example is hydroxylated

at the 1-, 3-, and 4- positions by CYP2D6 (Eiermann et al., 1998).

2. Pharmacogenetics of Humnn Cytochromes P450

The most common approach for classi@hg P450 gene alterations is

differentiating between monogenic and mukigenic variants- Ln muitigenic variants, two

or more genes are involved in producing the altered phenotype. Aiso, environmental

factors may contribute to the mdtigenic trait. Monogenk varia-, as seen in plasma

cholinesterase, are explained by a specific alteration in a single gene (Kalow and Grant,

1998; Britt, 1991). Monogenic variants are considered poIymorphic if the lowest

occurruig phenotype has a fiequency greater than 1% in a given population.

Polymorphisms have been reported for human Cytochromes P450 CYPIAI, CYPIA2,

CYPIBI, CYP2A6, CYPZA7, CYP2C9, CYPtCI8, CYP2C19, CYP2D6, CYP2E1,

CYP3A4 and CYP3A5 (Table 1). Important allelic members of the CYPlA family are

described in the following section.

2.1 CYPlA

The cytochrome P450 CYPlA family has two members, CYPl Al and CYPlA2.

The genes for these proteins are located on chromosome 15q22-qter (Jaiswal et al.,

1987). Although these proteins are highly homologous, they have discrete but

overlapping substrate specificities (Ikeya et al., 1989). CYP 1 A 1 favours polycyclic

aromatic hydrocarbons while CYP 1 A2 metabolizes heterwyclic and polyaromatic

variabüity in level, fold -100 40 - 30 ?

50 - 25 - - 100

>IO00 20 ?

20 >IO0

?

+ + + + +

none reported none reported

+ none reported

+ + + i-

none reported + +

none reported

P450

Table 1. Members of the human Cytochromes P450 Superfamily. Adapted from Guengerich, 1995.

Extent of Chromosome Polymorphism

amines and amides preferentiaily (Nedelcheva and Gut, 1994). Another important

difference is that while CYPlA2 is expressed predominantly in the liver and at very low

Ievels extrahepaticaily, CYPl Al is not present in the non-induced liver (Pastrakuljic et

al., 1997; Shimada, 1989). Aiso, both CYP 1 Al and CYP 1 A2 are regdated by the aryl

hydrocarbon receptor (Whïtlock, 1993).

There are currently seven known CYPIAI mutations and six known CYPIA2

mutations (Ingelman-Sundberg et al_, 1999). Of special interest here are CYPIAP2A

(Spurr et al., 1987), CYPIAI *2B (Hayashi et al., 1991), CYPIAI *2C (Hayashi et al.,

1991; Zhang et al., 1996; Persson er al., 1997), and CYPIA2*I C (Nakajima et al., 1999).

These mutant alleles either are associated with increased cancer susceptibility or alter

gene expression.

CYPIAP2A allele resdts fiom a thymidine to cytosine substitution at base pair

3801, creating an UrpI restriction site in the 3'-flanking region of this gene. The

CYPIAP2C allele (2455A>G) resdts in the substitution of Isoleucine for Valine at

amino acid 462. The CYPIAI *2B ailele contains both of the aforementioned mutations.

Interestingly, in Caucasian and Japanese popdations, either of these aileles has been

correlated with increased Iung cancer risk (Kawajiri et al., 1986; Nakachi et al., 1991; Xu

et al., 1996).

The CYPIA2*IC allele r e d t s fiom a guanine to adenine substitution in the

S'regulatory region (-3585) of this gene. Nakajima et al. reported that this mutation is

inherited. Furtherrnore, the CYPIA2*IC allele resdted in a significant decrease in the

rate of caffeine 3-demethylation, an indicator of CYPIAî activity.

3. CYP3A Famiiy

The cytochrome P450 CYP3A family consists of an estimated 41 membew

(Nelson, 1999). CYP3A members are found in species such as rat (CYP3A2), rabbit

(CYP3A6), and monkey (CYP3A8). Of the five human members, there are three

expressed genes, CYP3A4, CYP3A5 and CYP3A7, and two pseudogenes, CYP3ASPI and

CYP3AjP2. CYPSAJ, which is 98% suniIar to CP3A4, has recently been removed from

the P450 Superfamily, as it is most likely a sequencing artefact (Nelson, 1999).

Human CYP3A enzymes metabolize a wide range of xenobiotics and endogenous

substances (Maurel, 1996). There is no apparent structural requirement for CYP3A

substrates as these proteins metabolize substances of varying size and composition. in

general, CYP3A substrates are large and highly hydrophobie, the Km for these enzymes

Vary fiom 1 to 500pM and their active sites are said to be promiscuous.

Even though human CYP3A proteins are highly homologous (lowest sequence

homology - 75%), each has their own unique tissue distribution and regdatory processes.

CYP3A7 is believed to be the major P450 expressed in the fetal liver and kidney

(Schuetz et al., 1994; Yang et al., 1994). Recent reports, however, indicate appreciable

levels of CYP3A7 mRNA in adult liver and kidney tissues as weil (Kolars et al., 1994;

Murray et al., 1999). Interestingly, Schuetz et al. (1 994) demonstrated polymorphic

CYP3A7 mRNA expression in adult Liver; 7 of 13 livers possessed CYP3A7 mRNA. In

a similar study, which investigated the ethnic ciifferences between adult Caucasians and

Japanese, CYP3A7 protein was detected in microsornes nom 14 of 15 Japanese

individuals and in only 10 of 15 Caucasians (Tateishi et al., 1999). CYP3A7 protein

Ievels, however, were much lower than its CYP3A4 counterpart.

CYP3A7 and CYP3A4 have similar metabolic profiles for many xenobiotics.

These CYP3A proteins equally metabolize AFBI, stengmatocystin, and aflatoxin G1 to

their toxic metabolites (JiIashimoto et al., 1995). Furthermore, the catalysis of

dehydroepiandrosterone 16a-hydroxylation and testosterone 6P-hydroxylation are carried

out by both CYP3A7 and CYP3A4. CYP3A7, however, has a higher affinity and

maximal velocity for dehydroepiandrosterone l6a-hydroxylation. In con- CYP3A4

is primarily responsible for the 6fbhydroxylation of testosterone (LaCroix et al., 1997;

Waxman er al., 1991).

CYP3AS is the major CYP3A isofom found in human kidney. Interestingly,

both CYP3A5 protein and mRNA are ubiquitously expressed in the kidney while

CYP3A4 protein and mRNA are present in about 30 - 40% of kidney samples (Haehner

et al., 1996). CYP3A5 is also the major CYP3A present in the lung and blood, where

CYP3A4 is present in 20% of lung samples and not at al1 in leukocytes (Antiia et al.,

1997; Janardan et al., 1996)

CYP3A5 shares a similar substrate profile with CYP3A4. CYP3A5, however, has

significantly lower activity for substrates like testosterone, progesterone, and

androstenedione (Waxmm et al., 1991). A~so, unW<e CYP3A4, CYP3A5 does not

metabolize erythromycin, quiniche and 17P-estradiol (Maurel, 1996).

Interestingly, a point mutation in exon 11 of CYP3A5 (12800A) has k e n

identified (Jounaidi et al., 1996). This mutation results in the substitution of threonine

for asparagine. Recently, the Human Cytochrome P450 (CYP) Allele Nomenclature

Committee has renamed the variant CYP3A5 gene and protein, CYP3ASf2 and

CYP3A5.2, respectively (ingelman-Sundberg, 1999). Jounaidi et al. (1996) speculate

that CYP3A5*2 may result in an unstable gene product since this ailele was found to

cosegregate with the absence of protein accumulation in 2 of 5 defective individuais.

3.1 CYP3A4

CYP3A4 is of major clinical importance (Maurel, 1996). It is the rnost abundant

human P450 present in the liver and intestine and is responsible for the metabolism of a

wide variety of drugs, ranging from macrolide antibiotics to vinfa alkaloids. In fa*

CYP3A4 metabolizes approximately 60Y0 of pharmaceuticaIs. Thus there is a possibility

of detrimental drugdrug interactions when multiple CYP3A4metabolized

pharmaceuticals are prescribed- A classic example is the CO-administration of terfenadine

and erythromycin (Wilkinson, 1 996).

CYP3A4 metabolizes endogenous hormones such as testosterone and

progesterone (Waxman et al., 1988). These hydroxylation reactions typically occur at the

6P position, however, CYP3A4 also produces smaller amounts of 2P-, 15P- and 16a-

hydroxymetabolites.

3.2 Varia bility in CYP3A4 Activity

One of the major characteristics of CYP3A4 substrates is that there is marked

inte~dividuai variability in the amount of metabolite produced (Maurel, 1996). The

general consensus is that the variation seen in CYP3A4 activity is multigenic (Wilkinson,

1996). Important to note, is that CYP3A4 is greatly Sected by a number of inhibitors

and inducers, some of which are substrattes for this enzyme as well. For example,

erythromycin, which is N-demethylated by CYP3A4, acts as an inhibitor of this enzyme

at micromolar concentrations. In fact, CYP3A4 inhibitors generally act in the

micromolar range, with the most potent king the antifhgal agent, ketoconazole (Pichard

et al., 1990)- Interestingiy, grapefruit juicc acts as a potent inhibitor of CYP3A4. Its

effects Vary considerably between individuals and appear to selectively inhibit intestinal

CYP3A4 over hepatic CYP3A4 (Lown et al., 1997; Rau et al., 1997). One of the active

agents in grapefruit juice that contributes to this inhibition appears to be the

furanocoumuin, bergamottin (He et al., 1998).

There are also a number of inducers of CYP3A4. These include rifarnpicin,

barbiturates, and gIucocorticoids ( W i n , 1996). These compounds act to elevate

CYP3A4 protein levels and thus this may lead to decreased plasma levels of CYP3A4

substrates. An example is the failuse of oral contraceptives when phenytoin is taken

concurrentiy (Janz and Schmidt, 1974).

The generally accepted view of CYP3A4 activity is that it is normally distributed

throughout the population (Wilkinson, 1996). There have, however, been several

indications of a non-normal distribution in the activity of CYP3A4. There was an

apparent bimodal distribution of the area under the h g concentration curve (AUC) in 53

healthy subjects after the oral administration of nifiedipine (Kleinbloesen et al., 1984). In

a second, larger study, however, this bimodality could not be corroborated (Schellens et

al., 1988). Interestingiy, the recoveries of the major metabolite of nifedipine for cystic

fibrosis patients and their parents are shifted to the lefi as compared to a control

population @aly et al., 1992).

Other studies have described interethnic Merences in the metabolism of

CYP3A4 substrates. Ethnic differences of CYP3A4 activity between Caucasians, South

Asian Indians, Chinese and Mexicans are common in the iiterature (Ahsan et ai., 1993;

Caraco et al., 1996; Lin et al., 1999; Hoyo-Vadillo et al., 1989), however dietary factors

may contribute somewhat to this variation (Castaneda-Hemandez, 1993).

3 3 Gene Structure of CW3A4

Human C I T A was initialiy assigned to chromosome 7 by somatic ce11

hybridization and in situ hybridization (Gonzalez et al., 1988). By fluorescence in situ

hybridization, CW3AI was later assigned to 7q22.1 (houe et al., 1992). CYP3A4 is

approximately 25 kb in length aad contains 13 exons and 12 introns (Hashimoto et ai.,

1993). Interestingly, both CYP3A7 and CYP3A2 have similar exonlintron structures to

CYP3A4 @ashimoto et al., 1993). These investigators also noted that the consensus

sequences for splitting junctiom, GT and AG, were present at the boundaries of ali

ïntrons. Furthennore, the initiator (ATG) and terminator (TGA) codons were present in

the fmt and 1s t exons, respectively.

Hashimoto et al., (1993) also reported 1105 base pairs of the 5' regulatory region

of CYP3A4. CYP3A4 promoter region contains a typical TATA (TATAAA) box and the

basic transcription element (BTE; Figure 1). The identification of putative binding sites

of transcriptional regulatory factors such as the estrogen receptor, progesterone

receptor/glucocorticoids receptor and P53 were also reported. Interestingly, these authors

also reported that the sequence of the 5' regulatory regions of CYP3A4 and CYP3A7 were

approximately 91% identical. There were, however, hvo regions that differed between

CYP3A4 and CYP3A7. CYP3A4 contained an extra 1 0-nucleotide region (-287 to -296),

which they narned P45û~pspecific element (NFSE), while CYP3A7 contained an

P53 binding motif

Figure 1 . S'Regulatory region of CYP3A4. ERE = Estrogen Response Element; PREIGRE = Progesterone and Glucocorticoid Response Element; ER6-PXRE = PXR binding element; BTE = Basic Transcription Element

additional 9-nucleotide element (-729 to -737), which was named P450HFLa-specinc

element (HFLaSE). The investigators speculated that these regions might be responsible

for the developmental control of CYP3A4 however they were unable to demonstrate any

factors that bound specificaiiy to either element.

Using chlorarnphenicol acetyltransferase (CAT) expression systems, varying

Iengtbs of the S'regulatory region of CYP3A4 were transfected into HepG2 cells

(Hashimoto et al., 1993). The results of these experiments indicate the presence of two

cis-acting elements between nucleotides -2900 to -445, which decreased transcription of

the CAT gene. The region between - 4 5 and -254 was crucial for the proper

transcription of the CAT gene in HepG2 ceils. Furthemore, the region between -362

and -94 may have enhancer activities.

In 1998, a possible main contributor to the enhancing properties of the region

between base pairs -362 and -94 of CYP3AI was discovered. Three novel human orphan

nuclear receptors capable of binding to the 5' regulatory region of CYP3A4 were

identified, hPXR (Lehmann et al., 1998)' SXR (Blumberg et al., 1998) and hPAR

(Bertilsson et al., 1998). These receptors are designated 'orphan' because their

endogenous ligand is unknown. Figure 2 reveals, via a sequence cornparison between

these three receptors and the mouse PXR (Kliewer et al., 1998)' that hPXR, SXR and

hPAR are most Iikely the same receptor. The only discrepancy appears to be that the

first amino acid is either a Leucine or a Methionine. Whether this is the result of a

sequencing error or these proteins are isoforms of a specific gene remains unclear.

PAR LEVRPKESWNH ADFVEICEDTE SVPGKPSVNA DEEVGGPQIC RVCGDKATGY EIFNVMTCEGC PXR/SXR MEVFSXESWNH ADFVBCEDTE SVPGKPSVNA DEEVGGPQIC RVCGDKATGY HFNVMTCEGC mPXR --=BESUSR VGLVQCEEAD ÇALBSP-INV BEEDGGLQIC RVCGDKANGY HFNVMTCEGC

PAR KGFFRRAMRR NARLRCPFRK GACEITRKTLZ RQCQACRLRK CLESGMKKEM IMSDEAVEER PXR/SXR KGFFRRAMKR NARLRCPFRK GACEITRKTX RQCQACRLRK CLESGMKKEM IMSDEAVEER mPXR KGFFRRAMKR NVRLRCPFRK GTCEITRKTR RQCQACRLRK CLESGMKKEM IMSDILAVEQR

PAR RALIKRKKSE RTGTQPLGVQ GLTEEQRMMI RELMDAQMKT FDTTFSBFKN F'RLPGVLSSG PXR/SXR RALIKRKKÇE RTGTQPLGVQ GLTEEQRMMI RELMDAQMKT FDTTFS- FRLPGVLSSG mP%R RALIKRKKRE KIEAPPPWQ GLTEEQQALI QELMDAQMQT FDTTPSffFKD FRLPAVIIISG

PAR CELPESLQAP SREEAAIWSQ VRKDLCSLKV SLQLRGEDGS VWNYKPPADS GGXEIPSLLP PXR/SXR CELPESLQAP SREEAAKWSQ VRKDLCSLKV SLQLRGEDGS VWNYKPPADS GGKEIFSLLP mPXR CELPEILQAS ELEDPATWSQ ïHKDRVFKKï SLQLRGEDGS IWNYQPPSXS DGREIXPLLP

PAR HMADMSTYMF KGIISFAKVI SYFRDLPIED QISLLKGAAF ELCQLRFNTV FNAETGTWEC PXR/SXR HMADMSTYMF KGIISFAKVI: SYFRDLPIED QISLLKGAAF ELCQLRFNTV FNAETGTWEC mPXR EiLADVSTYMF KGVINFAKVI SYFRIDLPIED QISfiLKGATF EWCXLRFNTN FDTETGTWEC

PAR GRLSYCLEDT AGGFQQLLLE PMLKFHYMLK KLQLHEEEYV LMQAISLFSP DRPG'HRV PXR/SXR GRLSYCLEDT AGGFQQLLLE PMLKFEIYMLK KLQLHEEEYV LMQAISLFSP DRPGVLQEiRV mPXR GRLAYCFEDP NGGFQKLLLD PWMFIfCMLK KLQLIIltEEYV LMQAISLFSP DRPGVVQRSV

PAR VDQLQEQFAI TLKÇYIECNR PQPAHRFLFL KIMAMLTELR SINAQHTQRL LRIQDIHPFA PXR/SXR VDQLQEQFAI TLKSYIECNR PQPAIfRFLFL KIMAMLTELR SINAQBTQRL LRIQDIHPFA WXR VDQLQERFAL TLKAYIECSR PYPAHRE'LFL KIMAVLTELR SINAQQTQQL LRIQDSHPFA

PAR TPLMQELFGI TGS PXR/SXR TPLMQELFGI TGS mPXR TPLMQELFSS TDG

Figure 2. Sequence alignment between 3 human nuclear receptors, PAR (AAC64557), PXR (AAD05436) and SXR (NP-003880), and the mouse mPXR (AAC39964). Sequence analysis reveals PAR, PXR and SXR are most Likely the same protein. Sequence differences are bolded.

hPXR forms a heterodimer with the 9-cis retinoic acid receptor (RXR) and binds

to the 5' regdatory region of CW3A4. This complex activates transcription through a

response element, termed Everted Repeat 6 PXR Response Element (ER6 PXRE), which

is located between nucleotides -170 and -153 of CYP3A4 (Lehmann et al., 1998). This

element consists of two copies of the AG(G/T)TCA motif, which is recognized by

memben of the nuclear receptor s u p e r f d y . Rifampicin, dexamethasow and other

compounds, wbich have been s h o w to alter CYP3A4 activïty, activate the hPXR/RXR

complex.

3.4 CYP3A4 mRNA

CYP3A4 mRNA exists in two forms, a 2.2kb transcript and a 3.0kb transcript

(Bork et al., 1 989). Both transcripts share identical 5' untranslated regions (5' UTR) and

coding regions. The 3' untranslated region (3'UTR) of the 3.0 kb transcript contains two

polyadenylation signals, while that of the 2.2 kb transcript contains only the fim

polyadenylation signal. When northern blots were performed with RNA fiom human

liver samples, the 2.2kb and 3.0kb t ra~~cripts were found to be expressed in a ratio of

approximately 10:l. Furthemore, Bork et al. (1989) suggested that altemate use of the

second polyadenylation signal might present a pretranslational control mechanisrn in the

regdation of CYP3A4 activity. Interestingly, both the rabbit CYP3A6 and rat CYP3A1

also contain two mRNA transcripts of different sizes (Kirita and Matsubara, 1993; Dalet

et al., 1988).

There are four reported sequences of CYP3A4 mRNA (Table 2). These four

transcripts are hPCN 1 (Gonzalez et al., l988), CYP3 (Spurr et al., 19891, NF25 (Beaune

Nucleotide change*

-290A>G change*

- 1 Eson 1 Accessioi Number

promoter

1 1

12

12

1

3

8

Table 2. Reported nucleotide and amino acid variations in CYP3A4. * As compared to hPCN 1 (Accession Number - Ml 8907; G o d e z et ai., 1988) or 5 ' promoter region of CYP3A4 (Accession Number - D 1 1 1 3 1 ; Hashimoto et al., 1 993). t Certain texts consider this the CYP3A4 consensus sequence.

et al., 1986) and NF 1 O (Bork et al., 1989). Interestingly, when WCNI, NF25 and NF10

were expressed in yeast, hPCNl and NF25 produced roughly equivalent catalytic

properties, while NFIO produced a defective and unstable gene product (Peyronneau et

al., 1993). It is unclear which of these genes code for wildtype CYP3A4. Furthennore,

whether two or more of these proteins exist in normal human Liver has yet to be

determined.

3.5 CYP3A4*lB (CP3A4-C7

Recently, a novel genetic variant of CYP3A4 bas been discovered, the CYP3A4-V

allele (Rebbeck et al., 1998). This allele has now been renamed CYP3A1*IB (Ingelman-

Sundberg, 1999). This variant consists of a substitution mutation (-290A>G) in the

putative regulatory element, NFSE. The ailele fiequency of the rnutated CYP3AJ gene,

as estimated with 94 male Caucasian Americans, is 9.6%. The ethnic distribution of

CYP3AI*IB in Afkican Arnencans and Taiwanese is 53% and O%, respectively (Walker

et al., 1998). Rebbeck et al. (1998) reported that the CYP3AQ'IB genotype was

associated with a higher tumour-lymph node-metastasis stage and Gleason grade. Aiso,

CYP3A4*IB genotype and tumour stage was most pronounced in men diagnosed at a

relatively old age who reported no family history of prostate cancer. Individuals in this

group with stage T3m4 tumours had the CW3APIB genotype in 46% of the cases. The

authors speculate that the mutation in NFSE, a putative transcriptional regulatory

element, may decrease the amount of CYP3A4 protein and therefore decrease 2B-, 6P

and 15B- testosterone oxidation. The decrease in this pathway may result in more

testosterone available for synthesis of dihydrotestosterone, the main androgen regulating

prostate ce11 growth and fhction, and thus may influence androgen-mediated prostate

carcinogenesis.

In a similar study, the association between de novo and treatment-related

leukemias and CYP3A4 genotype was examined (Felk et ai., 1998). The investigators

observed that 19% of de novo and 3% of treatment-related leukemias carried at least one

CYP3A?*IB dele. These scientists suggested that individuals with the CYP3A4

wildtype (CYP3AI*IA) genotype were at a greater risk of developing îreatment-related

leukemia.

4. Allele S pecific Amplification

Allele Specific Amplification (ASA) is a simple and reproducible method to

detect known point mutations and mial1 deletions or insertions (reviewed in Bottema and

Sommer, 1993). In fact, estimated matched-to-mismatched signal-to-noise ratios of 1 O00

have been reported (Carpenter et al., 1996). Synonymous names for ASA include PCR

Amplification of Specific AUeles (PASA), Allele-Specific PCR (ASP) and the

Amplification Refiactory Mutation System (ARMS; Nelson, 1998).

Theoretically, ASA is quite simple. Essentidy, primea are designed so that the site

of the mutation occurs immediately across fiom the 3'end of the primer. Optimized PCR

conditions allow for the specific match between the desired primer and allele, while the

sarne primer mismatches with the other dele. Thus the desired allele, if present, is

amplified while the other allele is not amplified. To ensure technical success of the ASA

reaction, another set of primers is included for the amplification of a ubiquitous gene.

The PCR product fiom this ubiquitous gene must then be present in al1 ASA reactions to

avoid false negatives and thus ensure correct reading of the individual's genotype.

ASA requires two separate PCR reactions for the amplification of the wildtype and

variant alleles. PCR Amplification of Multiple Specific Meles (PAMSA) attempts to

circumvent this by a m p m g both deles in a single reaction. One of the allele-specific

primers is designed with 30 or more non-complementary bases to aiiow for the

amplification of a longer PCR product. Optimization, however, may become

complicated as one of the allele specific primers may allow for the more efficient

amplification of PCR product (Bottema and Sommer, 1993).

Other techniques for the identification of known mutations include Single Strand

Conformation Polymorphism (SSCP) and Conformation Sensitive Gel Electrophoresis

(CSGE). In SSCP, primers are designed so that the site of the mutation occurs near the

middle of the PCR amplified product. The double stranded PCR product is denatured to

form single stranded DNA and each strand assumes a specific folded conformation (2"

structure), which is based on intemal base pairing. A single base mutation can cause a

change in folded conformation and thus change the mobility of the PCR product through

the polyacrylamide gel. Thus, PCR products containing mutations form a separate and

distinct band as compared to those amplified fiom wild type DNA (Orita et al., 1989).

CSGE is an extension of SSCP with some similarity to ASA. In CSGE, double

stranded PCR product is generated as is done in SSCP. In addition, amplification of

known homozygous variant DNA is also perfomed in a separate reaction. For each

subject two samples are loaded onto a polyacrylarnide gel. The first lane contains 6pl of

unknown PCR sample and 6p1 of a PCR-generated hgment from an individual that is

homozygous variant, in the second lane, 12jil of unknown sample is loaded. Based on

the banding pattern, homoygous wildtype, heteroygous and homozygous variant

individuals may be differentiated (Ganguly et al., 1994; Rebbeck et al., 1998).

5. Alu Famiiy

Approximately 1% of the human genome is required to code for al i necessary

proteios. The rest of the genome contains 'junk' or 'fïiler' DNA as well as families and

subfamilies of repetitive elements (Mighell et al., 1997).

Transposons are a group of repetitive elements found withïn the human genome

(reviewed in Szmdewicz et al., 1998 and Migheil et al., 1997). These elements are

capable of translocating fiom one part of the genome to another via retrotransposition,

That is, transposons are transcribed to an RNA intermediate, reverse-transcribed into

DNA and then incorporated into a different part of the genome. Repetitive elements that

are incorporated in this manner are called retroposons Furthemore, retroposons can be

classified into viral and non-viral family members.

Short interspersed repetitive elements (SINE) are a subfamily of the Non-Wal

Retroposons Family. SINEs range fiom 75 to 500 base pairs and contain split intemal

RNA polymerase III promoters. The largest and best-characterized subfamily of SINEs

is the primate specific Alu family of repetitive elements. This family was named afler the

Mu1 restriction enzyme that cleaves most A h sequences. Alu sequences comprise about

6-13% of the human genome, with a copy number of approximately 0.5-1.1~10~

sequences (Houck et al., 1979).

Consensus A h sequences, which are approximately 280 base pairs, are composed

of two monomeric sequences separated by an adenine-rich region. The right monomer

contains a 3 1 base pair insert that is not present in the left one. The left portion contains a

two-box (Box A and B) RNA polymerase iII promoter. While bot . Box A and B are

essentiai for effective transcription, Box A determines transcription strength and accuracy

(Fuhrman et al., 198 1). Most Alu sequences, however, are unable to retrotranspose as

they are generally tnincated at the S'end and usually contain ody 70-80% homology to

their respective consensus sequences (Deninger er al., 1992).

There are currently 12 Alu Subfamilies (Figure 3). Statistical analysis of

nucleotides in specinc Iocations of AIu sequences was instrumental Ï n determining not

ody the number of subfamilies but also, with the help of cornputer modelling, the

estimation of Alu subfamily age (Batzer et al., 1996; Bailey and Shen, 1993; Britten,

1994; Kapitonov and Jurka, 1996). Of these, Alu-J members are the oldest and Alu-Y

members are the youngest. Alu-Sx s u b f d y was responsible for almost half of the

identified A h sequences in one report; 295 out of 628 analyzed sequences (Jurka and

Milosavljevic, 1991). uiterestuigly, Alu-Sx shares greatest sequence homology with Alu-

Sq (Jurka and MiIosavljevic, 199 1).

In the early 1980s, A h sequences were considered 'junk' or 'selfish' DNA, which

served no usefiil purpose @oolittle and Sapienza, 1980; Orge1 and Crick, 1980). There

are now numerous examples of Alu sequences regulating gene expression, altering

normal splicing, and resulting in the initiation and progression of multiple disease States

(Smulewicz et al., 1998). Interestingly, a recently discovered subclass of A h sequences

functions as estrogen receptor-de pendent enhancers (Noms et al., 1 995). Furthemore,

First A h Sequence

Figure 3. A h family tree. Numbers represent the age (millions of years) of the specinc A h subfamily member. Adapted fiom Mighell et al., 1997

an A h element in the 3' UTR of P53 mRNA has k e n irnplicated as a cis-acting repressor

of translation (Fu et al., 1996).

Rationale

In an in vitro study performed in our laboratory, 48 human liver microsomes were

phenotyped for the prototypical CYP3A4 catalytic activity, 6fbhydroxylation of

testosterone (Sy, 1998). There was a distinct deviation fiom a normal distribution as

analyzed by both a Probit and NTV plot- Furthemore, a detailed kinetic study of 6 iïver

microsomes indicated a 36-fold variation in the formation rate of testosterone 6B-

hydroxylation. The variation in testosterone 6P-hydroxylation was attributed to

differences seen in V,,, but not in Km.

A similar in vitro study perfomed in our Iaboratory indicated approximately 250-

fold variation in aryl hydrocarbon hydroxylase activity (Pastrakuijic, 1996). This activity

is generally due to CYPl Al, CYP2B6 and CYP3A (Shirnada et al., 1994). Since both

CYP 1 A 1 and CYP2B6 are expressed in very low levels hepaticaily, the majorïty of this

variation is likely due to CYP3A. In fact, we found a significant correlation (r=0.87,

p<0.00 1 ) between aryl hydrocarbon hydroxylase activity and testosterone 6B-

hydroxylation activity (unpublished results; See Appendix 1).

The genetic contribution to the overall variability of CYP3A4 is believed to be

approximately 2 to 4-fold (Wilkinson, 1996). Recently, in a comparison between the

intra- and inter-individual variability in the metabolism of a variet. of CYP3A4

substrates, a high genetic component value ( r d . 8 3 ) was observed (Kalow et al., 1999).

This genetic component value indicates that the metabolism by CYP3A4 may rely to a

greater extent on individual genetic factors. InterestingIy, a twin study also demonstmted

a high heritability (H2=0.88) for antipyrine 4-hydroxylation, another indicator of

CYP3A4 activity (Pemo et al., 1981; Kalow et cd., 1999).

In 1998, a novel genetic variant of CYP3A4 was discovered, the CW3A4-V

(CYP3AJ*IB) allele (Ftebbeck et al., 1998). This variant corresponds to a substitution

mutation (-290A1G) in the putative regulatory element NFSE. This mutation is

suspected to alter the binding of factors to the putative regulatory element, NFSE, thus

resulting in the decreased production of CYP3A4 protein.

Hypot hesis

Alterations in the regulation of CYP3AI contribute to the variability in

testosterone 6B-hydroxylation.

Objectives

The source of CYP3A4 variability remains unclear. The k t objective of this

study is to determine whether the observed testosterone 6p-hydroxylation differences

seen in our liver bank are due to variable expression of CYP3A4 protein.

The second objective of this study is to CO- the presence of CYP3Al*lB by

developing an independent genotype assay that has the ability of differentiating between

the CYP3A3 wildtype and variant alleles.

The third objective of this study is to determine whether CYP3A#*lB is

associated with the low testosterone 6P-hydroxylation phenotype observed in certain

samples of our liver bank.

The last objective of this study is to determine whether altered regulation of

CYP3A4 can be explained by the presence of unknown sequence differences in the

5 ' regulatory region and 3'untraaslated region of CYP3AI.

Materials and Methods

Caucasian Human Liver Tissue

Caucasian human liver samples used in this midy were generously provided by

Dr. Eve Roberts at the Hospital for Sick Children (L-senes livers) and by Richard

Voorman at Pharmacia & Upjohn (V-series livers). Both the L- and V-series liver

tissues were obtained fkom Liver grafts used in reduced liver transplants. AU liver

samples were negative for Hepatitis B and H I V and were stored at -70°C. Appendïx 2

and 3 presents the avaiiable data for these livers.

Immuno blot Andysis

Microsomes used for immunoblot experiments were prepared by Mr. Sherwin

K.B. Sy and Ms. Aleksandra Pastrakuljic from L-series livers as described (Sy, 1998).

The Mmunoblot protocol for the detection of CW3A4 was perfonned according to the

Mini-PROTE AN II Electrophoresis Ceil Instruction Manual (Bio-Rad). Each blot

contained 10 lanes. Lanes 2 - 10 were loaded with microsornes (IOpg) derived from L-

senes livers. The first Ime, which contained 35pmoVmg of purified CYP3A4 protein

(Gentest), was used as an extemal standard. Microsornal protein (IOpg), d e r king heat-

denatured for 10 minutes at 10O0C, was loaded onto an SDS-denaturing polyacrylamide

gel. The upper Stacking Gel contained 4% (wk) acrylamide, 0.36% (w/v) N'N'-bis-

methy lene-acry lamide, 0.1 % (w/v) sodium doedecy lsulphate (SDS), 0 .OS% (w/v)

ammonium p e r d fate, and 0.1 % (v/v) TEMED in 0 . l Z M Tris-HCl (pH 6.8). The lower

Separating Gel contaiwd 12% (w/v) acrylamide, 1.07% (wh) N'NY-bis-methylene-

acxylamide, 0.1% (w/v) sodium doedecylsulphate (SDS), 0.05% (wlv) ammonium

persulfate, and 0.05% (v/v) TEMED in 0.375M Tris-HCl (pH 8.8). The samples were

then electrophoresed with Running BuEer (25mM Tns-HCl, pH 8.3, O.2M Glycine and

0.5% SDS) in a Mini-PROTEAN II Electrophoresis Ce11 (Bio-Rad) for approximately 1

hour at 150 volts.

M e r electrophoresis, the polyafsrlamide gel was removed and submerged in

Transfer BufYer (25mM Tris-HCl, pH 8.3, 0 2 M Glycine in 20% methanol). The

rnicrosomal proteins, which were electrophoresed through the polyacrylamide gel, were

transferred ont0 nitrocellulose paper via a Mini Trans-Blot Ce11 Transfer Apparatus (Bio-

Rad) at 100 volts for 1 hour. The nitrocellulose membranes were then stained with

Ponceau S (Sigma) for 10 minutes at room temperature to ensure successful tramfer. The

membranes were then blocked ovemight at room temperature with 5% (wlv) skim miik

powder in M T (20mM Tris-HCl, pH 7.6, O.12M NaCl, 0.01% (vh) Tween-20)

contaïning 0.05% (w/v) thyrnerosol.

CYP3A4 was detected on nitrocellulose membranes according to the ECL

western blotting protocol (Amersham Life Science). Dr. Wei Li performed experiments

that determined the optimum antibody concentration of the goat antihuman CYP3A4

antibody (Daiichi) and the sheep antigoat horseradish peroxidase conjugated antibody

(Daiichi) used here. Following the ovemight blocking treatrnent, the nitrocellulose

membranes were subjected to three 15-minute washes with TNT by agitation. The

membranes were then incubated on a shaker with the goat antihuman CYP3A4 antibody

(1 : 1500) containhg 5% skim milk powder in TNT for 1 hour. The membranes were

washed again with three 15-minute washes with TNT by agitation. The secondary-

horseradish peroxidase antibody (1: 15000) in 5% skim milk powder was then added to

the membranes and the membranes were shaken for 1 hour. Following three 15-minute

washes, the membranes were incubated with the ECL reagents (3x111 ECL Detection

Reagent 1 and 3ml ECL Detection Reagent 2) for 1 minute. The membrane was drained

of excess ECL reagent, inserted into a film case and exposed to Hyperfilm ECL film

(Amersham Life Science) for 30s to 2 minutes. The exposed film was scanned (Umax

SuperVista S-12) and the band of interest was then quantitïed via densitometric andysis

using IPLabgel (Sind Analytics). Immunoblotted CYP3A4 was quantifïed according to

the ECL Western blotting protocol (Amersham Life Science). That is, the optical density

of immunoreactive CYP3A4 for each sample was compared to that in lane 1, which

contained 35pmoVmg of purified CYP3A4 protein.

RNA/DNA Extraction

DNA and total RNA were extracted fkom L- and V-series human liver tissues

according to the TRIzol reagent (Gibco/BRL) protocol. Approximately 100 mg of liver

tissue was homogenized for 30 seconds in 1 ml TiUzol using a Brinkman polytron

homogenizer. Homogenized samples were then incubated at room temperature for 5

minutes to allow the complete dissociation of the nucleoprotein complexes. Chlorofonn

(0.2 ml chloroform / ml TRIzol reagent used for initial homogenization) was then added

to the samples. After the samples were shaken vigorously by hand for 15 seconds, they

were incubated for 2-3 minutes at room temperature. The samples were then centrifiqged

at 12,000g in a Sorvail RCZ-B centrifuge at 4°C for 15 minutes. Following

centrifiigation, the mixture separated into a colourless upper aqueous phase (contains

RNA), an interphase, and a lower red phenol-chloroform phase (contains DNA).

For RNA extraction, the upper aqueous phase, which contains the RNA, was

carehlly removed and transferred to a new tube. The RNA was then precipitated with

isopropyl alcohol ( 0 S d / ml TRIzol reagent used for initial homogenization). Samples

were incubated at room temperature for 10 minutes and centrifiged 12,000g for 10

minutes at 4OC. The RNA precipitate fonned a gel-W<e pellet on the bottom of the

eppendorf tube. The supernatant was gently suctioned and the RNA pellet was washed

with ice-cold 75% ethanol in DEPC-treated water (lm1 75% ETOH / ml of TRIzol

reagent used for initial homogenization). Samples were mixed by vortexing and then

centrifùged at 7,500g at 4OC for 5 minutes, The supernatant was removed and the RNA

pellet was left for 10 minutes to air dry. The pellet was then redissolved in 1 5 ~ 1 DEPC-

treated water containhg lp1 of DNase I (Phannacia) and incubated at 37°C for 15

minutes. The DNase 1 enzyme was then inactivated by incubating the samples at 55°C

for 1 0 minutes.

To detemine the relative purity and total RNA concentration, a Beckman Du-7

Spectrophotometer was used to measure the opticd demity at 260 and 280 nm. To

determine the integrity of the RNA, via the 285 and 18s band assessrnent test, 1-2 pg

total RNA was electrophoresed for 1 hour at 90 volts through a 1% agarose (GibcoBRL)

gel with 0.01% ethydium bromide. The 28s and 18s bands were visuaiized using a UV

transilluminator. The RNA samples were stored in a concentrated form at -70°C.

For DNA extraction, the remaining upper aqueous phase, which was used for

RNA extraction, was completely removed fiom the eppendorf tube. To precipitate the

DNA, 100% ethanol (0.3 ml 100% ETOH / ml TRIzol reagent used for initiai

homogenization) was added to the eppendorf tube. The samples were then mixed by

inversion and incubated at room temperature for 2-3 minutes. The samples were then

centrifuged at 2,000g for 5 minutes at 4OC. The phenol-ethanol supernatant was removed

and the DNA pellet was then washed twice with sodium citrate solution (1 ml 0.1M

sodium citrate in 10% ETOH / ml of TRIzol reagent used for initiai homogenization) at

room temperature for 30 minutes. Foliowing centrifugation (2,000g for 5 minutes at

4"C), the DNA was suspended in 75% ethanol in double distilled water (1.5-21111 75%

ethanol / ml TRIzol reagent used for initial homogenization) for 10-20 minutes at room

temperature. The samples were once again centrifuged at 2,000g for 5 minutes at 4OC.

The supematant was removed nom the eppendorf tube and DNA pellet was

briefly dned for 5 - 10 minutes. To redissolve the DNA pellet, it was passed through a

pipette with an adequate amount of 8mM sodium hydroxide (0.3-0.61111 8mM NaOH I ml

TFüzoI reagent used for initial homogenization). The DNA preparation was then

centnfuged at 12,000g for 10 minutes to separate m y gel-Iike material fkom the DNA in

solution. The supematant was then transferred to a fresh tube and the optical density at

260 and 280 n m was measured using a Beckman Du-7 Spectrophotometer.

Altemate Method of DNA Extraction

The TiUzol DNA extraction method outlined above was only used when both

DNA and RNA was needed fiom a particula. üver tissue sample. For DNA extraction in

al1 other instances, a method based on the Stratagene DNA Extraction Kit was used.

Approximately, 200mg human liver tissue was cut out and weighed. Using a

Brinkman Polytron homogenïzer, the samples were homogenized for 30 seconds in 12ml

of a solution containhg 0.32M sucrose, lOmM Tris-HCL (pH 7.5), 1% Triton X-100,

5mM MgC12, 0.02% sodium azide. To degrade proteins, the homogenate was incubated

with lmg/mi pronase for 3 hours at 55°C in a shaking water bath and then chilled on ice

for 10 minutes. Following the addition of 6M NaCl (Sml), the solution was mixed by

inverting the eppendorf tube several times and then incubated on ice for 5 minutes. Next,

the samples were centrifuged at 12,000g in a Sorvd RC2-B centifbge for 15 minutes at

4°C. The supernatant, once transferred to a sterile Falcon 50m.l conical tube, was

incubated with 20pg/ml RNase (Gibco/BRL) for 15 minutes at 37°C. The samples were

treated with saturated phenol (10ml) and then centrifuged at 12,000g for 5 minutes at

4°C. The aqueous upper layer, which contained the DNA, was transferred to a fiesh tube.

The DNA was precipitated with the addition of 2 volumes of 100% ethanol. The samples

were centrifbged at 12,000g for 10 minutes at 4°C and the supernatant was then

discarded. The DNA pellet was washed with Iml 70% ice-cold ethanol. Mer

centrifugation (12,000g for 5 minutes at 4OC), the overlying ethanol was discarded and

the DNA pellet was air-dried for 10 minutes. The DNA was resuspended with 150-300pl

TE-8 (1Orn.M Tris, O.lmM EDTA, pH 8) and stored at 4°C.

Reverse Transcription of mRNA

The reverse transcription of mRNA to cDNA nom L-senes liver was achieved by

using M-MLV reverse transcriptase (GibcdBRL). For each sample, total RNA (lpg)

was diluted in 0.1% DEPC-treated water for a nnal volume of 7.5~1 and heated at 95°C

for 5 minutes to denature the RNA. The samples were then cooled on ice and 12.5p1 of

the reverse transcription master mix was added for a total volume of 20~1. Each reverse

transcription sample contained 1 pg total RNAy 1 X reaction buEer (50mM Tris-HCl (pH

8.3), 75mM KCI, 3mM MgC12), 20mM D m y 1mM of each dNTP (GibcoiBRL), 20units

RNA Guard @Nase inhibitor; GibcolBRL), 140pmol pd@& primer (Phannacia

Biotech), and 200units M-MLV reverse transcriptase. The samples were incubated at

room temperature for 10 miniutes and then incubated at 37°C for 1 hour. The cDNA

samples were then stored at -20°C until needed,

PCR Amplification of P-Actin cDNA

A 450bp fiagrnent of the B-Actin cDNA was amplified f?om L-Series livers using

a forward primer (SXTACAATGAGCTGCGTGTGG-3') and a reverse primer (5'-

TAGCTCTTCTCCA

GGGAGGA-3'). The PCR reaction mixture (50~1) of each sample was as follows;

0SpM of each oligonucleotide, 1X reaction b e e r (20mM Tris-HC1 (pH 8.4), 50mM

KCl), 100pM of each dNTP, 1SmM MgCli, 2 .5~1 of reverse transcriptase generated

cDNA, and 2.5units of Taq DNA polymerase (hot start; Gibco/BRL). The PCR

conditions were as follows; initial denaturation of 95°C for 5 minutes, 25 cycles of 20

seconds denaturation at 94OCY 20 seconds annealing at 5S0CY and 40 seconds extension at

72"C, followed by an additional 7-minute extension at 72°C. This reaction was

performed in a Gene Amp PCR Syaem 2400 Thermal Cycler (Perkin-Elmer). The

amplification product (10~1) was loaded onto a 1% agarose gel (0.01% ethydium

bromide), electrophoresed for 1 hour at 90 volts and then visualized with a W

transiiluminator-

PCR Amplification of CYP3A4 3'UTR (cDNA and genomic DNA)

The 3'untranslated region of CYP3A4 cDNA was amplified using the following

fonvard primer (5 ' -GTGAAAGTTAATCC ACTGTG-3') and reverse primer (5 ' -GAGCT

TCAGTTCTTTGTTAC-3') producing a 402bp fragment The PCR mixture and

conditions were exactly as those presented for the amplification of B-Actin.

The region on the C P 3 A 4 gene that corresponds to its 3'untransIated region was

also amplified, however, the foilowing forward primer (5'-AGGTTGAGTCAAGGG

ATGGC-3') and reverse primer (5'-GAGCTTCAGTTCTTTGTTAC-3') produced an

1159bp hgment. The PCR reaction mixture (50~1) of each sample was; 0.5pM of each

primer, 100pM of each dNTP, 1.5mM MgCh, -200ng of genomic DNA, and 2.5units of

Taq DNA polymerase (hot start; GibcoIBRL) in 1X reaction buffer. The PCR conditions

were an initial denaturation of 95°C for 5 minutes, 25 cycles of 45 seconds denaturation

at 94OC, 30 seconds annealhg at 60°C, and 1 minute and 30 seconds extension at 72"C,

followed by an additional 15-minute extension at 72OC.

PCR Ampiifkation of S'promoter region of CW3A4

The S'promoter region of CYP3A4 was arnplined using the forward primer (5'-

ATTGCTGGCTGAGGTGGTTG-3') and reverse primer (5'-TAGAGGAGCACCAGG

CTGAC-3') producing a 1253bp h g m e n t The PCR reaction mixture was exactly the

same as that for the region of the CYP3A4 gene that corresponds to its 3'untranslated

region descnbed above. There was, however, a slight change in the PCR conditions;

initial denaturation of 95°C for 5 minutes, 30 cycles of 45 seconds denaturation at 94OC,

30 seconds annealhg at 6S°C, and 1 minute and 30 seconds extension at 72OC, followed

by an additional 10-minute extension at 72°C. PCR-generated hgments fiom the

S'region of CYP3A4 and nom the region of CYP3A4 that corresponds to its

3'untranslated region were prepared for automated sequencing as described in the

following sections.

Extraction and Purification of PCR Products From Agarose Gels

DNA was extracted fiom agarose gels and purified according to the Qiagen

QIAquick Gel Extraction Kit Protocol. Amplified DNA was loaded (30pVwell) onto a

1% agarose gel (0.01% ethydium bromide) and electrophoresed for 1 hour at 90 volts.

The band of interest was excised fiom the agarose gel with a clean, sharp scaipel. The

gel slice was added to an empty eppendod tube and weighed. Three volumes of B&er

QXl were added for every 1 volume of gel slice. M e r a 10-minute incubation at 50°C,

the samples were treated with isopropyl alcohol(1 gel volume) and then vortexed.

The samples were added to a QIAquick spin column, which was inserted into a

2ml collection tube, and centrifuged at 10,000g for 1 minute. Once the flow-through was

discarded, one gel volume of Buffer QX1 was added to the column and centrifuged for 1

minute at 10,000g. The QIAqukk column was placed into a clean eppendod tube. To

dilute the DNA, 30-60~1 of lOmM Tris-HCl @H 8.5) was added to the column. M e r a

1 -minute incubation at roorn temperature, the samples were centrifuged for 1 minute at

10,000g. The purified DNA was stored at -20°C.

Su bcloning and Transformation

Subcloning and transformation was performed via the TOPO-Cloning protocol.

To generate plasmid DNA for the S'regulatory region and 3'UTR of CYP3A4, the PCR

amplified product that were gel purified were inserted into the plasmid vector pCR2.1-

TOPO (Invitrogen).

The gel extracted PCR product (4~1) was added to an empty eppendorf tube.

Following the addition of 1 pl of pCR.2. LTOPO plasmid, the solution was gently stirred

with a pipette tip and then incubated for 5 minutes ot room temperature. The samples

were then bnefly centrifûged and placed on ice.

"One Shot Competent Cells" were thawed on ice. 0.5M B-mercaptoethanot (2 pi)

was added to the competent cells by gentle stimng. Following the addition of the cloning

reaction (2~1) to the competent celis, the samples were then hcubated for 30 minutes at

4°C. The samples were then heat-shocked for 30 seconds at 42°C md then immediately

incubated for 2 minutes at 4°C. Room temperature SOC media (250~1) was added to the

samples. The samples were then shaken at 220rpm for 30 minutes at 37°C in a G24

Environmental Incubator Shaker (New Brunswick Scientific Co. Inc.) and then placed on

ice.

Luria-Bertani (LB) agar plates (1 .O% Tryptone, 0.5% Yeast Extract, 1.0% NaCl,

pH 7, 15g/L agar, 50pg/ml ampicillin) were prewarmed for 20 minutes at 37°C. The

plates were then treated with X-gal(40pl of 40mg/ml stock; Gibco/BRL) and let dry for

15 minutes at 37OC. For each sample, 50p1 and 1 5 0 ~ 1 of transformation reaction was

spread on the LB agar plates and then incubated ovemight at 37°C. The following day,

18 white colonies were picked. These colonies were streaked on LB plates and cultured

ovemight in LB medium (3rd) in a shaking incubator (220rpm, 37°C)-

Prepawtion of Plasmid DNA

The protocol for the isolation of plasmid DNA was taken fiom Sambrook et al

(1 390). Following the ovemight incubation in LB medium, 1 -5 ml of the bacterial colony

was transfened to an eppendorf tube and centrifuged at 12,0000 for 1 minute in a Biohige

A centrifuge (Caniab). The medium overlying the bacterial pellet was removed by

aspiration. To resuspend the bacterial pellet, 100p1 of ice-cold Solution 1 (5Om.M

glucose, 25mM Tris-Cl, lOmM EDTA, pH 8.0) was added and the samples were

vortexed vigorously. Next, Solution II (200~1; 0.2N NaOH, 1% SDS) was added to the

bacterial cultures and then mixed by rapidly inverting the tubes five times. The cultures

were then treated with 150pt of ice-cold Solution XII (5M acetate, 3M potassium),

vortexed for 10seconds and then stored on ice for 5 minutes. Foliowing a centrifugation

at 12,000g for 5 minutes at 4"C, the supernatant was transferred to a fiesh tube. The

samples were then treated at room temperature with 2 volumes of ethanol, to precipitate

the plasmid DNA, and vortexed briefly. After a centrifûgation at 12,000g for 5 minutes

at 4OC, the supematant was aspirated away. Following the addition of 1 ml of ice-cold

70% ethanol, the tube was again centrifuged at 12,000g for 5 minutes at 4°C. The

supematant was aspirated and the DNA pellet was lefi to dry for 10 minutes. The DNA

was dissolved in 50p1 TE-8 (1Om.M Tris, O.lmM EDTA, pH 8) containing 20pg/ml

RNase (Gibco/BRL).

Restriction Analysis

Restriction analysis of plasmid DNA was performed to confirm the insertion of

the DNA of interest. The pCR2.1-TOPO plasmid contains an EcoRJ site flanking each

side of the multiple clonhg site. The restriction reaction (1 Opl), which contained Zunits

of EcoRl (GibcolBRL), 1X REact 3 Buffer, and Spi1 of isolated plasmid DNA, was

incubated for 1 hour at 37°C. The restriction product was loaded onto a 1% agarose gel

(0.0 1% ethydium bromide) and electrophoresed for 1 hour at 90 volts.

The plasmid DNA, once quantified by measuring the optical density at 260 and

280 nm, was either used as a control for AUele Specific Amplification reactions (PCR

fragment fiom S'region; see next section) or sent off for automated sequencing.

Allele Specific Amplification

In allele specific amplification, separate PCR reactions are required to amplify the

variant or normal allele. The assay described here requires two forward primers, one

each for the variant and normal alleles, and a common reverse primer (Figure 4). The

forward wildtype CYP3A4 primer (5'-AGCCATAGAGACAAGGGCAA-3') and the

forward variant CYP3A4 primer (5'-AGCCATAGAGACAAGGGCAG-3 ') differed by

only a single nucleotide at the 3 k n d of the primers. This change corresponds to the -

290A>G mutation in the NFSE domain of the 5'-regdatory region of CYP3A4 (Rebbeck

et al., 1998). The reverse CYP3A4 primer (5'-TAGAGGAGCACCAGGCTGAC-3')

resides in the coding region of this gene.

ASA Wildtype Reaction

Double Stranded Genomic DNA

+ CYP3A4

Wildtype Forward P)

CYP3A4 Reverse

&Adin Forward -

Genotype Homozygous Wildtype

ASA Variant Reaction

Double Stranded Genornic DNA

+ CYP3A4

Variant Forward

CYP3A4 Reverse

1 PCR +

Gel Electro phoresis

Heterozygous Honlozygous Variant

W = PCR amplified products from wildtype reaction V = PCR amplifieci products from variant readion

Figure 4. Schematic of allele specific amplification of genomic DNA using CYP3A4 *IA and CYP3A4 *I B specific primers. Adapted fiom Kard (1 999).

To ensure the absence of false negatives, each reaction contained primers for a

gene that wodd be readily amplified. In this case, we chose the P-Actin gene. The

forward primer (5'-CCCAGCCATGTACGTTGCTA-3') and the reverse primer (5'-

AGGGAGGAGCTGGAAGCAG-3') were added to d l genotype reactions. The presence

of the P-Actin band was an indicator of successfid PCR amplification. Therefore, for

every sample, two PCR reactions were m. One reaction selectively, amplified the

wildtype allele and the other amplified the variant allele. A~so, ai l genotype assays

contained the P-Actin primers (Figure 4).

The ASA wildtype reaction mixture (50~1) contained 1X reaction buffer (20mM

Tris-HCI (pH 8.4), S0mM KCI), 0SpM of the forward wildtype CYP3A4 primer, OSpM

of the reverse CYP3A4 primer, OSpM of each of the f3-Actin primers, 100pM of each

dNTP, 1.5m.M MgC12, 200ng-lpg of genomic DNA and 2-5 units of Platinum Taq DNA

Polymerase (Gibco/BRL). For the ASA variant reaction, the same ingredients listed

above were added except that the forward wildtype CYP3A4 primer was substituted with

the forward variant CYP3AI primer (0.5 PM).

The PCR conditions for this ASA assay were as follows; initial denaturation of

95°C for 5 minutes, 25 cycles of 30 seconds denaturation at 94"C, 30 seconds annealing

at 67OC, and 30 seconds extension at 72"C, foilowed by an additional 7-minute extension

at 72OC. This reaction was performed in a Gene Amp PCR System 2400 Thermal Cycler

(Perkin-Eher). The amplification product (20~1) was loaded ont0 a 2% agarose gel

(0.01% ethydium bromide), electrophoresed for 1 hou. at 90 volts and then visualized

with a W transilluminator. For every set of samples, wildtype and variant positive

controls were run dong with a negative control, which contained al1 reaction components

except for the genomic DNA sample.

Data Analysis

Statisticd analyses were performed using the cornputer program, Statistica The

correlations between immunoreactive protein, testosterone 69-hydroxylation, V,,, I&,,

and CYP3A4 mRNA were calculated via Speamian rank order tests. Chi square analyses

were perfomed to compare CYP3AI allele fkquencies between populations.

Results

Immuno blot Analysis

Microsornes from selected livers were used to detemiine the optimal loading

amount of microsomal proteins for the detection of immunoreactive CYP3A4. Resuits

fkom L24, a microsomal sample with intermediate amount of CYP3A4 protein, are

presented in Figure 5. A linear relationship exists between 2.5 - 30pg of microsomal

protein loaded and optical density of the CYP3A4 protein band (r=0,98, p-0.004). Thus,

immunoblot experiments were perfonned using 1Opg of microsornd protein per sample.

The amount of immunoreactive CYP3A4 was detennined for forty-eight L-series

liver microsomes. CYP3A4 was present in al1 liver microsornes and the level of this

protein varied approximately 60 foid (Table 3). Probit (Bliss, 1934) and NTV (Endrenyi

and Patel, 199 1) analyses revealed a non-normal distribution with 38% of the population

below an apparent antirnode of 2lpmol CYP3A4 / mg microsomal protein (Figure 6).

Correla tion Analyses

We have previously tested our L-series human liver samples for testosterone 6p-

hydroxylation activity and mRNA content (detennined via RT-PCR; Sy, 1998). We

found a 60-fold variation in testosterone 6P-hydroxylation for 48 L-series livers (Figure

7). The V,, and K,,, for these liver samples varied 100- and 3-fold, respectively. Sy

(1998) estimated the V,, and Km values by extrapolation fiom Eadie-Hofstee Plots

based on observations with two testosterone concentrations (25 and 100CiM). CYP3A4

mRNA levels were also measured for 20 L-Series livers. There was a 80-fold variation in

the amount of mRNA.

Figure 5. hunoblotted CYP3A4 Standard Curve. Immunoblotted microsornai protein (0.5-40pg) fiom L24, a microsornai sample with intemediate amount of CYP3A4 protein. A linear relationship exists between microsoma1 protein loaded (2.5-30pg) and optical density of immunoreactive CYP3A4 (-98, p<0.005)

Table 3. Immunoreactive CYP3 A4 levels of L-series livers.

log pmoVmg Immunoreactive CY P3A4

Cumulative Frequency

Figure 6. Probit (A) and NTV (B) Plots of immunoreactive CYP3A4. Probit and NTV analysis revealed a non-nonnal distribution with 38% of the population below an apparent antimode of 2 1 pmol CYP3A4 / mg microsomal protein.

log Testosterone @-Hydmnylation

Figure 7. Probit Plots of testosterone 6B-hydroxylation (initial testosterone was 100 PM; A) and V,, (B). Low (L25, L37 & L70), intemediate (L23, L20) and high (L27, L21) activity livers are highlighted (Sy, 1998). V, was estimated by extrapolation based on observations with two testosterone concentrations (25 and 100pM; Sy, 1998)

We performed multiple correlation analyses between testosterone 6P-

hydroxylation, V,, Km, immunoreactive CYP3A4 and CYP3A4 mRNA (Table 4).

Immunoreactive CYP3A4 and testosterone 6P-hydroxylation signifïcantly correlated

(rs=O. 89, p<O.00 1 ; Figure 8A). V,, and immunoreactive CYP3A4 also significantly

correlated (r,=0.85, @.O0 1 ; Figure SB). Tmmunoreactive CYP3A4, however, did not

correlate with Km. Furthermore, CYP3A4 mRNA and immunoblotted protein also

correlated (r,=0.524, p<0 -05)-

Sequence of C W A 4

To ascertain whether a mutation in CYP3A4 could account for the wide range of

variation in CYP3A4 activity, Dr. Wei Li, working as a postdoctoral fellow in Dr. Allm

Okey's laboratory, sequenced the entire coding region ( 1 5 12 base pairs) of CYP3A4. Of

the six L-series Iiver cDNAs sequenced, L8, L12, L20, L25, L37 and L70, there were no

mutations detected. Furthermore, al1 six sequences matched up with the hPCNl sequence

reported by Gonzalez et al. (1 988).

Allele Specific Amplification Assay

A mutation in the 5' regdatory region of CP3A4 has recently been described

(Rebbeck et al., 1998). This variant allele, termed CP3A4-V (CYP3A4VB) results fiom

a substitution mutation (-290A>G) in the W S E domain of CYP3A4. To determine the

fiequency of CYP3A4 *IB in the population, Rebbeck et al., deveioped a genotype assay

Testosterone 6p-OH

vmax

Km

fmmunoreactive CYP3A4

CYP3A4 mRNA

I V Immunoreactive

Table 4. Multiple correlation analysis between testosterone 6f3-hydroxylation, V,,, K,,,, irnrnunoreactive CYP3A4 and CYP3A4 niRNA (determined via RT-PCR). *** p<0.001, p<o.05

0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0

Immunonrctive CW3A4 (pmollmg)

0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0

Immunoreactive C W W (pmollmg)

Figure 8. Correlation analyses between immunoreactive CYP3A4 and testosterone 6P- hydroxylation (A) and V,, (B). Both V,, and testosterone 6P-hydroxylation significantly correleated with immunoreactive CYP3A4 (P<0.00 1).

based on conformation sensitive gel electrophoresis. They reported a C P 3 A 4 * i B ailele

fiequency of 9.6% in the adult Caucasian male population.

in an attempt to verfi the existence of this mutant allele and to genotype our liver

bank, we developed a novel assay for CKWAPl B, based on allele specific amplification.

Allele specific amplification has been widely used as a method to genotype ùidividuals

with polyrnorphisrns in not only Cytochromes P450 but in other genes as well (Blum et

al., 199 1; Daly et uL, 1994; Bottema and Sommer, 1993)- Genomic DNA fiom

homozygous wildtype, heterozygous and homozygous variant individuals were kindly

provided by Dr. Timothy Rebbeck (University of Pennsylvania School of Medicine).

These samples served as positive controls for the development of the allele specific

amplification assay.

Figure 9 illustrates the three possible genotypes. For every genomic DNA

sample, two reactions were run. The first lane for each sample represents the wildtype

reaction and the second represents the variant reaction. The lower band is a PCR-

amplified fragment of the P-Actin gene and the upper band is a PCR-amplified £kgment

of CYP3A4 The f3-Actin PCR product, which served as an intemal control, is present in

al1 samples. In the Homozygous Wildtype Control, only the wildtype reaction arnplified

a CYP3AI gene product. in the Heterozygous Control, both the wildtype and variant

reactions arnplified CYP3A4 gene products. Finally, in the Homozygous Variant Control,

only the variant reaction arnplified a CYP3A4 gene product. These allele specific

amplification reactions were both specific and reproducible. Figure 10 represents the

sequence of a selected homozygous variant individual. The A + G substitution mutation

is highlighted.

Figure 9. AUele specific amplification of CYPilA4. Each sample is amplified with the wildtype and variant CYP3A4*IB primers in two separate reactions. The top band is a PCR-generated fiagrnent of CYP3A4 and the lower is a PCR-generated hgment of p- Actin (internai control). The wildtype, heterozygous and variant genotypes are highlighted.

590 600 610 62 0 63 O 64 O 650

, T A A A G A G G A A A G A G G A C A A T A G G A T T G C A T G A C G G G G A T G G A A A G T G C C C A G C G G A G G A A X T G G '

-- - -

700 7 1 0 7 20 730 74 0 750

, G A A G G CTCTG T C T G T C T G G G T T T G G A A G G A T G T G T A C G A G T C T T C T A G G G G G C X C A G G C A C A C T

Figure 10. Sequeme of CP3A4*1B. The guanine corresponding to the A + G mutation in NFSE is highlighted.

Genotype Frequency Estimation

To detennine the allele fiequency of CYP3A4*1B, we genotyped 1 16 individuals

using the allele specifïc amplification assay. The genomic DNA samples used hem were

kindly provided by Dr. D. Grant (Hospital for Sick Children) and were comprised of

predominantly Caucasian individuals (Appendk 4). For every set of samples that were

genotyped, a positive wildtype control, positive variant control and a negative control,

which contaiaed ai i PCR components except the genomic DNA, were amplified and

electrophoresed.

Of the 1 1 6 individuals, there- were 1 O3 homoygous wildtypes, 12 heterozygotes,

and 1 homozygous variant individual. Thus the allele fiequencies were 0.94 for the

wildtype allele and 0.06 for the variant allele.

When the data was reanalyzed by including only Caucasian individuals (IOO),

there were 88 homoygous wildtypes, 12 heterozygotes and O homozygous variant

individuals. The d e l e ftequencies were not changed; 0.94 for the wildtype dlele and

0.06 for the variant allele (Table 5).

When the data was M e r segregated into 51 male and 49 female Caucasian

individuals, an interesthg result was observed. For male Caucasians, there were 43

homozygous wildtypes and 8 heterozygous Uidividuals. This corresponded to an allele

fiequency of 0.92 for the wildtype allele and 0.08 for the variant allele. In the female

Caucasian group, there were 45 homozygous wildtypes and 4 heterozygous individuais.

FemaIe Caucasian individuais had an allele fiequency of 0.96 and 0.04 for the wildtype

and variant allele, respectively. Even though the CW3A1*1B allele frequency differed

Caucaalan Population (ASA test) Expected

Caucasian Fernale (ASA test) Expected

Caucasian Mali, (ASA test) Expected

Published (Caucasian Male) Expected

&

Number of lndividuals Heterozygote Variant 1 p value q value

0.06

0.01

0.08

0.1

Statistics f

Table 5. CYP3A4*I B allele frequencies. Predicted Hardy-Weinberg values for Caucasians (male andlor female) observed here and male Caucasian population fiom Rebbeck et al, 1998. Chi square analysis reveals CYP3A4*1B genotypes, fiom al1 populations above, follow Hardy- Weinberg principle.

approxirnately two fol4 a chi square adys is revealed no significant ciifference between

male and fernale Caucasians for the occurrence of the variant allele (X2 = 1.25, ~=0.26).

CIIP3Ad*IB Genotype and Testosterone 6~4Sydroxylation

Since CYP3A4*IB has k e n suggested to lead to decreased CYP3A4 mRNA

levels, we decided to investigate whether there was an association between CP3A4*IB

genotype and testosterone 6f3-hydroxylation. We genotyped samples fkom the L- and V-

series livers. Both these liver series were previously phenotyped for testosterone 6P-

hydroxylation (Sy, 1998; Voorman unpubiished results). For the L-series livers, we

genotyped three livers with low activity phenotype, two livers with intermediate activïty

phenotype and three livers with high activity phenotype as determined by Sy (1 998). The

f m MO lanes represent the CYP3AI wildtype and variant amplification reactions of the

Negative Control (Figure 11). Lanes 3 and 4 are the amplification reactions of a

Wildtype Control. Lanes 5 and 6 are the amplification reactions of a Variant Control.

The last 16 lanes are the selected L-series samples. The low (L25, L37, L70),

intermediate (L20, L23) and the high (L 12, L21, L27) activity liver microsomes were al1

CYP3A4 wildtype.

Genotyping for CYP3AJ*iB was also performed on V-series livers. The

testosterone 6f3-hydroxy lation activity of these livers is presented in Appendix 3. Probit

and NTV analysis revealed a non-normal distribution with 38% of the population below

an apparent antimode of 1500 pmoVmg!min testosterone 6B-hydroxylation (Figure 12).

Sy (1998) reported a similar antimode of 40% for testosterone 6P-hydroxylation in L-

series liver microsomes.

Figure 1 1 . Phenotype-Genotyp Cornparison. Genotyping results of microsomal samples with low (L25, L37 & L70), intermediate (L20, L23) and high (L21, L27) testosterone 6P-hydroxylation. For every PCR set, negative, wildtype-positive and variant-positive controls were amplified and electrophoresed alongside DNA samples.

Log Testosterone 6P-hydroxyladion

Cumulative Frequency

Figure 12. Probit (A) and NTV (B) Plots of testosterone 6P-hydroxylation fiom V-series livers. Probit and NTV analysis reveded a n o n - n o d distribution with 38% of the population below an apparent antirnode of 1500 pmol/mg/min testosterone 6B- hydroxylation-

CYP3A4 allele fkquencies for the V-series Livers were 0.06 for the variant allele

and 0.94 for the wildtype aiiele. For male Caucasians, the frequencies were 0-07 and

0.93 for the variant and wildtype alieles, respectively. For female Caucasians the variant

and wildtype alleles were 0.04 and 0.96, respectively. These ailele fkquencies are

similar to those detennined above. Of the twenty-six V-series liver samples genotyped,

there were three hetero zygotes, one in each activity phenotype (Appendix 3). Thus, there

appears to be no correlation between testosterone 6B-hydroxylation and CYP3A4 *I B

genotype.

Sequence of the Sregion of CW3A4

To explain the low activity phenotype seen in our liver bank, we next sequenced

the S'regulatory region of CYP3AI for two livers with low testosterone GD-hydroxylation

activity (L25 & L37) and one with high activity (L21). For al1 three liver samples, we

found a common set of nucleotide ciifferences between base pairs - 726 to -716 as

compared to the sequence of the published CYP3A3 S'regulatory region (Accession

number - Dl 1131). These differences corresponded to a guanine insertion between

nucleotides -726 and -725, an adenine insertion between nucleotides -721 and -720, and

an adenine deletion at nucleotide -7 17.

Found Here -727 5'-AGGGATGACAT 4 - 3 ' -7 16 Published -726 S'-A -GGATG -CATAG-3' -7 1 6

These differences were dso seen when genomic DNA fkom a homotygous CY;PJAI*IB

individual was sequenced (data not shown).

Sequence of the SUTR of CYP3A4

To investigate whether a mutation in the 3'UTR of CYP3A4 could account for

decreased expression of this protein, we sequenced the genomic DNA that corresponded

to the 3'UTR of CYP3A4 mRNA of L25, L37 (low activity Livers) and L2 1 (hi& activity

liver). We found two sequence clifferences, which were common to al1 three samples.

These corresponded to adenine insertions between nucleotides 1997 and 1998, and

nucleotides 2067 and 2068 (numbering based on CYP3A4 sequence in Accession

Found Here 1994 5'- CTATAAG'tTTT-3' 2004 Published 1994 5'- CTAT -AGITIT-3' 2003

Found Here 2063 5'- AGGAGAAATCT-3' 2073 Published 2063 5'- AGGAG -AATCT-3' 2072

After closer inspection of the 3'UTR, we found a putative A h sequence within the

3'UTR of CYP3A4 (Figure 13). The putative Alu sequence is only present in the larger

CYP3A4 transcript. This A h sequence has approximately 78% homology with AIu-Sq

(Accession number - U14573). Also, as with most Alu sites, the Alu sequence in

CYP3A4 is approximately 50 base pairs shorter than its consensus family member, Alu-

Sq. Thus, it may no longer be able to retrotrampose into other parts of the genome.

Expression of CYP3A4 mRNA transcripts

To corroborate the finding that there are two CYP3A4 transcripts, we perfomed

RT-PCR reactions on selected L-series livers. When liver samples for our human liver

tissue bank were coilected, necessary precautions were taken to ensure enzyrnatic

activity. These precautions, however, may not have been s a c i e n t to ensure RNA

2.2 kb AAAAAA

7 4 5' UTR Coding Region 3' UTR

3.0 kb Altr site AAAAAA

Pol yadenylat ion signals

Figure 13. CYP3A4 mRNA. The two transcripts of CYP3A4 share their S'UTRs and coding regions. The 3'UTR of the two transcripts differ via the use of an alternate polyadenylation signal. The putative Alu site is only expressed in the 3.0 iranscript and shares approximately 78% homology with Ah-Sq. For, Rev are the locations of the forward and reverse primers used for the specific amplification of the 3.0kb transcript.

integrity. In addition to the 28s and 18s assessrnent test performed after the isolation of

total RNA, a 450bp segment of P-Actin cDNA, which is ubiquitously expresse& was

arnplified for each sample. This amplification was necessary to assess the success of

reverse transcription reaction and thus to ensure that the quality of the cDNA produced

was adequate for PCR amplification. For the 7 livers tested, we found similar amounts of

arnplified PCR hgments f?om B-Actin cDNA (Figure 14).

We next amplined a section of CYP3A4 cDNA that is present in only the longer

transcrïpt of this P450. Qualitatively, we were able to show the presence of this hgment

in al1 liver samples tested and thus the presence of the longer transcript of CYP3A4

(Figure 14).

Figure 14. RT-PCR amplification of P-Actin and the 3.0kb transcript of CYP3A4. Primers were designed to a m p l e only the portion of CYP3A4 mRNA specific to the 3.0kb transcript, Al1 Livers tested contained similar amounts of B-Ath mRNA. Furthemore, the 3.0kb transcript was present in al1 livers tested.

Discussion

Immunoblot Analysis

There is an approximately 60-fold interindividual variation in the amomt of

irnmunologically detectable CYP3A4 present in microsornes derived fiom our human

liver bank. As expected, CYP3A4 was present in aü livers tested. Probit and NTV

analyses of CYP3A4 revealed a non-normal distribution with approxhately 38% of the

population below an apparent antimode of Zlpmol CYP3A4 /pg rnicrosomal protein.

These results suggest that 38% of the population have decreased leveis of hepatic

CYP3A4. Sy (1998) reported that 40% of individuais in our L-series liver bank had

testosterone 6P-hydroxylation activity below an apparent antimode. Interestingly, a

Probit and NTV analysis of testosterone 6P-hydroxylation for V-series livers indicated

that 38% of the population was below an apparent antimode of 1500 pmol/mg/min

testosterone 6B-hydroxylation. Thus we believe 40% of Caucasian Uldividuals form a

somewhat distinct subpopulation whereby the expression and activity of hepatic CYP3A4

is reduced.

We perfomed multiple correlation analyses between testosterone 6P-

hydroxylation, V,, Km, immunoreactive CYP3A4, and CYP3A4 mRNA levels (Table

4). Immunoreactive CYP3A4 correlated with testosterone 6P-hydroxylation ( r4 .89 ,

p<O.00 1) and V,, (rs=0.85, p<O.001). Km, which is a measure of the buiding aflhity of

substrate to enzyme, did not correlate with any of the aforementioned parameters. Thus

we are able to conclude that the hi& variation seen in testosterone 6B-hydroxylation is

mostiy due to a high variation Ui the amount of CYP3A4 protein.

Evidence in the Literature also points to a high correlation between CYP3A4

activity and immunoreactive CYP3A4 levels. Shirnada et al. (1994) characterized 30

Caucasian and 30 Japanese livers for the expression and activity of CYPlA2, CYP2A6,

CYP2C, CYP2D6, CYP2El and CYP3A. They reported that CYP3A significantly

correlated with arylhydrocarbon hydroxylase (r=0.76), nifedipine oxidase (~0.79)' and

testosterone 6B-hydroxylase ( ~ 0 . 8 1).

The correlation between immunoreactive CYP3A4 and CYP3A4 mRNA reported

here is also statistically signifiant (rs=0.524, ~ 0 . 0 5 ) . This correlation, however, is not

as convincing as those mentioned above. There are several possible explanations for

these resuits. Firstly, mechanisms may exist whereby CYP3A4 protein levels decrease

via increased protein degradation. Grapefit juice, for example, acts by irreveaibly

binding to CYP3A4 thereby reducing the pool of active protein (Chan et al., 1998). in

fact, after the oral administration of grapenuit juice, the concentration of enterocyte

CYP3A4 protein decreased -60% while mRNA levels remained constant (Lown et al.,

1997). It is possible that similar inhibitors may act to decrease hepatic CYP3A4 protein

levels as well. Interestiingly, increased degradation of CYP3A4 was seen in a recent

study investigating the effects of alcohol on iiver P450 enzymes (Lytton et al., 1999).

These investigators report the presence of CYP3A4 autoantibodies in livers derived from

alcoholics. These antibodies may exist in 'normal' subjects and may contribute to the

variability in CYP3A4 activity-

Secondly, the mediocre correlation between immunoreactive CYP3A4 and

CYP3A4 mRNA levels may reflect an alteration in mRNA translation. Factors in the

3'UTR of mRNA transcripts may decrease translation and thus result in situations where

mRNA levels are normal but protein levels are reduced As an example, an A h element

in the 3'UTR of P53 has been irnplicated as a cis-acting repressor of translation (Fu et al.,

1996). We have identifled an A h element in the 3'UTR of CYP3A4 that may also affect

CYP3 A4 translation.

Lady , the correlation between immunoreactive CYP3A4 and CYP3A4 mRNA

levels may imply a defect in the normal transcription of CYP3A4. The S'regulatory

region of CYP3A4 has been reported and putative regulatory domains have ken

identified (Hashimoto et al., 1993). We decided to pursue this avenue by investigating

the effects of CYP3APIB on testosterone 6B-hydroxylation.

AUele Specific Amplification

A mu-tation in the 5' regulatory region of CYP3A4, CYP3AI*IB, has recently

been described by Rebbeck et al. (1998). This allele resuits fiom an A + G substitution

in NFSE, a putative regulatory element Ushg CSGE, these investigators determined a

9.6% allele fiequency in male Caucasians. in another study nom that laboratory, Waker

et al. (1998) determined the ethnic distribution of CYP3A4VB in e c a n Americans

(53%) and Taiwanese (0%).

To veriQ the presence of CYPilAPIB and to determiae its contribution to the

variation seen in our liver bank, we developed a novel, simple and reproducible genotype

assay based on allele specific amplification. Primers for CYP3Al f lA and CYP3A1*IB

bind to NFSE and selectively ampl* their respective alieles. Furthemore, CYP3AS and

CYP3A7 do not contain NFSE and thus the allele specific primers are CYP3A isoform

specific as well. This assay requires that each sample DNA be arnplified in two

independent reactions. The £kst reaction specif?cally amplifies the wildtype d e l e while

the second amplifies the variant ailele. Prirners for f5-Actin, a ubiquitous gene, are

included in each reaction to ensure technical success of the assay. ASA has been

successfdly used to determine the genotype of various mutations in other drug

metabolizing enzymes such as N-acetyltransferase 2, CYP 1Al and CYP2D6 (Blum et al.,

1 99 1 ; Daly et al., 1994).

Genotype Frequency Estimation

We report here a CW3AI*IB allele fiequency of 6% for 100 unrelated Caucasian

individuals. The allele fkquency for male Caucasians is 896, while that for femde

Caucasians is 4%. A chi square analysis revealed no significant difference between

males and females for CYP3AJ*IB (X2=1.25, p=0.26). These allele fiequencies were

lower than those previously reported for male Caucasian individuals (9.6%) and for

Caucasians (9%) in general (Rebbeck et al., 1998; Waker et al., 1998).

We believe the CGSE assay developed by Rebbeck et al. (1998) may

overestimate the number of homozygous variant individuals and thus inflate the allele

fiequency of CYP3AI*IB. We present two arguments to support this statement. Firstly,

in an atternpt to determine whether CYP3APIB is fixed in the genome (T'able 5), we

performed Hardy-Weinberg caicuiations not only on our data but also on the data by

Rebbeck et al. (1998). For Caucasian males andlor females, the number of homozygous

wildtype, heterozygous, and homozygous variant individuals determined by our ASA

assay were not statistically different fiom expected values determined by the Hardy-

Weinberg Equation (Table 5). When we perforrned similar cdculations on the data by

Rebbeck et al. (1 W8), a trend away k m the Hardy-Weinberg Rule was observed. The

number of heteroygotes appeared to be under-represented while the nurnber of

homozygous variant individuais appeared to be dramaticaily over-represented (3-fold).

A second, more signifïcant explmation is presented in a very recent study

investigating the effects of CYP3A4*lB (Westlind et al., 1999). By direct sequencing of

the S'regulatory region for a limited number of çamples (39 individuals), these

investigators found only three heterozygotes. This resulted in an d e l e fiequency of

3.8%. It is important to note that these authors did not detemiine the genotype of al1

samples in their liver bank, as this limited sample size may have Sected their allele

fiequency. Also, the ethnic ongins of the liver samples were not specined.

Thus, when comparing the aliele frequencies detennined in three independent

laboratories, it appears our allele fiequency estimation may more adequately represent the

hue CYP3AI*ZB allele fiequency in the Caucasian population.

CYP3A4*IB Genotype and Testosterone 6p-Hydroarylation

Since CYP3A4*iB is suspected to alter the binding of NFSE transcriptional

elements and thus produce decreased CYP3A4 protein levels, we decided to genotype our

liver bank to determine whether CYP3A4*lB contributes to our low activïty

(testosterone-6f3hydroxylation) livers. We tested 3 Livers with low activity (L25, L37,

L70), 2 livers with intermediate activity (L20, L23) and 3 livers with high activity (L12,

L2 1, L27) and found no variant alleles in these samples (Figure 1 1). Thus, CYP3APIB

does not contribute to the decreased testosterone 6f3-hydroxylation activity obsemed in

L25, L37 and L70.

A genotype-phenotype cornparison was also performed on V-series livers to

detennine whether there was an association between CYP3A4*iB genotype and

testosterone 6P-hydroxylation. The ailele frequencies for CYP3A4*IB in V-senes livers

was similar to that reported for the 100 Caucasian individuals genotyped via our ASA

assay. For V-series livers (26 samples), low, intermediate and hi& testosterone 6P-

hydroxylation activity pbenotypes each contained only one heterozygote. Thus, with this

smaii data s e t we are unable to provide an association between CYP3A4*IS genotype

and testosterone 6p-hydroxylation-

In accordance with our study, Westlind et al. (1999) were also unabte to show an

association between CYP3APIB genotype and testosterone 6B-hydroxylation. These

investigators perf'ormed gel shift assays to determine the importance of NFSE. Via

cornpetition experiments, they were unable to £ind any nuclear proteins that specifically

bound to NFSE. Thus both our data and that of Westiind suggest that CYP3A4*1B does

not significantly affect CYP3A4 expression.

Sequence of Non-Coding Regions

A modest correlation between CYP3A4 protein and mRNA levels suggested a

possible variation of CYP3A4 transcription. Since we found no mutations in the coding

region of CYP3A4 for L25 a d L37 and the CYP3A4 * l B allele was not present in these

liven, we next decided to sequence the 5' and 3' regions of this gene. We found three

comrnon sequence differences in the 5' region and two common sequence differences in

the 3' region of CYP3A4 for L25, L37 and L21. We suspect that these differences

represent sequence errors when the 5' and 3' regions of CYP3A4 were orighally reported

by Hashimoto et al. (1993) and S p m et al., (1989), respectively.

Conclusion

In summary, we were able to demonstrate that CYP3A4 protein levels are non-

norrnally distributed. Furthennore, approximately 40% of individuals had decreased

hepatic CYP3A4 protein levels fomllog a subpopulation. A similar subpopulation was

seen in testosterone 6P-hydroxylation activity for both the L-series (Sy, 1998) and V-

senes livers. Thus, we were able to relate the high variation seen in testosterone 6P-

hydroxylation to variable CYP3A4 protein levels.

A genotype assay, able to distinguish between C Y P 3 A P I A and C Y P 3 A P I B

alleles, was created. The genotype fiequency of C Y P 3 A P I B for the Caucasian

population is 6%. Lady, we were able to provide evidence that CYP3A4VB may not

influence CYP3A4 activity as has been suggested by Rebbeck et al. (1998).

Future Studies

CYP3A4 mRNA exists as a 2.2kb transcript and a 3.0kb transcript (Bork et al.,

1989). The 2.2kb transcript is expressed approximately 10-fold higher than the 3.0kb

transcript. By analyzing the 3'UTR of CYP3A4, we found an Alu element present oniy

in the 3.0kb transcript This Alu element shares 78% sequence homology with AluSq.

Fu et al. (1996) reported that an Alu element was able to repress translation in viîro. A

possible friture study would be to determine whether the A h element in CYP3A4 has any

effect on translation. Thus, individds with over-expressed 3.0kb transcript may have

decreased CYP3A4 protein levels.

Recently, an orphan nuclear receptor (hPXR) capable of activating CYP3A4 has

been described (Lehmann et al., 1998). Another possible study would be to perform

immunoblot experiments on our iiver bank to detemiine whether levels of hPXR protein

are associated with CYP3A4 levels.

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Appendices

O 1 O0 200 300

Aryl Hydrocarbon Hydroxylase (pmollmglmin)

Appendix 1. Correlation analysis between testosterone 6P-hydroxylation and aryl hydrocarbon hydroxylase. Testosterone 6P-hydroxylation and aryl hydrocarbon hydroxylase significantly correlated (r,=û.87, p<0.001).

Appendix 2- Characteristics of L-series livers. nd = not detennined

79

number 24 36 29 34 38 30 9 15 22 17 16 26 35 19 14 27 13 11 10 12 37 40 18 20 5

23

Asthrna

Parkinson's Alzheimer's A& Schlr

Hyp.T. Thromb. C.

Peptic Ulcer CP, CF

Hyp. T.

Seizures Arth.

9rthr.Hyp.T.

R.Arthr.

Genotype' HL Sex Age 1 Diseaw

N

Former

N N ? N N

N N Y N ? ? N

Y N Y N N

N

Social

N N N N N

Y N N N ? ? N

Social Y N N Y

Smoker

Appendix 3. Characteristics and CYP3A4 *I l? genotype of 26 Caucasian V-senes livers. (Unpublished data fiom Dr. Vooman at Pharmacia & Upjohn) * Testosterone 6B-hydroxylation (pmoVmg/min)

Alcohol CYP3A4*

Our Label 1 2 3 7 9 10 11 12 13 14 15 16 17 18 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 55 56 57 58

Original Label 001VC 003CB 004BP O 1 3AR O29N J 034AG 039TW 040SS 042DS 043PV 044YA 045SD 046TN 047MC 049ED 052MS 053lR 054SV 055SJ 056RF 057AP 058KM 059MH 061 DM 062KA 0630s 064LC 065BC 066ST 067lT 068SH 070BT 071BW 072MM 073JK 074CB 075JL 076DM 077RS 082RW 083CD 084LP 0851 L

086MS 088MH 089AZ 090PL 091PW 097CC 099AK 1 OOLS 102AB

Race C C C C M C C C C I C C O C C C C

OC C C O C C C C C C C C C C I C C C C C C C C C C O C C C C C O C O I

sex F F M F F F F F M F F M F F M F F F F F F M M F F F M M M M F F F F F M M M M F M F F M F M F F F F F F

81

Variant

Our Label 59 60 62 63 64 65 66 67 68 69 70 71 72 73 75 76 77 79 80 8 1 82 83 85 86 87 88 89 90 91 94 95 96 97 98 1 00 101 102 103 104 105 lo6 I07 lo8 109 110 111 112 113 114 115 116 117 118 119

Race C C C C C C C C C 0 I C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C 0 C C C C C C C C C C C C

I CYP3A4 Genotype 1 I sex

M F F F M M F M F F F M M M F M F M F M M M F M M M M F M F F F M M M M M M M M F M M F F M F M M F M M M M

82

I Variant

121 1 22 123 124 126 127 128 130 131

Total

Appendix 4. Characteristics of Human DNA sampIes and CiT3A4*IB genotype. Genomic DNA was kindly provided by Dr. Grant. B = Black, C = Caucasian, 1 = Indian, M = Mixed, O = Oriental

184HS 186DQ 190LM 191NR 1921L 193TA 1 94TG 198RG 201 JH

116

Our Label 120

CYP3A4 Gonotype Original Label

183SS Race

B C C C O O C C C C

Variant V

Wildtype Hetomzygous Sex F F F F M F F F M F

Aga 30

1 03

20 22 24 22 33 26 21 35 28

12

W W W W W W

W 1

H

H

AHH pmoVmgîmin

122.8 9 3 15.8 10.4 32.2 5 1.2 14.9 22.4 20.4 106.3 18.0 22.9 59.4 43 -3 68.7 47.4 49.5 44.7 284.6 46.7 40.6 83 .O 30.9 28.2 73 -3 20.4 40.4 52.1 159.0 74.7 37.2 23.1 2 1.2 78.7 1.2

58.6

1 251.7 L-series livers (Sy

V m a pmoumg

lmin 181 0.7 47.1 699.5 39.7

71 3.7

580.2 1489.7 31 0.2 155.5 295.1 137.2 386.7 91 9.2 181.3 550.2 3605.4 1281 -8 1 89.7 965.0 59.7 821.1 2442.7 142.3 431 .O 740.8 1404.9 1 124.7 320.5 238.1 154.0 548.5 35.4 104.5 41 6.9 21 2.6 499.3 640.9 167.8 730.5 606.8 61.6 234.5 167.0 331 .O 514.3

akuijic, AHH = arylhydrocarbon hydroxy lase.

84

@ Department of Pharmacology

M Irri.olJ University of Toronto Allan B. Okey, P~.D., Professor and Chair

June 24,1999

Dr. AIlan Okey Professor and Chair Department of Pharmacology University of Toronto

Dear Dr, Okey,

1 am completing a Master of Science thesis at the University of Toronto entitled '?nvestigation of the intenndividual variability in hepatic Cytochrome P450 CYP3A4: Contribution of CYP3A4*IB". 1 would like permission to allow inclusion of the following matenal in the thesis and permission for the National Library to make use of the thesis (Le., to reproduce, loan, distribute, or seU copies of the thesis by any means and in any fom or format).

These rights will in no way restrict republication of the material in any other fom by you or by othen authorized by you.

The excerpts to be reprinted are: The section entitled "Sequence of CYP3AQ" in the Results section. Paragraph 3 of "Imunoblot Anaiysis" section in "Materials and Methods"

If these arrangements meet with your approval, please sign this letter where indicated below. Please note that this letter will be included at the end of the thesis. T h d you for your assistance in this matter.

Yours sincerely, t

/ - C - tonio Ciaccia

PERMISSION GRANTED FOR THE USE REQUESTED ABOVE:

au Print Name Date

Medical Sciences Building, Toronto, Canada M5S 1 A8 Telephone: (41 6) 978-2728 Fax: (41 6) 978-6395

Department of Pharmacology

University of Toronto Allan B. Okey, P~.D,, Professor and Chair

June 24, 1999

Dr. Wei Li Research Associate Department of Phannacotogy University of Toronto

Dear Dr. Li,

I am completing a Master of Science thesis at the University of Toronto entitled Ynvestigation of the interindividual vhability in hepatic Cytochrome P450 CYP3A4: Contribution of CYP3APIB". 1 wodd like permission to allow inclusion of the fol Iowing matenal in the thesis and permission for the National Library to make use of the thesis (Le., to reproduce, loan, distribute, or seli copies of the thesis by any meam and in any form or format).

These rights will in no way restrict republication of the material in any other fom by you or by others authorized by you.

The excerpts to be reprinted are: * The section entitled "Sequence of CYP3AJ" in the Results section. 0 Paragraph 3 of "Immunoblot Analysis" section in "Materials and Methods"

If these arrangements meet with your approvai, please sign this letter where indicated below. Please note that this Ietter wil1 be included at the end of the thesis. Thank you for your assistance in this matter.

Yours sincerely,

1-G- Antonio Ciaccia

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Signature Print Name Date

Medical Sciences Building, Toronto, Canada M5S 1 A8 Telephone: (41 6) 978-2728 Fax: (41 6) 978-6395