Transcriptomic variation of pharmacogenes in multiple human tissues and lymphoblastoid cell lines.

Variation in the expression level and activity of genes involved in drug disposition and action ('pharmacogenes') can affect drug response and toxicity, especially when in tissues of pharmacological importance. Previous studies have relied primarily on microarrays to understand gene expression differences, or have focused on a single tissue or small number of samples. The goal of this study was to use RNA-sequencing (RNA-seq) to determine the expression levels and alternative splicing of 389 Pharmacogenomics Research Network pharmacogenes across four tissues (liver, kidney, heart and adipose) and lymphoblastoid cell lines, which are used widely in pharmacogenomics studies. Analysis of RNA-seq data from 139 different individuals across the 5 tissues (20-45 individuals per tissue type) revealed substantial variation in both expression levels and splicing across samples and tissue types. Comparison with GTEx data yielded a consistent picture. This in-depth exploration also revealed 183 splicing events in pharmacogenes that were previously not annotated. Overall, this study serves as a rich resource for the research community to inform biomarker and drug discovery and use.

Interindividual variability of CYP2C19-catalyzed drug metabolism due to differences in gene diplotypes and cytochrome P450 oxidoreductase content.

Large interindividual variability has been observed in the metabolism of CYP2C19 substrates in vivo. The study aimed to evaluate sources of this variability in CYP2C19 activity, focusing on CYP2C19 diplotypes and the cytochrome P450 oxidoreductase (POR). CYP2C19 gene analysis was carried out on 347 human liver samples. CYP2C19 activity assayed using human liver microsomes confirmed a significant a priori predicted rank order for (S)-mephenytoin hydroxylase activity of CYP2C19*17/*17 > *1B/*17 > *1B/*1B > *2A/*17 > *1B/*2A > *2A/*2A diplotypes. In a multivariate analysis, the CYP2C19*2A allele and POR protein content were associated with CYP2C19 activity. Further analysis indicated a strong effect of the CYP2C19*2A, but not the *17, allele on both metabolic steps in the conversion of clopidogrel to its active metabolite. The present study demonstrates that interindividual variability in CYP2C19 activity is due to differences in both CYP2C19 protein content associated with gene diplotypes and the POR concentration.The Pharmacogenomics Journal advance online publication, 1 September 2015; doi:10.1038/tpj.2015.58.

Prediction of CYP3A4 enzyme activity using haplotype tag SNPs in African Americans.

The CYP3A locus encodes hepatic enzymes that metabolize many clinically used drugs. However, there is marked interindividual variability in enzyme expression and clearance of drugs metabolized by these enzymes. We utilized comparative genomics and computational prediction of transcriptional factor binding sites to evaluate regions within CYP3A that were most likely to contribute to this variation. We then used a haplotype tagging single-nucleotide polymorphisms (htSNPs) approach to evaluate the entire locus with the fewest number of maximally informative SNPs. We investigated the association between these htSNPs and in vivo CYP3A enzyme activity using a single-point IV midazolam clearance assay. We found associations between the midazolam phenotype and age, diagnosis of hypertension and one htSNP (141689) located upstream of CYP3A4. 141689 lies near the xenobiotic responsive enhancer module (XREM) regulatory region of CYP3A4. Cell-based studies show increased transcriptional activation with the minor allele at 141689, in agreement with the in vivo association study findings. This study marks the first systematic evaluation of coding and noncoding variation that may contribute to CYP3A phenotypic variability.

Identification of glucocorticoid-inducible cytochromes P-450 in the intestinal mucosa of rats and man.

We used monoclonal antibodies and complementary DNAs (cDNAs) to glucocorticoid-inducible liver cytochromes P-450 in rats (P-450p) and in man (HLp) to search for related cytochromes in intestinal mucosa. In rat enterocytes, we found two dexamethasone-inducible proteins related to the steroid-inducible liver cytochromes P-450. Induction of these proteins in enterocytes was associated with increases in the amount of a P-450p-related messenger RNA and of erythromycin demethylase, an activity highly characteristic of P-450p and HLp. Similar studies on human jejunal enterocytes revealed a microsomal protein indistinguishable from HLp on immunoblots and an abundance of RNA hybridizing with HLp cDNA. In human enterocytes the specific concentration of the HLp-related cytochrome (measured immunochemically or as erythromycin demethylase activity) was similar to that found in human liver and could account for all of the CO-binding hemo-protein detected. We conclude that the intestinal mucosa contains prominent form(s) of cytochromes P-450 similar to liver cytochrome P-450p in their structure, function, and some regulatory characteristics.

The xenobiotic compound 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is an agonist ligand for the nuclear receptor CAR.

A wide range of xenobiotic compounds are metabolized by cytochrome P450 (CYP) enzymes, and the genes that encode these enzymes are often induced in the presence of such compounds. Here, we show that the nuclear receptor CAR can recognize response elements present in the promoters of xenobiotic-responsive CYP genes, as well as other novel sites. CAR has previously been shown to be an apparently constitutive transactivator, and this constitutive activity is inhibited by androstanes acting as inverse agonists. As expected, the ability of CAR to transactivate the CYP promoter elements is blocked by the inhibitory inverse agonists. However, CAR transactivation is increased in the presence of 1,4-bis[2-(3, 5-dichloropyridyloxy)]benzene (TCPOBOP), the most potent known member of the phenobarbital-like class of CYP-inducing agents. Three independent lines of evidence demonstrate that TCPOBOP is an agonist ligand for CAR. The first is that TCPOBOP acts in a dose-dependent manner as a direct agonist to compete with the inhibitory effect of the inverse agonists. The second is that TCPOBOP acts directly to stimulate coactivator interaction with the CAR ligand binding domain, both in vitro and in vivo. The third is that mutations designed to block ligand binding block not only the inhibitory effect of the androstanes but also the stimulatory effect of TCPOBOP. Importantly, these mutations do not block the apparently constitutive transactivation by CAR, suggesting that this activity is truly ligand independent. Both its ability to target CYP genes and its activation by TCPOBOP demonstrate that CAR is a novel xenobiotic receptor that may contribute to the metabolic response to such compounds.

Identification of a new P-glycoprotein-like ATP-binding cassette transporter gene that is overexpressed during hepatocarcinogenesis.

The liver is remarkably insensitive to a variety of cytotoxins and expresses a number of known drug resistance genes. To isolate new P-glycoprotein (Pgp)-related genes, we screened a normal rat liver cDNA library at low stringency with a MDR1 cDNA fragment containing the P-loop and ATP binding site. We isolated a novel cDNA closely related to the Pgps that is dramatically increased in hepatic neoplasia and refer to it as P-glycoprotein-related protein (PRP). The predicted protein shows PRP to be a member of the ATP-Binding Cassette (ABC) family of proteins, and a multisequence comparison of the nucleotide binding domain and the ABC family signature sequences reveals that PRP sequences are highly conserved with the greatest similarity to the yeast heavy metal transporter encoded by hmtl. However, the hydropathy plot analysis suggests that PRP does not have any prominent membrane-spanning domains and thus is not typical of ABC transporters. The PRP transcript is detected in many normal tissues. In the H35 hepatoma cell line, PRP was overexpressed compared to normal liver. Southern blot analysis of DNA from the H35 rat hepatoma cells reveals that the PRP gene was amplified compared to normal liver. The orotic acid model of hepatocarcinogenesis was used to determine if during stepwise progression to liver cancer, PRP changed with hepatocarcinogenesis. At the hyperplastic nodule stage, PRP expression was increased over its expression in normal surrounding liver. More dramatic increases in PRP expression were found in frank hepatic carcinomas. Cumulatively, these studies are the first to link a novel ABC family member to the hepatic neoplastic process, a role that may be recapitulated in other cells, considering the ubiquitous expression of PRP.

Mdr1b facilitates p53-mediated cell death and p53 is required for Mdr1b upregulation in vivo.

The mdr1b gene is thought to be a "stress-responsive" gene, however it is unknown if this gene is regulated by p53 in the whole animal. Moreover, it is unknown if overexpression of mdr1b affects cell survival. The dependence of mdr1b upon p53 for upregulation was evaluated in p53 knockout mice. Wild-type (wt) or p53-/- mice were treated singly or in combination with gamma irradiation (IR) and/or the potent DNA damaging agent, diethylnitrosoamine (DEN). Both IR and DEN induced mdr1b in wild-type animals, but not in the p53-/- mice. IR also upregulated endogenous mdr1b in the H35 liver cell line, and the mdr1b promoter was activated by IR and activation correlated with p53 levels; moreover activation required an intact p53 binding site. Colony survival studies revealed that co-transfection of both mdr1b and p53 dramatically reduced colony numbers compared to cells transfected with either p53 or mdr1b alone and cells microinjected with both mdr1b and p53 had a more dramatic loss in viability compared to cells injected with either expression vector alone. Further studies using acridine orange and ethidium bromide to measure apoptosis revealed that mdr1b caused apoptosis and this was enhanced by p53, however the increased apoptosis required a functional p53 transactivation domain. These studies indicate that mdr1b is a downstream target of p53 in the whole animal and expression of mdr1b facilitates p53-mediated cell death.

Isolation of rat pgp3 cDNA: evidence for gender and zonal regulation of expression in the liver.

Distinct differences exist in the function and regulation of the individual p-glycoprotein (pgp) members in many species. In order to study regulation of individual pgp mRNA isoforms it is, therefore, necessary to have probes that can distinguish between the various pgp isoforms. However, to date few studies examining hepatic gene expression in rat liver have used pgp gene specific probes. Towards this end we screened a cDNA library constructed from a normal rat liver with a human pgp1 cDNA and isolated a partial cDNA for class III pgp, rat pgp3. By comparison of the sequence of this new rat pgp3 cDNA with genomic and cDNA sequences for rat pgp1 and rat pgp2 we selected oligonucleotide probe sequences that would allow us to differentiate between the highly homologous rat pgp2 and pgp3 genes on Northern blots and by polymerase chain reaction (PCR). We found that pgp3, for both male and female rats, was the predominant form of pgp expressed in normal rat liver with males consistently expressing several-fold lower levels of pgp3 than females. Because many genes are zonally expressed in the hepatic acinus we examined the possibility that pgp3 might show heterogeneous distribution as well. We found, by in situ hybridization of paraformaldehyde-fixed rat liver sections that pgp3 was distributed non-uniformly across the hepatic acinus with a gradient that showed the highest expression toward the terminal hepatic venule. We confirmed this finding by selectively isolating hepatocytes from either the terminal hepatic venular or periportal zones using a digitonin/collagenase perfusion procedure. Application of specific pgp3 PCR primers to RNA isolated from hepatocytes from these areas confirmed that pgp3 mRNA was the predominant form in the hepatocytes surrounding the terminal hepatic venule. Finally, we examined pgp3 expression in a variety of tissues by Northern blot analysis and found that pgp3 was most highly expressed in the liver and gastrointestinal tract (with a gradient of expression from small to large intestine), while low levels were found in the kidney, heart and brain. Pgp3 mRNA was undetectable in the adrenal gland and in skeletal muscle. In summary, using rat pgp gene specific oligonucleotide probes we found that pgp3 gene expression is regulated by anatomic location with the highest mRNA expression in organs that are involved in drug detoxification. Our results also demonstrate heterogeneity of hepatic rat pgp3 gene expression, which is influenced by both gender and by acinar location.

The human pregnane X receptor: genomic structure and identification and functional characterization of natural allelic variants.

The pregnane X receptor (PXR)/steroid and xenobiotic receptor (SXR) transcriptionally activates cytochrome P4503A4 (CYP3A4) when ligand activated by endobiotics and xenobiotics. We cloned the human PXR gene and analysed the sequence in DNAs of individuals whose CYP3A phenotype was known. The PXR gene spans 35 kb, contains nine exons, and mapped to chromosome 13q11-13. Thirty-eight single nucleotide polymorphisms (SNPs) were identified including six SNPs in the coding region. Three of the coding SNPs are non-synonymous creating new PXR alleles [PXR*2, P27S (79C to T); PXR*3, G36R (106G to A); and PXR*4, R122Q (4321G to A)]. The frequency of PXR*2 was 0.20 in African Americans and was never found in Caucasians. Hepatic expression of CYP3A4 protein was not significantly different between African Americans homozygous for PXR*1 compared to those with one PXR*2 allele. PXR*4 was a rare variant found in only one Caucasian person. Homology modelling suggested that R122Q, (PXR*4) is a direct DNA contact site variation in the third alpha-helix in the DNA binding domain. Compared with PXR*1, and variants PXR*2 and PXR*3, only the variant PXR*4 protein had significantly decreased affinity for the PXR binding sequence in electromobility shift assays and attenuated ligand activation of the CYP3A4 reporter plasmids in transient transfection assays. However, the person heterozygous for PXR*4 is normal for CYP3A4 metabolism phenotype. The relevance of each of the 38 PXR SNPs identified in DNA of individuals whose CYP3A basal and rifampin-inducible CYP3A4 expression was determined in vivo and/or in vitro was demonstrated by univariate statistical analysis. Because ligand activation of PXR and upregulation of a system of drug detoxification genes are major determinants of drug interactions, it will now be useful to extend this work to determine the association of these common PXR SNPs to human variation in induction of other drug detoxification gene targets.

6',7'-Dihydroxybergamottin in grapefruit juice and Seville orange juice: effects on cyclosporine disposition, enterocyte CYP3A4, and P-glycoprotein.

BACKGROUND: 6',7'-Dihydroxybergamottin is a furanocoumarin that inhibits CYP3A4 and is found in grapefruit juice and Seville orange juice. Grapefruit juice increases the oral bioavailability of many CYP3A4 substrates, including cyclosporine (INN, ciclosporin), but intestinal P-glycoprotein may be a more important determinant of cyclosporine availability. OBJECTIVES: To evaluate the contribution of 6',7'-dihydroxybergamottin to the effects of grapefruit juice on cyclosporine disposition and to assess the role of CYP3A4 versus P-glycoprotein in this interaction. METHODS: The disposition of oral cyclosporine was compared in healthy subjects after ingestion of water, grapefruit juice, and Seville orange juice. Enterocyte concentrations of CYP3A4 were measured in 2 individuals before and after treatment with Seville orange juice. The effect of 6',7'-dihydroxybergamottin on P-glycoprotein was assessed in vitro. RESULTS: Area under the whole blood concentration-time curve and peak concentration of cyclosporine were increased by 55% and 35%, respectively, with grapefruit juice (P < .05). Seville orange juice had no influence on cyclosporine disposition but reduced enterocyte concentrations of CYP3A4 by an average of 40%. 6',7'-Dihydroxybergamottin did not inhibit P-glycoprotein at concentrations up to 50 micromol/L. CONCLUSIONS: 6',7'-Dihydroxybergamottin is not responsible for the effects of grapefruit juice on cyclosporine. Because the interaction did not occur with Seville orange juice despite reduced enterocyte concentrations of CYP3A4, inhibition of P-glycoprotein activity by other compounds in grapefruit juice may be responsible. Reduced enterocyte CYP3A4 by 6',7'-dihydroxybergamottin could be important for other drugs whose bioavailability is less dependent on P-glycoprotein.

Identification of functionally variant MDR1 alleles among European Americans and African Americans.

MDR1 (P-glycoprotein) is an important factor in the disposition of many drugs, and the involved processes often exhibit considerable interindividual variability that may be genetically determined. Single-strand conformational polymorphism analysis and direct sequencing of exonic MDR1 deoxyribonucleic acid from 37 healthy European American and 23 healthy African American subjects identified 10 single nucleotide polymorphisms (SNPs), including 6 nonsynonymous variants, occurring in various allelic combinations. Population frequencies of the 15 identified alleles varied according to racial background. Two synonymous SNPs (C1236T in exon 12 and C3435T in exon 26) and a nonsynonymous SNP (G2677T, Ala893Ser) in exon 21 were found to be linked (MDR1*2 ) and occurred in 62% of European Americans and 13% of African Americans. In vitro expression of MDR1 encoding Ala893 (MDR1*1 ) or a site-directed Ser893 mutation (MDR1*2 ) indicated enhanced efflux of digoxin by cells expressing the MDR1-Ser893 variant. In vivo functional relevance of this SNP was assessed with the known P-glycoprotein drug substrate fexofenadine as a probe of the transporter's activity. In humans, MDR1*1 and MDR1*2 variants were associated with differences in fexofenadine levels, consistent with the in vitro data, with the area under the plasma level-time curve being almost 40% greater in the *1/*1 genotype compared with the *2/*2 and the *1/*2 heterozygotes having an intermediate value, suggesting enhanced in vivo P-glycoprotein activity among subjects with the MDR1*2 allele. Thus allelic variation in MDR1 is more common than previously recognized and involves multiple SNPs whose allelic frequencies vary between populations, and some of these SNPs are associated with altered P-glycoprotein function.

Induction of cytochromes P450.

Humans and rodents are exposed to many foreign compounds in their diet (e.g., herbal supplements such as St. John's wart), in their environment (e.g., organochlorine pesticides and polychorinated biphenyls), and as clinically prescribed drugs (e.g., rifampin and phenobarbital). In response to these exposures mammals have evolved mechanisms to induce proteins involved in xenobiotic detoxification. Metabolism by Phase I enzymes, particularly the heme containing monooxygenases cytochromes P450 is frequently the first line of defense against such xenobiotics.

Biphasic regulation of cytochrome P450 2B1/2 mRNA expression by dexamethasone in primary cultures of adult rat hepatocytes maintained on matrigel.

We have demonstrated recently that although rat hepatocytes rapidly lose their cytochrome P450 mRNA content following their introduction into primary culture, hepatocytes cultured on Matrigel, a reconstituted basement membrane, subsequently spontaneously "reexpress" the mRNAs of some constitutive P450 forms (Kocarek et al., Mol Pharmacol 43: 328-334, 1993). In the present study, we used the Matrigel cell culture system to examine the dose-dependent effects of dexamethasone (DEX) treatments on the mRNAs for two of the P450 forms that are reexpressed spontaneously between days 3 and 5 in culture, 2B1/2 and 2C6. Treatment of cultured hepatocytes with low doses of DEX (10(-9) to 10(-8) M) that induced the mRNA for tyrosine aminotransferase, a model glucocorticoid-inducible gene, suppressed the spontaneous appearance of 2B1/2 mRNA while having little or no effect on the level of 2C6 mRNA or on beta-actin mRNA. However, treatment of the hepatocyte cultures with high doses of DEX (10(-6) to 10(-5) M) that induced P450 3A1 mRNA increased the amounts of the 2B1/2 and 2C6 mRNAs (4.1- and 2.4-fold, respectively, at 10(-5) M DEX). In contrast to the suppressive effects on the spontaneous increases in 2B1/2 mRNA, low doses of DEX (10(-8) to 10(-7) M) enhanced the induction of 2B1/2 mRNA by phenobarbital (2.5-fold at 10(-7) M DEX). Treatment of the hepatocyte cultures with triamcinolone acetonide, another potent glucocorticoid, suppressed spontaneous 2B1/2 mRNA expression at low doses, but did not induce 2B1/2 mRNA at high doses. Treatments with steroids of other classes, including dihydrotestosterone, 17 alpha-ethinylestradiol, fludrocortisone or R-5020, failed to suppress 2B1/2 mRNA levels at low doses. Additionally, treatment with RU-486, a glucocorticoid/progestin receptor antagonist, induced 2B1/2 mRNA at high doses (10(-6) to 10(-5) M). The suppressive effects of DEX on spontaneous 2B1/2 mRNA expression observed at low doses are consistent with a classical glucocorticoid-mediated mechanism, while the high-dose inductive effects of DEX appear to be exerted through a nonclassical mechanism, perhaps akin to that for induction of 3A1.

Regulation of cytochrome P450 2B1/2 genes by diallyl sulfone, disulfiram, and other organosulfur compounds in primary cultures of rat hepatocytes.

Our previous study demonstrated that diallyl sulfide (DAS), a compound derived from garlic, transcriptionally activated the P450 2B1/2 genes in rat liver. In the present study, rat primary hepatocytes were used to determine the effects of DAS and its metabolite, diallyl sulfone (DASO2), on the expression of the P450 2B1/2 genes. Freshly isolated adult rat hepatocytes were cultured in a serum-free medium on a reconstituted basement membrane matrix "matrigel" that enabled the hepatocytes to maintain expression of numerous liver-specific genes for more than 1 week. After 48-hr of acclimation, 0.1, 0.5, and 2.0 mM concentrations of DAS or DASO2 were added to the culture medium and the cells were harvested at 4, 12, 24, or 36 hr after the treatment for the preparation of microsomes and RNA. Cytotoxicity was not observed by morphological examinations after DAS and DASO2 treatments. In contrast to the in vivo results, there was only a slight increase in the levels of P450 2B1/2 mRNA and protein in DAS-treated cells. However, DASO2 treatment (2 mM) resulted in 11-, 21-, and 22-fold increases in P450 2B1/2 mRNA levels at 12, 24, and 36 hr after the treatment, respectively. P450 2B1/2 protein levels were also increased markedly in DASO2-treated cells. Co-incubation of the rat hepatocyte cultures with a physiological concentration of growth hormone significantly blocked the induction of P450 2B1/2 mRNA by DASO2. Northern blot analysis using oligonucleotide probes specific for 2B1 and 2B2 demonstrated that DASO2 induced mRNA levels of both 2B1 and 2B2, with a greater induction of 2B1 mRNA. For comparison, the effects of disulfiram (DSF) and its metabolite, diethyldithiocarbamate (DDTC), on P450 2B1/2 mRNA expression were also examined in the cultured rat hepatocytes. Both DSF and DDTC caused a significant increase in P450 2B1/2 mRNA level with the highest induction at 0.5 mM. Addition of growth hormone to the culture effectively suppressed the P450 2B1/2 mRNA induction by DSF but had little effect on the induction by DDTC. Neither mRNA nor protein levels of P450 2E1 in cultured hepatocytes were affected by all the organosulfur compounds tested. These results suggest that DASO2, DSF and DDTC selectively modulate P450 isozymes in cultured rat primary hepatocytes and that the induction of P450 2B1/2 by DAS in rat liver may be mediated by its metabolite, DASO2.

The effect of atovaquone on etoposide pharmacokinetics in children with acute lymphoblastic leukemia.

PURPOSE: The use of trimethoprim/sulfamethoxazole in the prevention of Pneumocystis carinii pneumonia in patients with acute lymphoblastic leukemia (ALL) may cause undesirable adverse effects: fungal overgrowth, neutropenia, and drug resistance. A possible alternative is atovaquone, a hydroxynaphthoquinone with anti-Pneumocystis carinii activity. However, it is not known if atovaquone alters the disposition or adverse effects of antileukemic drugs. METHODS: Using a crossover study design, we compared the pharmacokinetics of etoposide and its CYP3A4-formed catechol metabolite when given as a 300 mg/m2 i.v. infusion following daily atovaquone versus trimethoprim/sulfamethoxazole in nine patients. RESULTS: The area under the concentration time curve (AUC) of etoposide, etoposide catechol and the catechol to etoposide AUC ratio were slightly higher (a median of 8.6%, 28.4%, and 25.9%) following atovaquone as compared to trimethoprim/sulfamethoxazole (P=0.055, P= 0.031 and P=0.023), respectively. In vitro analysis in human liver microsomes showed modest inhibition of etoposide catechol formation in the presence of atovaquone. Using uptake of 3H-vinblastine in L-MDR1 cells, atovaquone was shown to inhibit P-glycoprotein with an apparent Ki of 95.6 microM. CONCLUSIONS: Although the effect of atovaquone on etoposide disposition was modest, in light of the fact that the risk of etoposide-related secondary acute myeloid leukemia has been linked to minor changes in schedule and concurrent therapy, we suggest caution with the simultaneous administration of atovaquone and etoposide, particularly if used with other CYP3A4/P-glycoprotein substrates.

Regulation of cytochrome P450 2B1/2 mRNAs by Kepone (chlordecone) and potent estrogens in primary cultures of adult rat hepatocytes on Matrigel.

We previously reported that when primary cultures of rat hepatocytes were treated with phenobarbital (PB) or one of several organochlorine pesticides, including Mirex, there was co-induction of cytochrome P450 2B1 and 2B2 mRNAs and immunoreactive proteins, whereas Kepone selectively induced 2B2 (Kocarek et al. (1991) Mol. Pharmacol. 40, 203-210). Indeed, Kepone treatment actively suppressed induction of 2B1 and 2B2 mRNAs in hepatocytes cotreated with phenobarbital. Because Kepone differs chemically from Mirex only in the replacement of 2 chlorine atoms with a ketone group, which exists in aqueous solution as a gem-diol and appears to confer weak estrogenic properties, we treated hepatocyte cultures with one of 3 potent estrogens, beta-estradiol, 17 alpha-ethinylestradiol or diethylstilbestrol. Treatment with each of these estrogens induced 2B1 and 2B2 mRNA only at very high doses (10(-4) M). Beta-Estradiol (10(-4) M) treatment also induced 2B1/2 mRNA in hepatocyte cultures prepared from a prepubescent female rat. The anti-estrogen tamoxifen failed to reverse 2B1/2 mRNA induction following beta-estradiol or Kepone treatment of adult hepatocyte cultures. High doses of beta-estradiol or 17 alpha-ethinylestradiol failed to induce 2B1/2 mRNA in treated rats. We also examined the effects of chloral hydrate, a simple gem-diol, on 2B1/2 mRNA induction in the hepatocyte cultures. Treatment with chloral hydrate (3 x 10(-3) M), like Kepone (10(-5) M), suppressed 2B1/2 mRNA induction following phenobarbital (10(-4) M) treatment, while Kepone alcohol (10(-5) M), which is not a gem-diol, produced less suppression. Our results suggest that selective induction by Kepone of 2B2 is unlikely related to its effects as a weak classical estrogen, while the ability of Kepone to suppress induction of 2B1 and 2B2 by PB may be related to its properties as a gem-diol.

Pharmacogenetic pathway analysis of docetaxel elimination.

The purpose of this study was to evaluate the affinity of docetaxel for 14 transporter proteins and assess the functional significance of 17 variants in five genes involved in drug elimination. Among the transfected models investigated, OATP1B3 (SLCO1B3) was identified as the most efficient influx transporter for docetaxel. None of the observed genotypes (SLCO1B3, ABCB1, and ABCC2) was related with docetaxel clearance in 92 white patients (P > 0.17). However, the simultaneous presence of the CYP3A4*1B and CYP3A5*1A alleles was associated with a 64% increase in docetaxel clearance (P = 0.0015), independent of both sex and CYP3A activity (as determined using the erythromycin breath test). This haplotype was also associated with increased midazolam clearance in another population (P = 0.0198). An analysis of the CYP3A locus among CEPH-HapMap samples revealed that CYP3A4*1B is present exclusively among a subset of CYP3A5 expressors. Therefore, future studies should first stratify the population on the basis of CYP3A5 genotype and then compare CYP3A activity between individuals with and without the CYP3A4*1B allele.