Genetic polymorphisms of microsomal and soluble epoxide hydrolase and the risk of Parkinson's disease.

Oxidative stress is hypothesized to play a major role in the destruction of dopaminergic neurons, which is associated with Parkinson's disease. Epoxides are potentially reactive intermediates formed through the oxidative metabolism of both exogenous and endogenous substances that contribute to cytotoxic damage mediated by oxidative stress. The microsomal (EPHX1) and soluble (EPHX2) epoxide hydrolases function to regulate the oxidation status of a wide range of xenobiotic- and lipid-derived substrates; therefore, interindividual variation in these pathways may mitigate epoxide-related cellular injury. In this investigation, we examined the potential association between the risk of Parkinson's disease and genetic variation within the EPHX1 and EPHX2 genes. Fluorescent 5' nuclease-based assays were developed to identify the allelic status of individuals with respect to specific single nucleotide polymorphisms in exons 3 and 4 of the EPHX1 gene and exons 8 and 13 of the EPHX2 gene. EPHX1 and EPHX2 genotype data were obtained from 133 idiopathic Parkinson's disease patients and 212 control subjects matched on age, gender and ethnicity. No statistically significant differences were found in the distribution of the reference and variant alleles between Parkinson's disease and control subjects, or when results were stratified by gender. Therefore, common polymorphisms within EPHX1 and EPHX2 do not appear to be important risk factors for Parkinson's disease.

PI3K inhibitors reverse the suppressive actions of insulin on CYP2E1 expression by activating stress-response pathways in primary rat hepatocytes.

Insulin-associated signaling pathways are critical in the regulation of hepatic physiology. Recent inhibitor-based studies have implicated a mechanistic role for phosphatidylinositol 3' kinase (PI3K) in the insulin-mediated suppression of CYP2E1 mRNA levels in hepatocytes. We investigated the dose dependence for this response and for the effects of insulin and extracellular matrix on PI3K signaling and CYP2E1 mRNA expression levels using a highly defined rat primary hepatocyte culture system. The PI3K inhibitors wortmannin and LY294002 stimulated stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation in a rapid and concentration-dependent manner that paralleled the inhibition of protein kinase B (PKB) phosphorylation. Although PI3K inhibitors reversed the suppressive effects of insulin on CYP2E1 expression, these effects only occurred at concentrations well in excess of those required to achieve complete inhibition of PKB phosphorylation. These same concentrations produced cytotoxic responses as evidenced by perturbed cellular morphology and elevated release of lactate dehydrogenase. Wortmannin-mediated activation of the SAPK/JNK and p38 MAPK pathways also resulted in the mobilization of activator protein-1 complex to the nuclear compartment. We conclude that the suppression of CYP2E1 mRNA expression by insulin is not directly associated with PI3K-dependent pathway activation, but rather is linked to a cytotoxic response stemming from acute challenge with PI3K inhibitors.

Sequence analyses of CYP2B genes and catalytic profiles for P450s in Qdj:Sprague-dawley rats that lack response to the phenobarbital-mediated induction of CYP2B2.

The Qdj:Sprague-Dawley (SD) rat is a mutant strain lacking in phenobarbital (PB)-mediated induction of CYP2B2. The presence of interindividual differences in the hepatic content of CYP2B proteins and testosterone 16beta-hydroxylase activity demonstrated that the breeding colony of Qdj:SD rats involves normal (+/+) and intermediate (+/-) phenotypes as well as mutant (-/-)-type rats. Although PB-treated Qdj:SD (-/-) rats expressed CYP2B1 normally, testosterone 16beta-hydroxylase activity in these rats was quite low. Analysis of regioselective metabolism of testosterone and 4-hydroxybiphenyl glucuronidation demonstrated normal catalytic activities associated with other forms of cytochrome P450s, including CYP2A, -2C, and -3A, as well as PB-inducible UDP-glucuronosyltransferase in Qdj:SD (-/-) rats. There were no serious mutations in the exons of the CYP2B1 gene in Qdj:SD (-/-) rats, demonstrating that this gene codes a functional CYP2B1. These observations suggest that CYP2B1 needs the interaction with CYP2B2 to exert the full function. The CYP2B2 gene in Qdj:SD (-/-) rats was the same as that in wild-type (+/+) rats in its length of the region containing all exon/introns and 5'-upstream up to -2.3 kilobase pairs. Malignant mutation such as stop codon formation was not observed in the exons, and no mutation was detected in the region containing the PB-responsive unit. These results strongly suggest that impaired induction of CYP2B2 in Qdj:SD (-/-) rats is attributable either to mutation at the region different from PB-responsive unit and exons or to absence or lowered expression of trans-acting factor(s) necessary for gene regulation.

Baculovirus vectors repress phenobarbital-mediated gene induction and stimulate cytokine expression in primary cultures of rat hepatocytes.

Baculovirus transfection strategies have proven successful at transferring foreign DNA into hepatoma cells and primary hepatocytes. When testing the utility of these methodologies in cultured hepatocytes, we discovered that the presence of baculovirus disrupts the phenobarbital (PB) gene induction process, a potent transcriptional activation event characteristic of highly differentiated hepatocytes, and repressed expression of the albumin gene. In concert with previous reports from our laboratory demonstrating that increased cAMP levels can completely repress the induction of specific cytochrome P450 (CYP) genes, cAMP concentrations and PKA activities were measured in the primary hepatocytes subsequent to baculovirus exposure. However, neither parameter was affected by the presence of the virus. To evaluate whether immune response modulation was triggered by baculovirus exposure, RNase protection assays were performed and demonstrated that baculovirus infection activates TNF-alpha, IL-1alpha and IL-1beta expression in the primary hepatocyte cultures. Immunocytochemical experiments indicated that the production of cytokines was likely due to the presence of small numbers of Kupffer cells present in the culture populations. Exogenously added TNF-alpha was also effective in repressing PB induction, consistent with other reports indicating that inflammatory cytokines are capable of suppressing expression of biotransformation enzyme systems. Comparative studies demonstrated the specificity of these effects since exposures of hepatocytes to adenoviral vectors did not result in down-regulation of hepatic gene responsiveness. These results indicate that baculovirus vectors enhance the expression of inflammatory cytokines in primary hepatocyte cultures, raising concerns as to whether these properties will compromise the use of baculovirus vectors for study of cytochrome P450 gene regulation, as well as for liver-directed gene therapy in humans.

Activation of cytochrome P450 gene expression in the rat brain by phenobarbital-like inducers.

Oxidative biotransformation, coupled with genetic variability in enzyme expression, has been the focus of hypotheses interrelating environmental and genetic factors in the etiology of central nervous system disease processes. Chemical modulation of cerebral cytochrome P450 (P450) monooxygenase expression character may be an important determinant of in situ metabolism, neuroendocrine homeostasis, and/or central nervous system toxicity resulting from exposure to neuroactive drugs and xenobiotic substances. To examine the capacity of the rat brain to undergo phenobarbital (PB)-mediated induction, we developed reverse transcription-polymerase chain reaction methods and evaluated the effects of several PB-like inducers on P450 and microsomal epoxide hydrolase gene expression. Animals treated i.p. with four daily doses of PB demonstrated markedly induced levels of CYP2B1, CYP2B2, and CYP3A1 mRNA in the striatum and cerebellum. In contrast, 1 or 2 days of PB treatment resulted in unchanged or even slightly decreased levels of CYP2B1 and CYP2B2 in the brain, although the latter treatments produced marked induction of the corresponding genes in the liver. Only slight increases in epoxide hydrolase RNA levels resulted in brains of PB-treated animals. Substantial activation of cerebral CYP2B1, CYP2B2, and CYP3A1 mRNA levels also resulted when animals were treated with the neuroactive drugs diphenylhydantoin and amitryptiline, and with the potential PB-like xenobiotic inducers trans-stilbene oxide and diallyl sulfide, whereas dichlorodiphenyltrichloroethane was less efficacious. Although the time course of the induction response is delayed in brain relative to that required for the liver, these results clearly establish that brain P450s are markedly PB inducible.

Fingerprinting of cytochrome P450 and microsomal epoxide hydrolase gene expression in human blood cells.

To examine the character and variability of human cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) gene expression in human blood cells, we used a highly sensitive, quantitative, competitive reverse transcriptase-coupled polymerase chain reaction (QC RT-PCR) assay to assess mRNA profiles for a battery of 8 genes, in peripheral lymphocytes isolated from 10 healthy donors. Of the genes profiled, in lymphocytes CYP2D6 was typically expressed at the highest levels (3.8 x 10(5) molecules/microg total RNA), with CYP2E1 and mEH also maintained at relatively high abundance (1.2 x 10(5) and 1.8 x 10(5) molecules/microg total RNA, respectively). CYP1A1 levels were approximately an order of magnitude lower (3.9 x 10(4) molecules/microg total RNA), followed by CYP2F1 and CYP3A levels that were near the detection limit of the assay. CYP1A2 and CYP2A6/7 mRNAs were not detected in any of the lymphocyte samples. Overall, relatively low levels of inter-individual variation (2- to 6-fold) existed among these endpoint parameters in the subjects tested. To test whether established human blood cell lines were suitable models to assess basal expression and chemical induction responsiveness of these genes, we determined that constitutive CYP and mEH mRNA profiles were essentially conserved across 4 established human blood cell lines, and highly analogous to the basal expression patterns identified in freshly isolated peripheral lymphocytes. mEH protein was detected in all of the cell lines using Western immunoblotting and chemiluminescent visualization, whereas CYP1A1, CYP2D6, CYP2E1 or CYP3A proteins were not detected in these analyses. When blood cell-derived cultures were exposed to the prototypical CYP1A and CYP3A inducers, i.e., beta-naphthoflavone (beta-NF), dexamethasone (DEX) or phenobarbital, generally little or no inductive response was manifested. Thus, the data obtained from this investigation indicate that, although human blood cell lines in general exhibit poor responsiveness to prototypical inducer exposures, the constitutive patterns of CYP and mEH expression in peripheral lymphocytes appear to exhibit relatively low levels of variation among individuals. In addition, these in vivo patterns of expression are well maintained in established cultured blood-cell lines.

Identification and functional characterization of human soluble epoxide hydrolase genetic polymorphisms.

Human soluble epoxide hydrolase (sEH), an enzyme directing the functional disposition of a variety of endogenous and xenobiotic-derived chemical epoxides, was characterized at the genomic level for interindividual variation capable of impacting function. RNA was isolated from 25 human liver samples and used to generate full-length copies of soluble epoxide hydrolase cDNA. The resulting cDNAs were polymerase chain reaction amplified, sequenced, and eight variant loci were identified. The coding region contained five silent single nucleotide polymorphisms (SNPs) and two variant loci resulting in altered protein sequence. An amino acid substitution was identified at residue 287 in exon 8, where the more common arginine was replaced by glutamine. A second variant locus was identified in exon 13 where an arginine residue was inserted following serine 402 resulting in the sequence, arginine 403-404, instead of the more common, arginine 403. This amino acid insertion was confirmed by analyzing genomic DNA from individuals harboring the polymorphic allele. Slot blot hybridization analyses of the liver samples indicated that sEH mRNA steady-state expression varied approximately 10-fold. Transient transfection experiments with CHO and COS-7 cells were used to demonstrate that the two new alleles possess catalytic activity using trans-stilbene oxide as a model substrate. Although the activity of the glutamine 287 variant was similar to the sEH wild type allele, proteins containing the arginine insertion exhibited strikingly lower activity. Allelic forms of human sEH, with markedly different enzymatic profiles, may have important physiological implications with respect to the disposition of epoxides formed from the oxidation of fatty acids, such as arachidonic acid-derived intermediates, as well in the regulation of toxicity due to xenobiotic epoxide exposures.

Epoxide hydrolase--polymorphism and role in toxicology.

Microsomal epoxide hydrolase is a critical biotransformation enzyme that catalyzes the conversion of a broad array of xenobiotic epoxide substrates to more polar diol metabolites. The gene has been shown previously to exhibit polymorphism, including variation in the coding region leading to amino acid substitutions at positions 113 (Y/H) and 139 (H/R). To better evaluate the phenotype associated with the structural region genetic polymorphisms associated with mEH, we performed enzymatic analyses using purified mEH proteins that were expressed using a baculovirus system, or with microsomal preparations obtained from liver tissues that were derived from individuals with homozygous mEH allelic status. Benzo[a]pyrene-4, 5-oxide and cis-stilbene oxide were employed as substrates for the enzymatic determinations. Results obtained with the purified enzymes suggested that the reaction velocity catalyzed by the wild type (Y113/H139) protein was approximately two-fold greater than the corresponding velocities for the variant forms of the enzyme. However, when reaction rates were analyzed using human liver microsomal preparations, the maximal velocities generated among the variant mEH proteins were not statistically different. Collectively, these results indicate that the structural differences coded by the mEH genetic variants may have only modest impact on the enzyme's specific activity in vivo.

Epoxide hydrolases: biochemistry and molecular biology.

Epoxides are organic three-membered oxygen compounds that arise from oxidative metabolism of endogenous, as well as xenobiotic compounds via chemical and enzymatic oxidation processes, including the cytochrome P450 monooxygenase system. The resultant epoxides are typically unstable in aqueous environments and chemically reactive. In the case of xenobiotics and certain endogenous substances, epoxide intermediates have been implicated as ultimate mutagenic and carcinogenic initiators Adams et al. (Chem. Biol. Interact. 95 (1995) 57-77) Guengrich (Properties and Metabolic roles 4 (1982) 5-30) Sayer et al. (J. Biol. Chem. 260 (1985) 1630-1640). Therefore, it is of vital importance for the biological organism to regulate levels of these reactive species. The epoxide hydrolases (E.C. 3.3.2. 3) belong to a sub-category of a broad group of hydrolytic enzymes that include esterases, proteases, dehalogenases, and lipases Beetham et al. (DNA Cell Biol. 14 (1995) 61-71). In particular, the epoxide hydrolases are a class of proteins that catalyze the hydration of chemically reactive epoxides to their corresponding dihydrodiol products. Simple epoxides are hydrated to their corresponding vicinal dihydrodiols, and arene oxides to trans-dihydrodiols. In general, this hydration leads to more stable and less reactive intermediates, however exceptions do exist. In mammalian species, there are at least five epoxide hydrolase forms, microsomal cholesterol 5,6-oxide hydrolase, hepoxilin A(3) hydrolase, leukotriene A(4) hydrolase, soluble, and microsomal epoxide hydrolase. Each of these enzymes is distinct chemically and immunologically. Table 1 illustrates some general properties for each of these classes of hydrolases. Fig. 1 provides an overview of selected model substrates for each class of epoxide hydrolase.

Lack of modulation by phenobarbital of cyclic AMP levels or protein kinase A activity in rat primary hepatocytes.

Results of previous studies have substantiated a negative modulatory role for cyclic AMP (cAMP) and protein kinase A (PKA) dependent processes on the phenobarbital (PB) induction response in hepatocytes. The current study was conducted to further examine the potential role of second messenger pathways in the initial phases of induction, specifically addressing the effects of PB on the expression of intracellular cAMP levels and associated PKA activity. Using a highly differentiated primary rat hepatocyte system, cells were exposed to PB for various intervals (30 sec to 48 hr), and levels of intracellular cAMP and subsequent PKA activity were determined. Although PB markedly induced CYP2B expression, exposure to this agent produced no detectable increases in cAMP levels and PKA activity at any of the times examined. These results demonstrated that the initial events stimulated by PB in rat hepatocytes do not include alterations of cAMP levels or associated PKA activities.

Development of digoxigenin-labeled PCR amplicon probes for use in the detection and identification of enteropathogenic Yersinia and Shiga toxin-producing Escherichia coli from foods.

By including digoxigenin-11-dUTP in a polymerase chain reaction (PCR), amplification products were produced that contained nonisotopic markers for use as DNA hybridization probes. Because these labeled amplicons encode pathogenic traits for specific foodborne bacteria, they can be used to detect the presence of potentially virulent organisms that may be present in foods. This technology allows the synthesis of a variety of shelf-stable probe reagents for detecting a number of foodborne microbes of public health concern. We used this technology to detect four genes in two potential pathogens: virF and yadA in enteropathogenic Yersinia and stx1 and stx2 in Shiga-like toxin-producing Escherichia coli. Results of DNA hybridizations of dot blots of 68 Yersinia strains and 24 of 25 E. coli strains were consistent with results of equivalent PCR analyses. DNA colony hybridization with nonisotopic virF probes of colonies arising on spread plates from artificially contaminated food homogenates was able to detect potentially pathogenic Y. enterocolitica. When compared with oligonucleotide probes, amplicon probes are much less sensitive to changes in hybridization and wash temperatures, allowing greater reproducibility. Labeled probe preparations were reused more than five times and have been stored at -20 degrees C for more than 8 months. This method conveniently generates probes that are safe, stable, inexpensive, reusable, and reliable.

Symposium overview: the role of genetic polymorphism and repair deficiencies in environmental disease.

A symposium of this title was presented at the 37th Annual Meeting of the Society of Toxicology held in Seattle, Washington during March of 1998. The symposium focused on heritable variations in metabolism, DNA replication, and DNA repair that may predispose humans to environmental diseases. Human metabolic, replication, and repair enzymes function in protective roles. Metabolic enzymes are protective because they detoxify a stream of chemicals to which the body is exposed. Replication and repair enzymes are also protective; they function to maintain the integrity of the human genome. Polymorphisms in the genes that code for some of these enzymes are known to give rise to variations in their protective functions. For example, functional polymorphisms of the N-acetyltransferases, paraoxonases, and microsomal epoxide hydrolases vary in their capacity to metabolize environmental chemicals. Specific isoforms of the N-acetyltransferases and microsomal epoxide hydrolases are increasingly associated with incidences of cancer attributable to exposure to these chemicals. Thus, maintenance of cellular-growth homeostasis, normally and in the face of environmental challenge, is dependent on an inherited assortment of metabolic isoforms. Since replication and repair are also protective cellular functions, and since mutations in genes that code for these functions are associated with tumorigenesis, one can reasonably speculate that common functional polymorphisms of replication and repair enzymes may also impart susceptibility to environmental disease.

Phenobarbital responsiveness conferred by the 5'-flanking region of the rat CYP2B2 gene in transgenic mice.

Phenobarbital (PB) is a prototype for a class of agents that produce marked transcriptional activation of a number of genes, including certain cytochrome P-450s. We used transgenic mouse approaches and multiple gene reporters to assess the functional consequences of specific deletions and site-specific mutations within the 2.5kb 5'-flanking region of the rat CYP2B2 gene. Protein-DNA interactions at the PBRU domain also were characterized. Using the transgenic models, we demonstrate that sequences between -2500 and -1700bp of the CYP2B2 gene are critical for PB induction; mice with 1700 or 800bp of 5'-flanking CYP2B2 sequence are not PB responsive. DNA affinity enrichment techniques and immunoblotting and electromobility shift assays were used to determine that nuclear factor 1 (NF-1) interacts strongly with a site centered at -2200bp in the PB responsive unit (PBRU) of CYP2B2. To test the functional contribution of NF-1 in PB activation, we introduced specific mutations within the PBRU NF-1 element and demonstrated that these mutations completely ablate the binding interaction. However, transgenic mice incorporating the mutant NF-1 sequence within an otherwise wild-type -2500/CYP2B2 transgene maintained full PB responsiveness. These results indicate that, despite the avidity of the respective DNA-protein interaction within the PBRU in vitro, NF-1 interaction is not an essential factor directing PB transcriptional activation in vivo.

Insulin-mediated modulation of cytochrome P450 gene induction profiles in primary rat hepatocyte cultures.

In this investigation, we examined the effects of insulin on gene induction responsiveness in primary rat hepatocytes. Cells were cultured for 72 hours either in the absence or presence of 1 microM insulin and then exposed to increasing concentrations of phenobarbital (PB; 0.01-3.5 mM). Culturing in the absence of insulin produced 1.5-2-fold increases in the induction magnitude of CYP2B1 and CYP2B2 mRNA expression resulting from PB exposures, without altering the bell-shaped dose-response curve characteristic of this agent. However, for the CYP3A1 gene, insulin removal led to a pronounced shift in both the PB-induction magnitude and dose-response relationships of the induction response, with higher levels of CYP3A1 expression resulting from exposures to lower concentrations of inducer. Insulin removal also reduced the time required to attain maximal induction of CYP2B1/2 and CYP3A1 gene expression. The insulin effects were not specific for PB induction, as insulin deprivation similarly enhanced both dexamethasone- and beta-naphthoflavone-inducible CYP3A1 and CYP1A1 expression profiles, respectively. In contrast, the level of albumin mRNA expression was reduced considerably in cells deprived of insulin. We conclude that insulin is an important regulator of inducible and liver-specific gene expression in primary rat hepatocytes.

Physiological and pathophysiological regulation of cytochrome P450.

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the April 1998 Experimental Biology '98 meeting in San Francisco. The presentations focused on the mechanisms of regulation of cytochrome P450 gene expression by developmental factors and by hormones and cytokines, as well as on the interplay between physiological and chemical regulation. Approaches and systems used to address these questions included conditional gene knockouts in mice, primary hepatocyte cultures, immunofluorescence imaging of cells, and cell lines stably expressing reporter gene constructs.

Identification of Listeria monocytogenes from unpasteurized apple juice using rapid test kits.

A microbiological survey of 50 retail juices was conducted in the fall of 1996. These juices were analyzed for Listeria monocytogenes, Escherichia coli O157:H7, Salmonella, coliforms, fecal coliforms, and pH. Two unpasteurized juices were positive for L. monocytogenes: an apple juice and an apple raspberry blend with a pH of 3.78 and 3.75, respectively. Three L. monocytogenes isolates were characterized. The colonies were typical for Listeria sp. on Oxford and lithium chloride-phenylethanol-moxalactam agars and were beta-hemolytic on sheep blood agar. The isolates required 5 days of incubation at 35 degrees C to produce a positive rhamnose reaction in a phenol red carbohydrate broth. This slow rhamnose utilization resulted in these isolates not being identified using the Micro-ID test strip (Organon Technika). However, the isolates were positive for L. monocytogenes using the API Listeria strip (BioMerieux) and a multiplex polymerase chain reaction for detection of the hemolysis (hyla) and invasion-associated protein (iap) genes.

Differential induction of cytochrome P450 gene expression by 4n-alkyl-methylenedioxybenzenes in primary rat hepatocyte cultures.

A well-characterized primary rat hepatocyte culture system was used to examine induction patterns of cytochrome 450 gene expression by a series of 4-n-alkyl-methylenedioxybenzene (MDBs) derivatives. Hepatocytes were treated for 24, 48, or 72 hours with 0-500 microM of the MDB compounds, and total cellular RNA and protein from each treatment was evaluated by hybridization and immunochemical techniques. Exposure to MDB congeners possessing increasing 4-n-alkyl side-chain length (C0-C8) resulted in dose- and structure-dependent activation of CYP2B1, 2B2, 3A1, 1A1, and 1A2 gene expression. At equivalent 100 microM concentrations, the C6 and C8 MDB congeners were more effective than the prototypical inducer phenobarbital (PB) with respect to induction potency of CYP2B1, CYP2B2, and CYP3A1 gene expression. In contrast to PB, longer side-chain-substituted MDBs effectively induced CYP1A1 and CYP1A2 gene expression, in addition to the CYP2B and CYP3A genes. At equivalent molar concentrations, the catechol derivative of C6-MDB was ineffective in its ability to induce CYP gene expression, indicating the importance of the intact methylenedioxy bridge in the induction mechanism. Levels of MDB-inducible CYP2B1 and CYP2B2 mRNA were highly correlated with CYP2B1/2 apoprotein levels, ascertained by immunoblot analysis of cultured hepatocyte S9 fractions. Compared with results from previous in vivo analysis (12), the current data indicate that pharmacodynamic factors may influence MDB induction profiles and that differences in MDB effects on CYP gene expression result depending on distinct structure-activity relationships.

Effects of chemical inducers on human microsomal epoxide hydrolase in primary hepatocyte cultures.

Human microsomal epoxide hydrolase (mEH; EC 3.3.2.3) is an important biotransformation enzyme and potential risk determinant for pathologies such as cancer and teratogenesis. Currently, the effects of chemical exposures on human mEH gene expression are largely unknown, but they may constitute a unique modifier of disease susceptibility. To examine this issue, we exposed cultures of primary human hepatocytes isolated from seven donors to prototypic chemical inducers [such as phenobarbital (PB), polyaromatic hydrocarbons, dexamethasone, butylated hydroxyanisole, and ciprofibrate]. Basal levels of mEH RNA and protein were detected readily in untreated cells. Chemical treatment of cultured hepatocytes resulted in variable mEH RNA and protein expression, but, in general, only modest modulatory effects were detected following these exposures. The maximum increase in mEH RNA expression observed was approximately 3.5-fold following Arochlor 1254 exposure. Immunochemical levels of mEH protein were quantified for all treatment groups in three cultures and demonstrated less overall variation and, in general, a lack of concordance with corresponding mEH RNA levels. Cytochrome P450 (CYP) 1A2 and 3A mRNA levels were measured before and following exposure to beta-naphthaflavone and PB, respectively, to permit independent evaluation of hepatocyte inducer responsiveness. Substantial increases in RNA expression levels for both the CYP1A2 and CYP3A genes demonstrated that the hepatocyte cultures were robust and highly responsive to inducer treatment. These results indicate that the mEH gene in human hepatocytes is only modestly responsive to chemical exposures.

Quantification of multiple human cytochrome P450 mRNA molecules using competitive reverse transcriptase-PCR.

We developed a quantitative competitive reverse transcriptase-polymerase chain reaction (QC RT-PCR) assay to measure mRNA levels of seven human cytochrome P450 (P450, CYP) genes and microsomal epoxide hydrolase (EH) simultaneously. This assay employs an exogenous recombinant RNA (rcRNA) molecule as an internal standard that shares PCR primer and hybridization probe sequences with CYP1A1, CYP1A2, CYP2A6/7, CYP2D6, CYP2E1, CYP2F1, CYP3A4/5/7, and EH mRNA. Because each rcRNA molecule contains several primer sequences, an entire battery of genes that exhibit differential responsiveness to various classes of xenobiotics may be measured simultaneously from one population of cDNA molecules. In this study, we demonstrated the precision and power of the assay using small amounts of human liver total RNA. We also report for the first time quantitative profiles of P450 and EH mRNA abundance in eight human livers. Cytochrome P450 2E1 mRNA maintained the highest abundance (average 6.67 x 10(7) molecules/microg of total RNA) and least variation (13 fold) in all livers examined. Cytochrome P450 1A2, CYP2A6/7, CYP2D6, CYP3A4/5, and EH mRNAs were approximately one order of magnitude less abundant than CYP2E1 transcripts, with CYP2D6 levels exhibiting the greatest variation (220 fold) between individuals. This QC RT-PCR assay should prove valuable for measuring basal and induced mRNAs in different cell types in vitro, as well as in biomonitoring applications where individuals are exposed or hypersusceptible to certain xenobiotic-initiated toxicities.