Genetic risk factors for major bleeding in patients treated with warfarin in a community setting.

The influence of warfarin pharmacogenomics on major bleeding risk has been little studied in long-term users and non-specialist care settings. We conducted a case-control study to evaluate associations between CYP2C9*2/*3, VKORC1(1173), and CYP4F2*3 variants and major bleeding among patients treated with warfarin in a community setting. We calculated major bleeding odds ratios, adjusting for race, duration of warfarin use, age, gender, and body mass index. In 265 cases and 305 controls with 3.4 and 3.7 mean years of warfarin use, respectively, CYP4F2*3 was associated with decreased major bleeding risk (odds ratio: 0.62; 95% confidence interval: 0.43-0.91). CYP2C9*2/*3 and VKORC1(1173) had null associations overall, but there was a nonsignificant increase in major bleeding risk in patients with duration <6 months (odds ratio: 1.30; 95% confidence interval: 0.60-2.83; odds ratio: 1.23; 95% confidence interval: 0.57-2.64, respectively). In summary, in the largest study of warfarin pharmacogenomics and major bleeding to date, we found a 38% lower risk in patients with CYP4F2*3, potentially reflecting interaction with warfarin and dietary vitamin K intake and warranting additional evaluation.

The effect of nicotinamide on gene expression in a traumatic brain injury model.

Microarray-based transcriptional profiling was used to determine the effect of nicotinamide on gene expression in an experimental traumatic brain injury (TBI) model. Ingenuity Pathway Analysis (IPA) was used to evaluate the effect on relevant functional categories and canonical pathways. At 24 h, 72 h, and 7 days, respectively, 70, 58, and 76%, of the differentially expressed genes were up-regulated in the vehicle treated compared to the sham animals. At 24 h post-TBI, there were 150 differentially expressed genes in the nicotinamide treated animals compared to vehicle; the majority (82%) down-regulated. IPA analysis identified a significant effect of nicotinamide on the functional categories of cellular movement, cell-to-cell-signaling, antigen presentation and cellular compromise, function, and maintenance and cell death. The canonical pathways identified were signaling pathways primarily involved with the inflammatory process. At 72 h post-cortical contusion injury, there were 119 differentially expressed genes in the nicotinamide treated animals compared to vehicle; the majority (90%) was up-regulated. IPA analysis identified a significant effect of nicotinamide on cell signaling pathways involving neurotransmitters, neuropeptides, growth factors, and ion channels with little to no effect on inflammatory pathways. At 7 days post-TBI, there were only five differentially expressed genes with nicotinamide treatment compared to vehicle. Overall, the effect of nicotinamide on counteracting the effect of TBI resulted in significantly decreased number of genes differentially expressed by TBI. In conclusion, the mechanism of the effect of nicotinamide on secondary injury pathways involves effects on inflammatory response, signaling pathways, and cell death.

Transcriptome correlation analysis identifies two unique craniosynostosis subtypes associated with IRS1 activation.

The discovery of causal mechanisms associated with nonsyndromic craniosynostosis has proven to be a difficult task due to the complex nature of the disease. In this study, differential transcriptome correlation analysis was used to identify two molecularly distinct subtypes of nonsyndromic craniosynostosis, termed subtype A and subtype B. In addition to unique correlation structure, subtype A was also associated with high IGF pathway expression, whereas subtype B was associated with high integrin expression. To identify a pathologic link between altered gene correlation/expression and the disease state, phosphorylation assays were performed on primary osteoblast cell lines derived from cases within subtype A or subtype B, as well as on primary osteoblast cell lines with novel IGF1R variants previously reported by our lab (Cunningham ML, Horst JA, Rieder MJ, Hing AV, Stanaway IB, Park SS, Samudrala R, Speltz ML. Am J Med Genet A 155A: 91-97, 2011). Elevated IRS1 (pan-tyr) and GSK3ß (ser-9) phosphorylation were observed in two novel IGF1R variants with receptor L domain mutations. In subtype A, a hypomineralization phenotype coupled with decreased phosphorylation of IRS1 (ser-312), p38 (thr-180/tyr-182), and p70S6K (thr-412) was observed. In subtype B, decreased phosphorylation of IRS1 (ser-312) as well as increased phosphorylation of Akt (ser-473), GSK3ß (ser-9), IGF1R (tyr-1135/tyr-1136), JNK (thr-183/tyr-187), p70S6K (thr-412), and pRPS6 (ser-235/ser-236) was observed, thus implicating the activation of IRS1-mediated Akt signaling in potentiating craniosynostosis in this subtype. Taken together, these results suggest that despite the stimulation of different pathways, activating phosphorylation patterns for IRS1 were consistent in cell lines from both subtypes and the IGF1R variants, thus implicating a key role for IRS1 in the pathogenesis of nonsyndromic craniosynostosis.

Functional variants of the lipoprotein lipase gene and the risk of preeclampsia among non-Hispanic Caucasian women.

Hypertriglyceridemia is an important pathophysiologic feature of preeclampsia, a common vascular disorder of pregnancy. Three well-documented functional variants (N291S, S447X, and D9N) of the lipoprotein lipase gene were related to hypertriglyceridemia. Results from the only two studies concerning the relationship between these variants and preeclampsia risk have been inconsistent. We investigated this relationship in a case-control study including 144 preeclamptic and 290 normotensive pregnant women (all non-Hispanic Caucasians). We estimated odds ratios (OR) and 95% confidence intervals (CI) adjusted for maternal age, pre-pregnancy body mass index, and parity. After adjusting for covariates, women with the 291 N/S or S/S genotype had significantly increased risk of preeclampsia (OR 6.9, 95% CI 1.9-25.4) compared with women with the common 291N/N genotype. The 447 S/X or X/X genotype was not significantly associated with preeclampsia risk. The frequency of the 9N variant allele was 1.8% in controls, while this allele was not observed among cases. Haplotype 9D/291S/447S was strongly associated with higher risk of preeclampsia as compared with the most common haplotype 9D/291N/447S (adjusted OR 6.6, 95% CI 1.7-25.0). Results from our study support the thesis that abnormal lipid metabolism is important in the pathogenesis of preeclampsia.

Phytochemical-induced changes in gene expression of carcinogen-metabolizing enzymes in cultured human primary hepatocytes.

1. The naturally occurring compounds curcumin (CUR), 3,3'-diindolylmethane (DIM), isoxanthohumol (IXN), 8-prenylnaringenin (8PN), phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) protect animals against chemically induced tumours. Putative chemoprotective mechanisms include modulated expression of hepatic biotransformation enzymes. However, few, if any, studies have used human primary cells as test models. 2. The present study investigated the effects of these phytochemicals on the expression of four carcinogenesis-relevant enzymes--cytochrome P450 (CYP)1A1 and 1A2, NAD(P)H:quinone oxidoreductase (NQO1) and glutathione S-transferase A1 (GSTA1)--in primary cultures of freshly isolated human hepatocytes. 3. Quantitative RT-PCR analyses demonstrated that CYP1A1 was up-regulated by PEITC and DIM in a dose-dependent manner. CYP1A2 transcription was significantly activated following DIM, IXN, 8PN and PEITC treatments. DIM exhibited a remarkably effective induction response of CYP1A1 (474-, 239- and 87-fold at 50, 25 and 10 microM, respectively) and CYP1A2 (113-, 70- and 31-fold at 50, 25 and 10 microM, respectively), that was semiquantitatively reflected in protein levels. NQO1 expression responded to PEITC (11 x at 25 microM), DIM (4.5 x at 50 microM) and SFN (5 x at 10 microM) treatments. No significant effects on GSTA1 transcription were seen. 4. The findings show novel and unexpected effects of these phytochemicals on the expression of human hepatic biotransformation enzymes that play key roles in chemical-induced carcinogenesis.

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.

Vitamin D receptor polymorphism and the risk of colorectal adenomas: evidence of interaction with dietary vitamin D and calcium.

Laboratory studies and epidemiological investigations suggest that vitamin D plays a role in the etiology of colorectal adenomas, possibly through a mechanism mediated by the vitamin D receptor (VDR). We conducted a clinic-based case-control study to examine the association between VDR polymorphisms and colorectal adenomas. We selectively identified a random subset of 393 cases of colorectal adenomas and 406 colonoscopy-negative controls from a clinic-based case-control study conducted in the metropolitan Minneapolis/St. Paul area during 1991-1994. A self-administered questionnaire was used to collect data on dietary and supplement intake of vitamin D and calcium, as well as on demographics, physical activity, medical information, lifestyle factors, reproductive history, and anthropometry. DNA was extracted from whole blood and assayed for the BsmI VDR polymorphism using an ABI 7700 TaqMan assay. Adjusted odds ratios (OR) and 95% confidence intervals (CIs) were evaluated using logistic regression. Compared with the bb genotype (33% of controls), neither the Bb (48.8% of controls) nor the BB (18.2% of controls) genotypes was strongly associated with risk of colorectal adenomas (OR = 0.86, CI = 0.63-1.19 and OR = 0.77, CI = 0.50-1.18, respectively). However, those with the lowest tertile of vitamin D intake and the BB genotype had a lower risk of colorectal adenoma (OR = 0.24, CI = 0.08-0.76) than those with the highest tertile of intake and the bb genotype. Similarly, those with the lowest tertile of calcium intake and the BB genotype had a reduced risk of colorectal adenoma (OR = 0.34, CI = 0.11-1.06). Although it has generally been shown that higher calcium and vitamin D intake are associated with a modestly reduced risk of colorectal neoplasia, our data suggest that those with the BB BsmI VDR genotype may be at reduced risk of colorectal adenoma in the presence of lower calcium and vitamin D intake.

Genetic polymorphisms of superoxide dismutase in Parkinson's disease.

Oxidative stress reactions may contribute to the pathogenesis of Parkinson's disease (PD). The superoxide dismutases potentially play significant roles in PD by detoxifying superoxide radical. We developed genomic DNA and cDNA-based sequencing assays to identify genetic variants in the copper/zinc superoxide dismutase (SOD1) and manganese superoxide dismutase (SOD2) genes. No genetic variants were detected in the gene encoding SOD1 in DNA from 45 idiopathic PD cases and 49 controls from a population-based case-control study. However, we identified a previously described polymorphism of the mitochondrial targeting sequence consisting of a C47T in exon 2 of SOD2, which results in an alanine to valine substitution. We analyzed this SOD2 variant in DNA from 155 cases and 231 controls from the same study, using an allele-specific fluorogenic 5' nuclease assay, and found no differences in the distributions of allelic frequencies. These results indicate that SOD gene variants do not contribute to PD pathogenesis.

Tissue specific changes in the expression of glutamate-cysteine ligase mRNAs in mice exposed to methylmercury.

Glutamate-cysteine ligase (GLCL), the rate-limiting enzyme in glutathione (GSH) synthesis is composed of two subunits, a catalytic (GLCLc) and a regulatory subunit (GLCLr). These two subunits are known to be differentially regulated in vitro, in different cell types and in response to various xenobiotic exposures. In this study, we examined whether these two subunits can also be differentially regulated in vivo. We found that GLCLc and GLCLr are differentially regulated at the transcriptional level in a tissue-dependent manner in female mice treated with methylmercury (MeHg). MeHg caused a downregulation of both subunit mRNAs in the liver, upregulation of both subunit mRNAs in the kidney and upregulation of only the catalytic subunit mRNA in the small intestine of female mice treated with a single dose of MeHg (6 mg/kg) by intraperitoneal injection. These results suggest that GLCLc and GLCLr can be differentially regulated in vivo, and that this regulation is tissue dependent in the mouse.

Pharmacogenetics of methotrexate: toxicity among marrow transplantation patients varies with the methylenetetrahydrofolate reductase C677T polymorphism.

This study investigated whether a polymorphism in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene (C677T) modifies responses to methotrexate (MTX) in patients undergoing bone marrow transplantation. About 10% to 12% of the population carry the MTHFR TT genotype (enzyme activity, 30% of wild type [CC]). Patients (n = 220) with chronic myelogenous leukemia underwent marrow allografts and were given a short course of MTX. MTX toxicity measures included the oral mucositis index (OMI), speed of engraftment (platelet and granulocyte counts), and bilirubin. Patients with lower MTHFR activity (TT genotype) had 36% higher mean OMI during days 1 to 18 (+5.7, P =.046) and 20% higher OMI between days 6 and 12 (+3.8, P =.27). Platelet counts recovered more slowly among patients with the TT genotype compared to wild type (24% slower recovery to 10 000 platelets/microL, P =.23; 34% slower to 20 000/microL, P =.08). Patients with decreased MTHFR activity appear at risk of higher MTX toxicity. Because of the high prevalence of the TT genotype, these results may have implications for MTX dosage.

Genetic polymorphisms as biomarkers of sensitivity to inhaled sulfur dioxide in subjects with asthma.

BACKGROUND: Individuals with asthma are sensitive to inhaled sulfur dioxide (SO2); decrements in pulmonary function occur after exposure to low concentrations even for a short duration of time. There is a great amount of interindividual variation in response to SO2. OBJECTIVE: It was our objective to determine whether one of the following polymorphism locations linked with asthma is associated with the bronchial hyperresponsiveness to SO2 observed in some asthmatic patients: the beta2-adrenergic receptor, interleukin-4 (IL-4) receptor alpha subunit, Clara cell secretory protein (CC16), TNF-alpha gene promoter, and first intron of the lymphotoxin alpha (LT-alpha) gene. METHODS: Subjects were volunteers with physician-diagnosed asthma requiring regular asthma medication. Spirometry was performed before and after a 10-minute exposure to 0.5 ppm SO2. Subjects were classified as SO2 responders if forced expiratory volume in 1 second (FEV1) decreased > or = 12%. DNA obtained from buccal cell samples was analyzed for genetic polymorphisms. RESULTS: Of the 62 subjects (21 male and 41 female), 13 had a 12% or greater decrement in FEV1 after SO2 exposure (range + 19% to -49%). Response to SO2 was associated with the wild-type allele of the TNF-alpha promoter polymorphism (12 of 12 SO2 responders versus 28 of 46 nonresponders; P < .05) but with no other polymorphisms. Medication category and atopic status showed no association with SO2 sensitivity. CONCLUSIONS: The wild-type allele of the TNF-alpha promoter polymorphism may be associated with mechanisms of asthmatic sensitivity to inhaled SO2.

Mitochondrial ND1 sequence analysis and association of the T4216C mutation with Parkinson's disease.

Mitochondrial dysfunction originating from mutations in Complex I genes may play a role in the pathogenesis of Parkinson's disease (PD). In this study, the entire ND1 coding sequence was sequenced in 84 newly diagnosed PD cases and 127 age/gender-matched controls. Numerous missense mutations were found at low frequency (<5%), whereas a thymidine to cytosine missense mutation at position 4216 that results in the replacement of tyrosine with histidine was found in 25% of the PD case samples and in 18% of the controls. When calculated according to gender, the 4216 mutation was observed in 26% of the male cases versus 16% of male controls (Odds Ratio [OR] = 1.85; 95% CI = 0.79-4.34). In contrast, females exhibited approximately equal frequencies among cases (22.5%) and controls (21%), yielding an OR of 1.08 (95% C.I. = 0.36-3.22). The findings indicate only a weak association of this genetic variant with PD.

Searching expressed sequence tag databases: discovery and confirmation of a common polymorphism in the thymidylate synthase gene.

Databases of expressed sequence tags (EST) can be used to screen rapidly for potential polymorphisms in candidate proteins. As part of this study, we screened the gene for the enzyme thymidylate synthase (TS). TS is important physiologically because it is essential for the synthesis of deoxythymidylate, a nucleotide required for DNA synthesis and repair. TS is also a major target for cancer chemotherapeutic drugs, especially the widely used 5-fluorouracil. Using sequence alignment of ESTs, we identified a candidate 6-bp variation at bp 1494 in the 3'-untranslated region of the TS mRNA. This sequence variation occurred in 21 of 34 aligned ESTs at this location, including ESTs from various tissue sources. The presence of this polymorphism was confirmed in a Caucasian population (n = 95) by polymerase chain restriction amplification/RFLP analysis. The allele frequency of the 6-bp deletion was found to be 0.29 (wildtype +6 bp/+6 bp, 48%; +6 bp/-6 bp, 44%; -6 bp/-6 bp, 7%). Although the function of this polymorphism has not yet been investigated, the 3'-untranslated region of a gene can play a role in mRNA stability and translation. This study illustrates an approach to polymorphism discovery in candidate enzymes of physiological interest by searches of publicly available sequence data, a rapid and inexpensive method. The potential functional relevance of the common 6-bp deletion in the TS gene needs to be investigated, because this enzyme is plausibly of major importance not only in cancer treatment but also in cancer prevention.

Genetic polymorphisms in Parkinson's disease.

The search for genetic polymorphisms relevant to Parkinson's disease etiology and pathogenesis has been motivated by recent thinking emphasizing the potential significance of gene-environment interactions. Especially influential to this research have been the MPTP model of PD induction, hypotheses concerning oxidative stressor reactions, and epidemiological observations of an inverse relation between cigarette smoking and PD risk. This brief review summarizes trends in genetic polymorphism research, with examples provided by investigations of cytochrome P450 enzymes, monoamine oxidase, superoxide dismutase, and mitochondrial genes.

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.

Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry.

Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and beta-naphthoflavone (beta NF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-microns thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from beta NF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur.

Expression of cytochrome P450 and microsomal epoxide hydrolase in cervical and oral epithelial cells immortalized by human papillomavirus type 16 E6/E7 genes.

Epidemiological evidence suggests that the presence of human papillomaviruses (HPV), when combined with smoking behaviors, considerably enhances the risk of developing oral, cervical, vulvar, and/or anal carcinomas. It is well established that the cytochrome P450 (CYP), microsomal epoxide hydrolase (mEH), and other biotransformation enzymes are important modulators of the bioactivation and detoxification of many environmental chemicals, including constituents of tobacco smoke such as certain nitrosamines and polycyclic aromatic hydrocarbons (PAH). Since there is little information regarding oral and cervical epithelial-specific expression of these genes, established primary and HPV-immortalized oral and cervical epithelial cell lines were analyzed for morphology, mRNA and protein expression patterns of specific CYPs and mEH. Primary human oral and cervical epithelial cells were immortalized using retroviral infection with HPV-16 E6/E7 genes. Primary human keratinocyte cells were immortalized by transfection of HPV-18 and made tumorigenic with nitrosomethylurea treatment. Expression profiles for mEH, CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP3A, and CYP2E1 were evaluated in these cultures in the presence or absence of a PAH inducer, using reverse transcriptase-coupled polymerase chain reaction analysis. mEH gene expression was evident in all cultures, while CYP2A6 mRNA was not detected in any of the cell lines, regardless of culture conditions. CYP2E1 mRNA expression was greatest in the oral epithelial cultures and detectable in all other epithelial cultures except for the HPV-18 immortalized keratinocyte cell line. Elevated levels of CYP2D6 mRNA existed in both oral epithelial cell lines and the HPV-16 immortalized cervical epithelial cells when compared to the other cell lines examined. CYP1A1 and CYP1A2 mRNAs were detected in all the cells and several cultures were inducible by PAH exposure. To corroborate the RT/PCR data, Western immunoblotting experiments were conducted on selected samples. Using these methods, CYP1A1 and CYP2E1 proteins were detected in primary and HPV-immortalized oral and cervical epithelial cultures. These data indicate that both primary and HPV immortalized cells appear to express certain biotransformation enzymes necessary for the activation of tobacco-specific nitrosamines and PAHs. Although the overall impact of HPV gene infection on expression of these systems remains to be fully elucidated, as in vitro system is characterized which should prove useful in examining interactive mechanisms of HPV with xenobiotic activation in the etiology of squamous cell carcinomas.

Expression of cytochrome P450s and microsomal epoxide hydrolase in primary cultures of human umbilical vein endothelial cells.

In view of the potential role of the cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) biotransformation enzymes in the metabolism of protoxicants in the circulatory system, we examined CYP and mEH expression in several primary cultures of human umbilical vein endothelial cells (HUVEC), each established from a different individual. Total RNA was isolated from untreated cells and cells 72 hr after exposure to dimethyl sulfoxide (DMSO), Arochlor 1254 (PCB), and beta-naphthoflavone (beta NF). Specific mRNA transcripts were examined by Northern blotting and reverse transcriptase-coupled polymerase chain reaction (RT/PCR) analyses. CYP2E1, CYP3A, and CYP1A2 mRNAs were not detectable in any of the cultures by Northern blot analysis with radiolabeled oligomer probes; however, CYP1A1 mRNA was detected using this procedure in HUVEC cultures exposed to beta NF for 72 hr. Using RT/PCR, constitutive levels of CYP1A1, CYP1A2, CYP2E1, and CYP3A gene expression in HUVEC cultures were evident; however, constitutive CYP2B6 mRNA was not detected. Constitutive CYP1A2 transcript levels were detected in four of six HUVEC cultures, but levels varied between individual cultures. CYP1A2 mRNA levels were also increased in HUVEC cultures exposed to PCB and beta NF. No increases in the levels of CYP2E1 and CYP3A mRNAs were observed in HUVEC cells subsequent to PCB or beta NF exposures. Constitutive CYP2E1 transcript levels were present in all HUVEC cultures examined and varied among individuals. All HUVEC cultures examined for mEH activity exhibited constitutive levels of mEH which varied 40% between individual cultures and produced on average, 1.51 pmol benzo[a]pyrene 4,5-dihydrodiol per milligram protein per minute of reaction. Thus, these results demonstrate that human endothelial cells express CYP and mEH gene products and suggest that these enzymes may play important roles in determining metabolic fates for circulating protoxicants.