Human Epoxide Research Articles

LC-MS/MS Analysis of the Epoxides and Diols Derived from the Endocannabinoid Arachidonoyl Ethanolamide.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a useful tool to characterize the behavior of natural lipids within biological matrices. We report a LC-MS/MS method developed specifically to analyze CYP products of the arachidonoyl ethanolamide (anandamide, AEA), the epoxyeicosatrienoic acid ethanolamides (EET-EAs) and their hydrolyzed metabolites, and the dihydroxyeicosatrienoic acid ethanolamides (DHET-EAs). This method was used to measure EET-EA biotransformation to DHET-EAs by two human epoxide hydrolases: the soluble EH (sEH) and the microsomal EH (mEH). In general, sEH and mEH substrate preference was similar, based on kcat/KM. The 14,15-EET-EA and 11,12-EET-EA were the most efficiently hydrolyzed, followed by 8,9-EET-EA and 5,6-EET-EA. The method was also used to detect endogenous levels of these lipids in mouse tissues, although levels were below the instrumental detection limit (0.1-3.4nM). Because both AEA and EETs are biologically active, the method described herein will be invaluable in revealing the role(s) of EET-EAs in vivo.

Synthesis and inhibitory activity of 1,3-(adamantan-1(2)-yl)- imidazolidine-2,4,5-triones and 3,3'-(adamantan-1-yl)- bis(1-alkylimidazolidine-2,4,5-triones)

A series of 1,3-(adamantan-1(2)-yl)imidazolidine-2,4,5-triones and 1,1'-(alkane-1,n-diyl)bis[3-(adamantan-1-yl)imidazolidine-2,4,5-triones] was synthesized via cyclization of 1,3-bis[adamantan-1(2)-ylureas] and 1,1'-(alkyl-1,n-diyl)bis[3-(adamantan-1-yl)ureas] with oxalyl chloride under mild conditions with high yields. All synthesized compounds were tested in vitro as inhibitors of soluble human epoxide hydrolase. A number of compounds have high inhibitory activity (IC50 1.6–650 nM), which makes them promising inhibitors of soluble epoxide hydrolase.

Tissue Expressions of Soluble Human Epoxide Hydrolase-2 Enzyme in Patients with Temporal Lobe Epilepsy.

We sought to simply demonstrate how levels of soluble human epoxide hydrolase-2 show changes in both temporal the cortex and hippocampal complex in patients with temporal lobe epilepsy. A total of 20 patients underwent anterior temporal lobe resection due to temporal lobe epilepsy. The control group comprised 15 people who died in traffic accidents or by falling from a height, and their autopsy findings were included. Adequately sized temporal cortex and hippocampal samples were removed from each patient during surgery, and the same anatomic structures were removed from the control subjects during the autopsy procedures. Each sample was stored at-80°C as rapidly as possible until the enzyme assay. The temporal cortex in the epilepsy patients had a significantly higher enzyme level than did the temporal cortex of the control group (P= 0.03). Correlation analysis showed that as the enzyme level increases in the temporal cortex, it also increases in the hippocampal complex (r2= 0.06, P= 0.00001). More important, enzyme tissue levels showed positive correlations with seizure frequency in both the temporal cortex and hippocampal complex in patients (r2= 0.7, P= 0.00001 and r2= 0.4, P= 0.003, respectively). The duration of epilepsy was also positively correlated with the hippocampal enzyme level (r2= 0.06, P= 0.00001). Soluble human epoxy hydrolase enzyme-2 is increased in both lateral and medial temporal tissues in temporal lobe epilepsy. Further studies should be conducted as inhibition of this enzyme has resulted in a significant decrease in or stopping of seizures and attenuated neuroinflammation in experimental epilepsy models in the current literature.

The Association between mrna Expression Levels of Ephx1 and Prognosis of Acute Myeloid Leukemia

The human epoxide hydrolase 1 (EPHX1) is a metabolism gene, which is responsible for the first phase of the xenobiotic metabolism. The product, microsomal epoxide hydrolase (mHE), plays an important role in the detoxification of carcinogenic polycyclic aromatic hydrocarbons. Many reports also have studied the correlation between EPHX1 gene polymorphisms and development and generation of cancers. However, few of studies on EPHX1 participating in chemotherapeutics metabolism were reported. The purpose of the present study was to evaluate the prognostic significance of the EPHX1gene expression in acute myeloid leukemia (AML). Using real time‑quantitative polymerase chain reaction (RT‑qPCR), mRNA expression levels of EPHX1gene was measured in bone marrow samples of newly diagnosed AML patients (n=46) and control group (n=12) without tumors. The levels expression of EPHX1 had a significant association with the treatment response and prognosis of AML patients. Additionally, the EPHX1 expression levels can aid for an improved understanding of the multidrug resistant mechanism in AML.

EH3 (ABHD9): the first member of a new epoxide hydrolase family with high activity for fatty acid epoxides

Epoxide hydrolases are a small superfamily of enzymes important for the detoxification of chemically reactive xenobiotic epoxides and for the processing of endogenous epoxides that act as signaling molecules. Here, we report the identification of two human epoxide hydrolases: EH3 and EH4. They share 45% sequence identity, thus representing a new family of mammalian epoxide hydrolases. Quantitative RT-PCR from mouse tissue indicates strongest EH3 expression in lung, skin, and upper gastrointestinal tract. The recombinant enzyme shows a high turnover number with 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid (EET), as well as 9,10-epoxyoctadec-11-enoic acid (leukotoxin). It is inhibited by a subclass of N,N'-disubstituted urea derivatives, including 12-(3-adamantan-1-yl-ureido)-dodecanoic acid, 1-cyclohexyl-3-dodecylurea, and 1-(1-acetylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea, compounds so far believed to be selective inhibitors of mammalian soluble epoxide hydrolase (sEH). Its sensitivity to this subset of sEH inhibitors may have implications on the pharmacologic profile of these compounds. This is particularly relevant because sEH is a potential drug target, and clinical trials are under way exploring the value of sEH inhibitors in the treatment of hypertension and diabetes type II.

Genetic Polymorphism of Microsomal Epoxide Hydrolase Enzyme Gene in Preeclamptic Females

IntroductionMicrosomal epoxide hydrolase enzyme is involved in xenobiotics detoxification. It catalyzes the phase I hydrolysis of epoxides and plays a role in the detoxification processes and in the metabolism of endogenous and exogenous compounds. Preeclampsia, which is one of the most serious complications of pregnancy, may be due to an imbalance between these compounds, such as lipid peroxides and oxygen-free radicals and detoxifying and scavenging substances. Two variants of human epoxide hydrolase enzyme with different enzyme activity have been described; exon 3 polymorphism is associated with lower enzyme activity whereas exon 4 polymorphism is associated with higher activity. The authors tried to investigate the association between these genetic polymorphisms and preeclampsia. MethodThirty preeclamptic females together with 30 normal pregnant females as controls were included in the study. Genotyping for exons 3 and 4 of microsomal epoxide hydrolase enzyme was done by polymerase chain reaction–restriction fragment length polymorphism. ResultsThere was no statistical significant difference in the distribution of exon 3 genotype between cases and controls (P= 0.4); on the other hand, a highly statistical significant difference was found between cases and controls as regard exon 4 genotype (P= 0.002). ConclusionThere may be an association between epoxide hydrolase enzyme polymorphism and the risk of preeclampsia.

EPHX1 Gene Polymorphisms in Alcohol Dependence and their Distribution among the Indian Populations

Background: The microsomal epoxide hydrolase is a phase II enzyme of the biotransformation. The human epoxide hydrolase 1 (EPHX1) gene lies in the chromosomal region 1q42.1 and exhibits polymorphism. Two single nucleotide polymorphisms (SNPs) have been described in the coding region of the EPHX1 gene that produces two protein variants. Subjects and methods: A total of 604 samples belonging to 13 Indian populations were included in this study. Based on the DSM-IV criteria, 184 individuals from Kota population were classified into alcoholism cases (100) and controls (84). Genotypes of Tyr113His and His139Arg polymorphisms in the EPHX1 gene were determined using PCR and sequencing. Associations were tested using Pearson’s χ2 test and haplotype analyses. Results: We found significant association between EPHX1 gene Tyr113His polymorphism and alcoholism in the Kota population (T vs. C: OR = .615, 95% CI = .399–.949, p = .027; TT vs. CC + CT: OR = .536, 95% CI = .297–.969, p = .038). The very slow activity haplotype CA (113His–139His) was also found to be associated with alcohol dependence (p = .048). Analysis of additional populations demonstrated that the Tyr113His polymorphism significantly deviated from Hardy–Weinberg equilibrium in four populations but only one population deviated for the His139Arg locus. All populations shared the four possible two-site haplotypes. Linkage disequilibrium between these two loci was not significant in any of the population studied. Conclusion: EPHX1 gene polymorphisms and haplotypes are associated with an increased risk for alcoholism in the Kota population. This is the first report from India that will serve as a template for future investigations of the prevalence of EPHX1 alleles in association with various clinical entities.

Identification of a new potential human epoxide hydrolase (ABHD7)

Two human EHs are well‐investigated. The microsomal EH (mEH) hydrolyzes a large variety of structurally different xenobiotic‐derived genotoxic epoxides and therefore is implicated in detoxification. The soluble EH (sEH) converts signalling molecules such as epoxyeicosatrienoic acids (EETs) to the corresponding dihydroxyeicosatrienoic acids (DHETs) and thus is involved in the regulation of physiological processes. Using the signature sequence RVIAPDLRGYGDSDKP that is reasonably conserved among epoxide hydrolases and some other structurally related enzymes of the α/β hydrolase fold superfamily, we could recently identify two new genes ABHD9 and ABHD7. All features so far identified to be necessary for a functional EH, including a catalytic triad with an aspartic acid in the position of the catalytic nucleophile, as well as two catalytic tyrosines, are conserved in the primary structures of the proteins predicted from these genes. The amino acid sequences of ABHD9 and ABHD7 share 42% identity and therefore belong to the same family being different from mEH and sEH (sequence similarity below 25%). Our previous studies show an EET converting activity of ABHD9. To elucidate the function of ABHD7 we are presently working on the recombinant expression and biochemical characterization of this new enzyme.The present work is supported by the Swiss National Fonds.

Unexpected DNA damage caused by polycyclic aromatic hydrocarbons under standard laboratory conditions

The genotoxicity of 15 polycyclic aromatic hydrocarbons was determined with the alkaline version of the comet assay employing V79 lung fibroblasts of the Chinese hamster as target cells. These cells lack the enzymes necessary to convert PAHs to DNA-binding metabolites. Surprisingly, 11 PAHs, i.e., benzo[ a]pyrene (BaP), benz[ a]anthracene, 7,12-dimethylbenz[ a]anthracene, 3-methylcholanthrene, fluoranthene, anthanthrene, 11 H-benzo[ b]fluorene, dibenz[ a,h]anthracene, pyrene, benzo[ ghi]perylene and benzo[ e]pyrene caused DNA strand breaks even without external metabolic activation, while naphthalene, anthracene, phenanthrene and naphthacene were inactive. When the comet assay was performed in the dark or when yellow fluorescent lamps were used for illumination the DNA-damaging effect of the 11 PAHs disappeared. White fluorescent lamps exhibit emission maxima at 334.1, 365.0, 404.7, and 435.8 nm representing spectral lines of mercury. In the case of yellow fluorescent lamps these emissions were absent. Obviously, under standard laboratory illumination many PAHs are photo-activated, resulting in DNA-damaging species. This feature of PAHs should be taken into account when these compounds are employed for the initiation of skin cancer. The genotoxicity of BaP that is metabolically activated in V79 cells stably expressing human cytochrome P450-dependent monooxygenase (CYP1A1) as well as human epoxide hydrolase (V79-hCYP1A1-mEH) could not be detected with the comet assay performed under yellow light. Likewise the DNA-damaging effect of r-7, t-8-dihydroxy- t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene ( anti-BaPDE) observed with the comet assay was only weak. However, upon inhibition of nucleotide excision repair (NER), which is responsible for the removal of stable DNA adducts caused by anti-BaPDE, the tail moment rose 3.4-fold in the case of BaP and 12.9-fold in the case of anti-BaPDE. These results indicate that the genotoxicity of BaP and probably of other compounds producing stable DNA adducts are reliably detected with the comet assay only when NER is inhibited.

Association of microsomal epoxide hydrolase gene polymorphism and pre‐eclampsia in Turkish women

To assess the association between human epoxide hydrolase exon 3 and 4 polymorphisms and pre-eclampsia by carrying out a case-control study in Turkish women. DNA was extracted from peripheral blood leukocytes, and genotype distribution of exon 3 and exon 4 of epoxide hydrolase gene (EPHX) was carried out in 271 patients and 155 controls. There was no statistically significant difference in the distribution of genotypes between pre-eclampsia without HELLP and pre-eclampsia plus HELLP cases and controls for the exon 3 and 4 polymorphism of EPHX. However, we found a significant association between the predicted enzyme activity level and pre-eclampsia (P = 0.018). The distribution of subjects with predicted high, intermediate and low microsomal epoxide hydrolase enzyme (EPHX) activity were 23.2, 38.8 and 38% in cases and 12, 47.3 and 40.7% in controls, respectively. Although we could not find any association between genetic variability in exon 3 and 4 of EPHX and pre-eclampsia, genetic variability in these two exons jointly modifies the predicted enzyme activity and may be a risk factor for pre-eclampsia.

A novel, sensitive method for determining benzo[a]pyrene-diones using high-performance liquid chromatography with post-column zinc reduction

A novel and sensitive high-performance liquid chromatography (HPLC) method was developed to analyze dione metabolites of benzo[a]pyrene (BaP). Because BaP-diones do not fluoresce, detection of low concentrations is difficult to achieve when analyzing these chemicals with a simple HPLC system. We developed a method to increase the detection sensitivities for BaP-diones using reduction by zinc after the chromatographic separation. A post-column zinc reducer was used to convert BaP-diones, in-line, to their corresponding fluorescent BaP-hydroquinones, which can be measured by fluorescence detection with high sensitivity. With 20-muL injections, the limits of detection for the BaP-diones tested (BaP-1,6-dione, BaP-3,6-dione, and BaP-6,12-dione) were all below 1.0 nM. In addition to the high detection sensitivity, this HPLC method provides a wide linear dynamic range for BaP-dione detection (1.0-220 nM). We also studied the extraction recovery of BaP-diones from recombinant human cytochrome P450 and epoxide hydrolase. To demonstrate the application of this method, the kinetics of BaP-dione formation was studied by incubating BaP with these recombinant enzymes. The present method enhances the detection sensitivity for BaP-diones by more than two orders of magnitude compared with traditional ultraviolet detection. Moreover, the method avoids the time-consuming derivatization or reduction steps required by other methods.

Kava does not display metabolic toxicity in a homogeneous cellular assay.

To determine whether kava (Kava kava, 'Awa, Yaqona, Piper methysticum Forst.), the popular herbal product associated recently with possible human hepatotoxicity, is bioactivated by cytochrome P450 enzymes to cytotoxic metabolites, three kava lactones (methysticin, yangonin, and desmethoxyyangonin) and an ethanolic extract of dried kava root were incubated over time in culture with MCL-5 cells, a human lymphoblastoid cell line that has been stably transfected with five human P450's (CYP 1A1, 1A2, 2A6, 2E1, and 3A4) and human epoxide hydrolase. Incubations were performed concurrently with a control cell line (cH2) that is derived from the same parental line as MCL-5, but transfected with two empty vectors. The kava compounds displayed varying degrees of toxicity (IC (50) values ranged from 50 to > 100 microM) to the MCL-5 and cH2 cell lines; however, both cell lines were equally sensitive to the test compounds. These results suggest that the parent compound for each of the four test compounds was primarily responsible for the observed cell toxicity and that CYP 1A1, 1A2, 2A6, 2E1, and 3A4 or epoxide hydroxylase did not appear to be involved. Thus, in vitro kava does not appear to be activated to toxic metabolites by enzymes known to be important in metabolic toxicity.

Structure of human epoxide hydrolase reveals mechanistic inferences on bifunctional catalysis in epoxide and phosphate ester hydrolysis.

The X-ray crystal structure of human soluble epoxide hydrolase (sEH) has been determined at 2.6 A resolution, revealing a domain-swapped quaternary structure identical to that observed for the murine enzyme [Argiriadi, M. A., Morisseau, C., Hammock, B. D., and Christianson, D. W. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10637-10642]. As with the murine enzyme, the epoxide hydrolytic mechanism of the human enzyme proceeds through an alkyl-enzyme intermediate with Asp-333 in the C-terminal domain. The structure of the human sEH complex with N-cyclohexyl-N'-(iodophenyl)urea (CIU) has been determined at 2.35 A resolution. Tyr-381 and Tyr-465 donate hydrogen bonds to the alkylurea carbonyl group of CIU, consistent with the proposed roles of these residues as proton donors in the first step of catalysis. The N-terminal domain of mammalian sEH contains a 15 A deep cleft, but its biological function is unclear. Recent experiments demonstrate that the N-terminal domain of human sEH catalyzes the metal-dependent hydrolysis of phosphate esters [Cronin, A., Mowbray, S., Dürk, H., Homburg, S., Fleming, I., Fisslthaler, B., Oesch, F., and Arand, M. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 1552-1557; Newman, J. W., Morisseau, C., Harris, T. R., and Hammock, B. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 1558-1563]. The binding of Mg(2+)-HPO4(2-) to the N-terminal domain of human sEH in its CIU complex reveals structural features relevant to those of the enzyme-substrate complex in the phosphatase reaction.

The ubiquitous aldehyde reductase (AKR1A1) oxidizes proximate carcinogen trans-dihydrodiols to o-quinones: potential role in polycyclic aromatic hydrocarbon activation.

Polycyclic aromatic hydrocarbons (PAHs) are metabolized to trans-dihydrodiol proximate carcinogens by human epoxide hydrolase (EH) and CYP1A1. Human dihydrodiol dehydrogenase isoforms (AKR1C1-AKR1C4), members of the aldo-keto reductase (AKR) superfamily, activate trans-dihydrodiols by converting them to reactive and redox-active o-quinones. We now show that the constitutively and widely expressed human AKR, aldehyde reductase (AKR1A1), will oxidize potent proximate carcinogen trans-dihydrodiols to their corresponding o-quinones. cDNA encoding AKR1A1 was isolated from HepG2 cells, overexpressed in Escherichia coli, purified to homogeneity, and characterized. AKR1A1 oxidized the potent proximate carcinogen (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene with a higher utilization ratio (V(max)/K(m)) than any other human AKR. AKR1A1 also displayed a high V(max)/K(m) for the oxidation of 5-methylchrysene-7,8-diol, benz[a]anthracene-3,4-diol, 7-methylbenz[a]anthracene-3,4-diol, and 7,12-dimethylbenz[a]anthracene-3,4-diol. AKR1A1 displayed rigid regioselectivity by preferentially oxidizing non-K-region trans-dihydrodiols. The enzyme was stereoselective and oxidized 50% of each racemic PAH trans-dihydrodiol tested. The absolute stereochemistries of the reactions were assigned by circular dichroism spectrometry. AKR1A1 preferentially oxidized the metabolically relevant (-)-benzo[a]pyrene-7(R),8(R)-dihydrodiol. AKR1A1 also preferred (-)-benz[a]anthracene-3(R),4(R)-dihydrodiol, (+)-7-methylbenz[a]anthracene-3(S),4(S)-dihydrodiol, and (-)-7,12-dimethylbenz[a]anthracene-3(R),4(R)-dihydrodiol. The product of the AKR1A1-catalyzed oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene was trapped with 2-mercaptoethanol and characterized as a thioether conjugate of benzo[a]pyrene-7,8-dione by LC/MS. Multiple human tissue expression array analysis showed coexpression of AKR1A1, CYP1A1, and EH, indicating that trans-dihydrodiol substrates are formed in the same tissues in which AKR1A1 is expressed. The ability of this general metabolic enzyme to divert trans-dihydrodiols to o-quinones suggests that this pathway of PAH activation may be widespread in human tissues.

A polymorphism in the gene for microsomal epoxide hydrolase is associated with pre-eclampsia

OBJECTIVEMicrosomal epoxide hydrolase is an important enzyme involved in the metabolism of endogenous and exogenous toxicants. Polymorphic variants of the human epoxide hydrolase gene vary in enzyme activity. We determined...

Human epoxide hydrolase is the target of germander autoantibodies on the surface of human hepatocytes: enzymatic implications.

Western countries observed an increased interest for the use of herbal medicines because of their supposed safety in contrast to chemical drugs. Wild germander (Teuchrium chamaedrys L) was traditionally used as a folk medicine for its for and antiseptic properties. In 1991, germander consumed to treat obesity, caused an epidemic of cytolytic hepatitis. Thirty cases of hepatotoxicity were first reported including cases with positive rechallengge. For these patients, an early recurrence was observed despite lack of other features of hypersensitivity (Castot and Larrey, 1992). In mice, germander toxicity required CYP3A-dependent metabolism (Loeper et al 1994). More specifically, the metabolic activation of the furan ring of the diterpenoid teucrin A (TA) (Kouzi et al 1994). TA- toxicity was via CYP3A-generated electrophilic metabolites that were detoxified by glutathione conjugation, depleted cellular thiols and caused apoptosis in isolated rat hepatocytes atocytes (Lekehal et al 1996; Fau et al 1997). However, these death processes did not explain the immunologial cal features observed in several cases of germander-induced hepatitis. To explain this immune process, patient’s sera consuming germander tea in great quantity were Tested by Western blot analysis. They contained autoantibodies directed against human microsomal epoxide hydrolase (hmEH), that was located both, in the endoplasmic reticulum and the plasma membrane (PM) of human hepatocyte and hmEH- expressing yeast. Germander-induced autoantibodies (GIAA) were shown to recognize hmEH on the cell surface. To implicate hmEH in the metabolic activation of TA we used a “humanized” yeast strain expressing human P450-reductase and cytochrome b5 transformed with CYP3A4 and/or hmEH cDNAs. TA was metabolized by CYP3A4 into a and hmEH with TA inactivated hmEH in a time-dependent-manner, in agreement with formation of reactive teuchrin A-metabolite that could covalently alter and inhibit hmEH. This modified enzyme may bypass the immunologic tolerance that normally exists for hmEH.

Structure of Aspergillus niger epoxide hydrolase at 1.8 Å resolution: implications for the structure and function of the mammalian microsomal class of epoxide hydrolases

Background: Epoxide hydrolases have important roles in the defense of cells against potentially harmful epoxides. Conversion of epoxides into less toxic and more easily excreted diols is a universally successful strategy. A number of microorganisms employ the same chemistry to process epoxides for use as carbon sources. Results: The X-ray structure of the epoxide hydrolase from Aspergillus niger was determined at 3.5 Å resolution using the multiwavelength anomalous dispersion (MAD) method, and then refined at 1.8 Å resolution. There is a dimer consisting of two 44 kDa subunits in the asymmetric unit. Each subunit consists of an α/β hydrolase fold, and a primarily helical lid over the active site. The dimer interface includes lid–lid interactions as well as contributions from an N-terminal meander. The active site contains a classical catalytic triad, and two tyrosines and a glutamic acid residue that are likely to assist in catalysis. Conclusions: The Aspergillus enzyme provides the first structure of an epoxide hydrolase with strong relationships to the most important enzyme of human epoxide metabolism, the microsomal epoxide hydrolase. Differences in active-site residues, especially in components that assist in epoxide ring opening and hydrolysis of the enzyme–substrate intermediate, might explain why the fungal enzyme attains the greater speeds necessary for an effective metabolic enzyme. The N-terminal domain that is characteristic of microsomal epoxide hydrolases corresponds to a meander that is critical for dimer formation in the Aspergillus enzyme.

Activation and detoxication of aflatoxin B 1

Aflatoxin B 1 (AFB 1) is a potent hepatocarcinogen in experimental animals and a hazard to human health in several parts of the world. Implementation of rational intervention plans requires understanding of aspects of the roles of individual chemical steps involved in its disposition. AFB 1 is activated to AFB 1 exo-8,0-epoxide primarily by cytochrome P450 (P450) enzymes, particularly P450 3A4. However, P450 3A4 and other P450s also oxidize AFB 1 to less dangerous products. The exo-epoxide is unstable in H 2O ( t 1 2 1 s at 25°C, k = 0.6 s −1) and the diol product undergoes base-catalyzed rearrangement to a dialdehyde that reacts with protein lysine residues. AFB 1 exo-8,9-epoxide reacts with DNA to give adducts in high yield (> 98%). This interaction is characterized by a K d of ∼ 1.4 mM, intercalation between base pairs, and rapid reaction with the guanyl N 7 atom ( k ∼ 40 s −1). A proton field on the periphery of DNA is postulated to catalyze hydrolysis and also conjugation. Rat and especially human epoxide hydrolase show very little rate acceleration of hydrolysis of AFB 1 exo- or endo-8,9-epoxide. However, glutathione transferases (GSTs) can catalyze AFB 1 exo-8,9-epoxide conjugation. Kinetic analysis indicates a range of ratios of k cat/ K d varying from 10 to 1700 s −1 M −1, with the polymorphic GST M1-1 having the highest activity of the human GSTs. Studies with human hepatocytes indicate a major role for GST M1-1 in AFB 1 conjugation and that the model chemoprotective agent oltipraz can act by both inducing GSTs and inhibiting P450s.

2,3-epoxy-4-hydroxynonanal, a potential lipid peroxidation product for etheno adduct formation, is not a substrate of human epoxide hydrolase.

Our previous studies have shown that 2,3-epoxy-4-hydroxynonanal, a reactive epoxy aldehyde capable of forming etheno adducts with DNA bases, is mutagenic and tumorigenic (Carcinogenesis, 14, 2073). The epoxy aldehyde can be generated from trans-4-hydroxy-2-nonenal, a lipid peroxidation product of omega-6 polyunsaturated fatty acids, by autoxidation or by incubation with fatty acid hydroperoxides or hydrogen peroxides (Chem. Res. Toxicol., 9, 306). These are plausible in vivo pathways for the formation of 2,3-epoxy-4-hydroxynonanal. The possibility that 2,3-epoxy-4-hydroxynonanal is a tumorigen of endogenous origin is suggested by recent observations that etheno bases are detected as background DNA lesions in untreated rodents and humans. A metabolic pathway critical for detoxification of 2,3-epoxy-4-hydroxynonanal involves the ring-opening by epoxide hydrolase, which abolishes its ability to form cyclic etheno DNA adducts. In this study, we examined whether 2,3-epoxy-4-hydroxynonanal is a substrate of cDNA expressed human epoxide hydrolase. Human epoxide hydrolase was expressed in TK- 143 cells (thymidine kinase-deficient human embryoblast) infected with recombinant vaccinia virus encoding human epoxide hydrolase cDNA. Controls consisted of the cells infected with vaccinia virus in the absence of human epoxide hydrolase cDNA. No hydrolysis occurred when [2,3-(3)H]2,3-epoxy-4-hydroxynonanal was incubated at 37 degrees C for 30 min at pH 7.4 with cells expressing human epoxide hydrolase, as indicated by the presence of a pair of radioactive peaks in reversed-phase HPLC chromatography, which comigrated with the UV standards of the two diastereomers of the epoxy aldehyde. The identity of these compounds as the intact epoxy aldehyde was further supported by derivatization to the 2,4-dinitrophenylhydrazones followed by reversed phase HPLC analysis. Similar results were observed with the control cells or with the heat deactivated human epoxide hydrolase. The epoxide hydrolase activity in the expressed cells was demonstrated by their ability to convert benzo[a]pyrene-4,5-dihydroepoxide to benzo[a]pyrene-trans-4,5-dihydrodiol under the same conditions. These results clearly indicate that 2,3-epoxy-4-hydroxynonanal is not a substrate of human epoxide hydrolase, and, thus strengthen its possible endogenous role in the formation of promutagenic exocyclic etheno adducts in vivo.

The role of human glutathione transferases and epoxide hydrolases in the metabolism of xenobiotics.

Human glutathione transferases (GSTs) are a multigene family of enzymes that are involved in the metabolism of a wide range of electrophilic compounds of both exogenous and endogenous origin. GSTs are generally recognized as detoxifying enzymes by catalyzing the conjugation of these compounds with glutathione, but they may also be involved in activation of some carcinogens. The memmalian GSTs can be differentiated into four classes of cytosolic enzymes and two membrane bound enzymes. Human epoxide hydrolases (EHs) catalyze the addition of water to epoxides to form the corresponding dihydrodiol. The enzymatic hydration is essentially irreversible and produces mainly metabolites of lower reactivity that can be conjugated and excreted. The reaction of EHs is therefore generally regarded as detoxifying. The mammalian EHs can be distinguished by their physical and enzymatic properties. Microsomal EH (mEH) exhibits a broad substrate specificity, while the soluble EH (sEH) is an enzyme with a "complementary" substrate specificity to mEH. Cholesterol EH and leukotriene A4 hydrolase are two EHs with very limited substrate specificity. The activities of either GSTs or EHs expressed in vivo exhibit a relatively large interindividual variation, which might be explained by induction, inhibition, or genetic factors. These variations in levels or activities of individual isoenzymes are of importance with respect to an individual's susceptibility to genotoxic effects. This article gives a general overview of GSTs and EHs, discussing the modulation of activities, determination of these enzymes ex vivo, and the polymorphic expression of some isoenzymes.