Epoxy Metabolites Research Articles

Separation of hydroxyeicosatetraenoic acids and leukotrienes originating from kidney tissue by C-18 reverse-phase high-pressure liquid chromatography.

AbstractMetabolism of arachidonic acid results in formation of eicosanoids including prostaglandins (PGs), thromboxanes (Txs), monohydroxyeicosatetraenocinoids (HETEs), leukotrienes (LTs), lipoxins (Lxs), epoxy metabolites, and hepoxilins (1). Most of these compounds are formed in minute quantities in response to specific stimuli and participate in modulation of the vascular tone or of the inflammatory process. We have been studying the formation of arachidonic acid metabolites in kidney tissue (mainly glomeruli) following immune injury. Normal glomeruli synthesize very small quantities of HETEs and LTs. Following immune injury, glomerular synthesis and levels of these eicosanoids increase (2). Leukotrienes (B4, C4, and D4), 5-HETE, and 12-HETE can be separated, detected, and quantitated by several analytical techniques, including immunoassay, bioassay, gas chromatography-mass spectrometry (GC-MS), thin-layer chromatography (TLC), ultraviolet spectroscopy, and high pressure liquid chromatography (HPLC).KeywordsHigh Pressure Liquid ChromatographyReverse Phase ColumnInjection PortHydroxyeicosatetraenoic AcidImmune InjuryThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Quantitative analysis of 1,3-butadiene‐induced DNA adducts in vivo and in vitro using liquid chromatography electrospray ionization tandem mass spectrometry

1,3-Butadiene (BD) is a high volume industrial chemical which is known as a multi-site rodent carcinogen and is classified as a probable human carcinogen. Covalent interactions of the reactive epoxy metabolites of BD with DNA lead to the formation of DNA adducts which may cause mutations and tumor formation. In the present work, liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was employed for analyses of BD-induced DNA adducts in vitro and in vivo. Selected reaction monitoring (SRM) using the fragmentation of the [M+H]+ ions of the adducts to the corresponding protonated nucleobases under collision-induced dissociation was performed. Quantitation was based on isotope dilution with 13C- and 15N-labeled internal standards. The methods were applied in vitro [calf thymus DNA and TK6 cell cultures treated with epoxy metabolites of BD, 3,4-epoxy-1-butene (EB) and diepoxybutane (DEB)] and in vivo [DNA isolated from tissues of BD-exposed laboratory animals]. Two regioisomers of N-7-EB-guanine adducts, N-7-(2-hydroxy-3-buten-1-yl)guanine (N-7-EB-Gua I) and N-7-(1-hydroxy-3-buten-2-yl)guanine (N-7-EB-Gua II) and two N-3-EB-adenine isomers, N-3-(2-hydroxy-3-buten-1-yl)adenine and N-3-(1-hydroxy-3-buten-2-yl)adenine (N-3-EB-Ade I and II), were found in EB-exposed samples. N-7-(2′,3′,4′-trihydroxybut-1′-yl)guanine (N-7-THB-Gua), N6-(2′,3′,4′-trihydroxybut-1′-yl)adenine (N6-THB-Ade), and N-3-(2′,3′,4′-trihydroxybut-1′-yl)adenine (N-3-THB-Ade) were detected in DEB-treated DNA. DNA isolated from liver and lung of rats and mice exposed to 1250 ppm BD for 2 weeks contained both regioisomers of N-7-EB-Gua and N-3-EB-Ade, as well as N-7-THB-Gua and N6-THB-Ade. The methods developed in this work provide the means to study accumulation, repair and dose–response relationships of BD–DNA adducts in vivo. Although less sensitive than gas chromatography/electron capture negative ionization high-resolution mass spectrometry (GC/ECNI-HRMS), LC/ESI+-MS/MS in the SRM mode is extremely useful for analysis of BD–DNA adducts, which are not amenable to GC and derivatization owing to the presence of several adjacent polar functional groups. Using LC/ESI-MS/MS and isotope dilution, multiple structurally diverse BD–DNA adducts can be analyzed simultaneously in the same sample with minimal sample preparation. © 1998 John Wiley & Sons, Ltd.

Quantitative analysis of 1,3-butadiene-induced DNA adducts in vivo and in vitro using liquid chromatography electrospray ionization tandem mass spectrometry.

1,3-Butadiene (BD) is a high volume industrial chemical which is known as a multi-site rodent carcinogen and is classified as a probable human carcinogen. Covalent interactions of the reactive epoxy metabolites of BD with DNA lead to the formation of DNA adducts which may cause mutations and tumor formation. In the present work, liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was employed for analyses of BD-induced DNA adducts in vitro and in vivo. Selected reaction monitoring (SRM) using the fragmentation of the [M + H]+ ions of the adducts to the corresponding protonated nucleobases under collision-induced dissociation was performed. Quantitation was based on isotope dilution with 13C- and 15N-labeled internal standards. The methods were applied in vitro [calf thymus DNA and TK6 cell cultures treated with epoxy metabolites of BD, 3,4-epoxy-1-butene (EB) and diepoxybutane (DEB)] and in vivo [DNA isolated from tissues of BD-exposed laboratory animals]. Two regioisomers of N-7-EB-guanine adducts, N-7-(2-hydroxy-3-buten-1-yl)guanine (N-7-EB-Gua I) and N-7-(1-hydroxy-3-buten-2-yl)guanine (N-7-EB-Gua II) and two N-3-EB-adenine isomers, N-3-(2-hydroxy-3-buten-1-yl)adenine and N-3-(1-hydroxy-3-buten-2-yl)adenine (N-3-EB-Ade I and II), were found in EB-exposed samples. N-7-(2',3',4'-trihydroxybut-1'-yl)guanine (N-7-THB-Gua), N6-(2',3',4'-trihydroxybut-1'-yl)adenine (N6-THB-Ade), and N-3-(2',3',4'-trihydroxybut-1'-yl)adenine (N-3-THB-Ade) were detected in DEB-treated DNA. DNA isolated from liver and lung of rats and mice exposed to 1250 ppm BD for 2 weeks contained both regioisomers of N-7-EB-Gua and N-3-EB-Ade, as well as N-7-THB-Gua and N6-THB-Ade. The methods developed in this work provide the means to study accumulation, repair and dose-response relationships of BD-DNA adducts in vivo. Although less sensitive than gas chromatography/electron capture negative ionization high-resolution mass spectrometry (GC/ECNI-HRMS), LC/ESI(+)-MS/MS in the SRM mode is extremely useful for analysis of BD-DNA adducts, which are not amenable to GC and derivatization owing to the presence of several adjacent polar functional groups. Using LC/ESI-MS/MS and isotope dilution, multiple structurally diverse BD-DNA adducts can be analyzed simultaneously in the same sample with minimal sample preparation.

Adenine adducts with diepoxybutane: isolation and analysis in exposed calf thymus DNA.

1,3-Butadiene (BD) is a high-volume industrial chemical and a common environmental pollutant. Although BD is classified as a "probable human carcinogen", only limited evidence is available for its tumorigenic effects in occupationally exposed populations. Animal studies show a surprisingly high sensitivity of mice to the carcinogenic effects of BD compared to rats (approximately 10(3)-fold), making interspecies extrapolations difficult. Identification and quantitation of specific BD-induced DNA adducts are important for improving our understanding of the mechanisms of BD biological effects and for explaining the observed species differences. Covalent binding of BD to DNA is probably due to its two epoxy metabolites: 3,4-epoxy-1-butene (EB) and 1,2:3,4-diepoxybutane (DEB). Both EB and DEB are direct mutagens producing frameshift and point mutations at both A:T and G:C base pairs. DEB is 100 times more mutagenic than EB and is found in quantity only in tissues of the most sensitive species (mouse). This has led to the suggestion that the higher sensitivity of mice to BD could be due to greater exposure to DEB. The present work was initiated in order to isolate and structurally characterize DEB-induced adenine adducts. The adducts were formed by reacting DEB with free adenine (Ade), 2'-deoxyadenosine (2'-dAdo), and calf thymus DNA followed by HPLC separation and analysis of the products by UV spectrophotometry, electrospray ionization mass spectrometry, and nuclear magnetic resonance. The adenine reaction resulted in three products which were identified as N-3-, N-7-, and N-9-(2'-hydroxy-3',4'-epoxybut-1'-yl)adenine. These adducts underwent acid-catalyzed hydrolysis to their corresponding (2',3',4'-trihydroxybut-1'-yl)adenines upon heating or storage. The 2'-dAdo reaction with DEB followed by acid hydrolysis yielded a single adduct, N6-(2',3',4'-trihydroxybut-1'-yl)adenine (N6-DEB-Ade). N-3-DEB-Ade and N6-DEB-Ade were also found in hydrolysates of calf thymus DNA exposed to DEB. The amounts of N-3-DEB-Ade (13/10(3) normal Ade) and N6-DEB-Ade (5/10(3) normal Ade) were slightly lower than those of the corresponding EB-induced adducts in similar experiments, suggesting comparable reactivity of the two epoxy metabolites of BD toward adenine in DNA. The findings of this study provide a basis for future analyses of BD-induced adenyl DNA adducts in vitro and in vivo.

In vivo and in vitro metabolism of cannabidiol monomethyl ether and cannabidiol dimethyl ether in the guinea pig: On the formation mechanism of cannabielsoin-type metabolite from cannabidiol.

Oxidative metabolism of cannabidiol monomethyl ether (CBDM), one of the components of marihuana, was studied in the guinea pig. Cannabielsoin monomethyl ether (CBEM) was found to be formed with hepatic microsomes by gas chromatography-mass spectrometry (GC-MS). Experiments using various modifiers of enzymatic reaction suggested that, as in the case of cannabielsoin (CBE) formation from canabidiol (CBD), CBEM was formed from CBDM by the monooxygenase system including cytochrome P450. When cannabidiol dimethyl ether (CBDD), in which phenolic hydroxyl groups of CBD are masked with methyl groups, was incubated with liver microsomes and an reduced nicotinamide adenine dinucleotide phosphate-generating system, 1S,2R-epoxy-CBDD was identified by GC-MS. The epoxy metabolite was also found in the liver of a guinea pig pretreated with CBDD (100 mg/kg, intraperitoneally) 1 h before sacrifice. Rate of 1S,2R-epoxide metabolism was slower than that of 1R,2S-epoxy-CBDD under the conditions, as in the microsomal oxidation of CBDD described above. These results indicate that 1S,2R-epoxides are formed from CBD, CBDM and CBDD and that the epoxides are quickly converted to elsoin-type metabolites in the cases of CBD and CBDM.

Difference in epoxides formation and their further metabolism between delta 9- and delta 8-tetrahydrocannabinols by human liver microsomes.

In vitro metabolism of delta 9- and delta 8-tetrahydrocannabinols (THCs) was studied using human liver microsomes. Delta 9- or delta 8-THC was incubated with microsomes in the presence of an NADPH-generating system. The metabolites formed were extracted with ethyl acetate, separated by preparative thin-layer chromatography, and identified as trimethylsilyl derivatives by gas chromatography-mass spectrometry. 9 alpha, 10 alpha-Epoxyhexahydrocannabinol (EHHC) together with four monohydroxylated metabolites was formed from delta 9-THC. The epoxide was found to resist the hydrolysis by epoxide hydrolase, and was further converted to several metabolites by monooxygenase system involving cytochrome P-450. On the other hand, 8 beta, 9 alpha-dihydroxyhexahydrocannabinol (diOH-HHC) instead of epoxy metabolites was formed from delta 8-THC under the conditions for monooxygenase. When 1,1,1-trichloropropene-2,3-oxide was further added to the incubation mixture, both of 8 alpha, 9 alpha-EHHCs were found to be formed from delta 8-THC. These epoxides of delta 8-THC were preferentially hydrolyzed to 8 beta, 9 alpha-diOH-HHC by epoxide hydrolase. These results indicate that 9 alpha, 10 alpha-EHHC formed from delta 9-THC is further metabolized not by epoxide hydrolase but by monooxygenase system involving cytochrome P-450, and that, on the contrary, 8 alpha, 9 alpha- and 8 beta, 9 beta-EHHCs derived from delta 8-THC may be metabolized by epoxide hydrolase rather than cytochrome P-450 in the human liver, forming 8 beta, 9 alpha-diOH-HHC.

Human liver microsomal oxidation of delta 8-tetrahydrocannabinol.

Human Liver microsomal oxidation products of Δ8-tetrahydrocannabinol (THC) were isolated and identified by thin-layer chromatography, gas chromatography and gas chromatography-mass spectrometry. As the results, five monohydroxylated metabolites including 11-hydroxy-Δ8-THC, and 8β, 9α-dihydroxyhexahydrocannabinol were identified as trimethyl-silyl derivatives. Isolation of the dihydroxylated metabolite suggests that epoxy metabolite (s) may be formed from Δ8-THC in humans.

Carbamazepine and its 10,11-epoxide in children and adults with epilepsy.

The plasma concentrations of carbamazepine and its 10,11-epoxide were measured in 37 children and 13 adults with epilepsy during maintenance therapy. The children formed relatively more of the epoxy metabolite than adults. The daily dose, expressed as mg/kg or mg/m2, showed a statistically significant correlation with blood level in children, but not in adults. The cerebrospinal fluid concentrations of carbamazepine and carbamazepine-10,11-epoxide in nine children were 33 and 41 per cent, respectively, of the concomitant plasma level.