Detection of DNA adducts derived from the reactive metabolite of furan, cis-2-butene-1,4-dial

Furan is a toxic and carcinogenic compound used in industry and commonly found in the environment. The mechanism of furan's carcinogenesis is not well-understood and may involve both genotoxic and nongenotoxic pathways. Furan undergoes oxidation by cytochrome P450 to cis-2-butene-1,4-dial, which is thought to mediate furan's toxic effects. Consistently, cis-2-butene-1,4-dial readily reacts with glutathione, amino acids, and nucleosides. To determine the importance of DNA alkylation in furan-induced carcinogenesis, we developed an assay for the detection of cis-2-butene-1,4-dial-derived DNA adducts. DNA samples were treated with O-benzyl-hydroxylamine, which reacts with the aldehyde functionality of the DNA adducts. Enzyme hydrolysates of these samples were then analyzed by capillary electrospray tandem mass spectrometry with selected reaction monitoring. The dCyd and dAdo adducts were detected in digests of DNA treated with nanomolar concentrations of cis-2-butene-1,4-dial. In addition, these adducts were present in DNA isolated from Ames assay strain TA104 treated with mutagenic concentrations of cis-2-butene-1,4-dial. These data support the hypothesis that cis-butene-1,4-dial is a genotoxic metabolite of furan. This method will allow us to explore the role of these adducts in furan-induced carcinogenesis.     

Trapping of cis-2-butene-1,4-dial to measure furan metabolism in human liver microsomes by cytochrome P450 enzymes

Furan is a liver toxicant and carcinogen in rodents. It is classified as a possible human carcinogen, but the human health effects of furan exposure remain unknown. The oxidation of furan by cytochrome P450 (P450) enzymes is necessary for furan toxicity. The product of this reaction is the reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). To determine whether human liver microsomes metabolize furan to BDA, a liquid chromatography/tandem mass spectrometry method was developed to detect and quantify BDA by trapping this reactive metabolite with N-acetyl-l-cysteine (NAC) and N-acetyl-l-lysine (NAL). Reaction of NAC and NAL with BDA generates N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine (NAC-BDA-NAL). Formation of NAC-BDA-NAL was quantified in 21 different human liver microsomal preparations. The levels of metabolism were comparable to that observed in F-344 rat and B6C3F1 mouse liver microsomes, two species known to be sensitive to furan-induced toxicity. Studies with recombinant human liver P450s indicated that CYP2E1 is the most active human liver furan oxidase. The activity of CYP2E1 as measured by p-nitrophenol hydroxylase activity was correlated to the extent of NAC-BDA-NAL formation in human liver microsomes. The formation of NAC-BDA-NAL was blocked by CYP2E1 inhibitors but not other P450 inhibitors. These results suggest that humans are capable of oxidizing furan to its toxic metabolite, BDA, at rates comparable to those of species sensitive to furan exposure. Therefore, humans may be susceptible to furan's toxic effects.     

Identification of a cis-2-butene-1,4-dial-derived glutathione conjugate in the urine of furan-treated rats

The hepatocarcinogen and toxicant furan requires metabolic activation to elicit its toxic effects. The available experimental evidence indicates that the overall metabolism of furan is initiated via cytochrome P450 catalyzed oxidation to cis-2-butene-1,4-dial. This alpha,beta-unsaturated dialdehyde reacts in vitro with protein and DNA nucleophiles. To determine if this compound is an in vivo intermediate in the metabolism of furan, rats were treated with either [(12)C(4)]furan or [(13)C(4)]furan, and urine was collected for 24 h. Capillary LC/MS/MS analysis of the urine indicated that one of the metabolites was a monoglutathione conjugate of cis-2-butene-1,4-dial. These results indicate that glutathione conjugation of the reactive metabolite of furan occurs in vivo. This metabolite may serve as a useful marker for furan exposure and metabolism in risk assessment studies.     

A reactive metabolite of furan, cis-2-butene-1,4-dial, is mutagenic in the Ames assay

Furan is classified as a nongenotoxic hepatocarcinogen. It is thought to be activated to a toxic metabolite, cis-2-butene-1,4-dial, which is acutely toxic to liver cells. The resulting cytotoxicity is followed by compensatory cell proliferation, increasing the likelihood of tumor production. We examined the genotoxic activity of cis-2-butene-1,4-dial in several strains of Salmonella typhimurium commonly used in the Ames assay. This reactive compound tested positive in TA104, a strain that is sensitive to aldehydes. Mutagenic activity was concentration-dependent (1000 +/- 180 revertants/micromol). Incubation of cis-2-butene-1,4-dial with glutathione prior to addition of bacteria inhibited both the acute toxic and genotoxic activity of this compound. No evidence of mutagenic activity was seen at nontoxic concentrations in TA97, TA98, TA100, and TA102. Our findings are consistent with the hypothesis that cis-2-butene-1,4-dial reacts with DNA to form mutagenic adducts. Our data suggest that cis-2-butene-1,4-dial may be an important genotoxic as well as toxic intermediate in furan-induced tumorigenesis.     

Identification and pathway mapping of furan target proteins reveal mitochondrial energy production and redox regulation as critical targets of furan toxicity

Furan, a heat-generated food contaminant, is hepatotoxic and carcinogenic in rodents. Furan is oxidized by cytochrome P450 2E1 to cis-2-butene-1,4-dial, a chemically reactive α,β-unsaturated dialdehyde, which has been identified as the key toxic metabolite of furan based on its ability to interact with tissue nucleophiles. In addition to genotoxicity, sustained cytotoxicity mediated through covalent binding of cis-2-butene-1,4-dial to critical target proteins is thought to play a key role in furan carcinogenicity. To identify putative protein targets of reactive furan metabolites, male F344/N rats (n = 5 per dose) were administered a single dose of [3,4-(14)C]-furan (20 mCi/mmol) at doses associated with hepatotoxicity following long-term exposure (0.1 and 2 mg/kg body weight [bw]). Liver proteins were separated by two-dimensional gel electrophoresis and protein spots carrying radiolabel were located by fluorography. In total, 83 discrete protein spots containing (14)C were consistently detected in livers of animals given [3,4-(14)C]-furan at 2.0 mg/kg bw, accounting for 4-5% of the proteome covered by our analyses. Protein spots were excised and digested in gel with trypsin for identification by protein mass spectrometry. Protein database search and subsequent pathway mapping identified 61 proteins localized predominantly in the cytosol and mitochondria, including structural proteins, mitochondrial enzymes involved in glucose metabolism, mitochondrial β-oxidation, and adenosine triphosphate synthesis, and proteins that participate in the maintenance of redox homeostasis and protein folding. Collectively, our data suggest that functional loss of several individual proteins and interference with pathways, most notably mitochondrial energy production, redox regulation, and protein folding, may combine to disrupt cell homeostasis and cause hepatocyte cell death.     

Polyamines are traps for reactive intermediates in furan metabolism

Furan is toxic and carcinogenic in rodents. Because of the large potential for human exposure, furan is classified as a possible human carcinogen. The detailed mechanism by which furan causes toxicity and cancer is not yet known. Since furan toxicity requires cytochrome P450-catalyzed oxidation of furan, we have characterized the urinary and hepatocyte metabolites of furan to gain insight into the chemical nature of the reactive intermediate. Previous studies in hepatocytes indicated that furan is oxidized to the reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA), which reacts with glutathione (GSH) to form 2-(S-glutathionyl)succinaldehyde (GSH-BDA). This intermediate forms pyrrole cross-links with cellular amines such as lysine and glutamine. In this article, we demonstrate that GSH-BDA also forms cross-links with ornithine, putrescine, and spermidine when furan is incubated with rat hepatocytes. The relative levels of these metabolites are not completely explained by hepatocellular levels of the amines or by their reactivity with GSH-BDA. Mercapturic acid derivatives of the spermidine cross-links were detected in the urine of furan-treated rats, which indicates that this metabolic pathway occurs in vivo. Their detection in furan-treated hepatocytes and in urine from furan-treated rats indicates that polyamines may play an important role in the toxicity of furan.     

Characterization of nucleoside adducts of cis-2-butene-1,4-dial, a reactive metabolite of furan

Furan is a hepatic toxicant and carcinogen in rodents. Its microsomal metabolite, cis-2-butene-1,4-dial, is mutagenic in the Ames assay. Consistent with this observation, cis-2-butene-1,4-dial reacts with 2'-deoxycytidine, 2'-deoxyguanosine, and 2'-deoxyadenosine to form diastereomeric adducts. HPLC analysis indicated that the rate of reaction with deoxyribonucleosides was dependent on pH. At pH 6.5, the relative reactivity was 2'-deoxycytidine > 2'-deoxyguanosine > 2'-deoxyadenosine whereas it was 2'-deoxyguanosine > 2'-deoxycytidine > 2'-deoxyadenosine at pH 8.0. Thymidine did not react with cis-2-butene-1,4-dial. The primary 2'-deoxyguanosine and 2'-deoxyadenosine reaction products were unstable and decomposed to secondary products. NMR and mass spectral analysis indicated that the initial 2'-deoxyadenosine and 2'-deoxyguanosine reaction products were hemiacetal forms of 3-(2'-deoxy-beta-D-erthyropentafuranosyl)-3,5,6,7-tetrahydro-6-hydroxy-7-(ethane-2'-al)-9H-imidazo[1,2-alpha]purine-9-one (structure 2) and 3-(2'-deoxy-beta-D-erythropentafuranosyl)-3,6,7,8-tetrahydro-7-(ethane-2'-al)-8-hydroxy-3H-imidazo[2,1-i]purine (structure 4), respectively. These adducts resulted from the addition of cis-2-butene-1,4-dial to the exo- and endocyclic nitrogens of 2'-deoxyadenosine and 2'-deoxyguanosine. The data provide support for the hypothesis that cis-2-butene-1,4-dial is an important genotoxic intermediate in furan-induced carcinogenesis.     

Electrophilic intermediates produced by bioactivation of furan

The industrial and environmental chemical, furan, is a liver toxicant and carcinogen in laboratory animals. It has been classified as a possible human carcinogen. The mechanism of tumor induction is unknown. However, toxicity is initiated by cytochrome P450 catalyzed oxidation of furan to an alpha,beta-unsaturated dialdehyde, cis-2-butene-1,4-dial. This metabolite reacts readily with protein and DNA nucleophiles and is a bacterial mutagen in Ames assay strain TA104. Metabolism studies indicate that this reactive metabolite is formed in vivo. It is also an intermediate leading to other metabolites whose role in furan-derived toxicities has yet to be explored.     

Electrophilic Intermediates Produced by Bioactivation of Furan

The industrial and environmental chemical, furan, is a liver toxicant and carcinogen in laboratory animals. It has been classified as a possible human carcinogen. The mechanism of tumor induction is unknown. However, toxicity is initiated by cytochrome P450 catalyzed oxidation of furan to an α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial. This metabolite reacts readily with protein and DNA nucleophiles and is a bacterial mutagen in Ames assay strain TA104. Metabolism studies indicate that this reactive metabolite is formed in vivo. It is also an intermediate leading to other metabolites whose role in furan-derived toxicities has yet to be explored.     

Toxicity and carcinogenicity of furan in human diet

Furan is formed during commercial or domestic thermal treatment of food. The initial surveys of furan concentrations in heat-treated foods, published by European and US authorities, revealed the presence of relatively high furan levels in coffee, sauces, and soups. Importantly, furan is consistently found in commercial ready-to-eat baby foods. Furan induces hepatocellular tumors in rats and mice and bile duct tumors in rats with a high incidence. Epidemiological studies are not available. It is assumed that cis-2-butene-1,4-dial, the reactive metabolite of furan, is the causative agent leading to toxicity and carcinogenicity. Based on this data, furan is classified as a possible human carcinogen. The initial exposure estimates revealed a relatively small margin (~2,000) between human exposure and those furan doses, which induce liver tumors in experimental animals. As this may give rise for concern, in this review, the currently available toxicological and mechanistic data of furan are summarized and discussed with regard to its applicability in assessing the risk of furan in human diet.     

Furan induction of DNA cross-linking and strand breaks in turkey fetal liver in comparison to 1,3-propanediol

Furan, a food contaminant formed by heating, is hepatocarcinogenic to rats and mice. Conflicting genotoxicity data exist on furan and its metabolite, cis-2-butene-1,4-dial and there are few data for the target organ, the liver. We assessed the abilities of furan and, as a positive control, 1,3-propanediol (PDO), to cause DNA damage in the livers of turkey fetuses in ovo using the alkaline comet assay. Single injections of furan (2-20 μmoles) into turkey eggs, at 23 days of incubation, when the liver is well developed, reduced the %DNA in the comet tail (%DNA-CT) in hepatocytes isolated from fetuses 24 h later indicating DNA cross links. Treatment of the hepatocytes with proteinase K, digest DNA-protein cross links (DPXLs), increased the %DNA-CT compared to the corresponding controls, indicating the presence of DNA single or double stand breaks (SB). PDO showed little toxicity and was used at high doses (up to 300 μmoles/egg), where it induced DPXLs at about 20 times the furan dose. Thus, furan produced dose proportional reductions in %DNA-CT in turkey liver fetal hepatocytes indicating the presence of DPXLs and, after proteinase K treatment, an increase in %DNA-CT, indicating the presence of DNA single and/or double SB.     

Evaluation of serum and liver toxicokinetics for furan and liver DNA adduct formation in male Fischer 344 rats

Furan is a food processing contaminant found in many common cooked foods that induces liver toxicity and liver cancer in animal models treated with sufficient doses. The metabolism of furan occurs primarily in the liver where CYP 2E1 produces a highly reactive bis-electrophile, cis-2-butene-1,4-dial (BDA). BDA reacts with nucleophilic groups in amino acids and DNA in vitro to form covalent adducts. Evidence for BDA-nucleoside adduct formation in vivo is limited but important for assessing the carcinogenic hazard of dietary furan. This study used controlled dosing with furan in Fischer 344 rats to measure serum and liver toxicokinetics and the possible formation of BDA-nucleoside adducts in vivo. After gavage exposure, furan concentrations in the liver were consistently higher than those in whole blood (∼6-fold), which is consistent with portal vein delivery of a lipophilic compound into the liver. Formation of BDA-2′-deoxycytidine in furan-treated rat liver DNA was not observed using LC/MS/MS after single doses as high as 9.2 mg/kg bw or repeated dosing for up to 360 days above a consistent background level (1-2 adducts per 10⁸ nucleotides). This absence of BDA-nucleoside adduct formation is consistent with the general lack of evidence for genotoxicity of furan in vivo.     

The formation of substituted 1,N6-etheno-2'-deoxyadenosine and 1,N2-etheno-2'-deoxyguanosine adducts by cis-2-butene-1,4-dial, a reactive metabolite of furan

Furan is an environmental chemical that induces liver toxicity and tumor formation in rodents, leading to its classification as a probable human carcinogen. cis-2-Butene-1,4-dial, the metabolite considered responsible for furan's toxicological effects, is mutagenic in the Ames assay and reacts with 2'-deoxycytidine (dCyd), 2'-deoxyadenosine (dAdo), and 2'-deoxyguanosine (dGuo) to form previously characterized diastereomeric adducts. The initially formed dCyd adducts are stable to rearrangement, while the dAdo and dGuo adducts are unstable and rearrange to form secondary products. On the basis of UV absorbance, fluorescence, 1H NMR, and mass spectral data, the rearrangement product of the dAdo adduct was identified as the substituted etheno-dAdo adduct, 1'-[3-(2'-deoxy-beta-D-erythropentafuranosyl)-3H-imidazo[2,1-i]purin-8-yl]ethane-2'-al. The NMR characterization of the O-methyloxime derivative of the secondary dGuo adduct, along with mass spectral and UV data on the underivatized adduct, allowed for its structural assignment as the substituted etheno-dGuo compound, 3-(2'-deoxy-beta-D-erythropentafuranosyl)imidazo-7-(ethane-2'-al)[1,2-alpha]purine-9-one. The characterization of the primary and secondary products formed in the reaction of cis-2-butene-1,4-dial with nucleosides is important for understanding the mechanism of furan-induced carcinogenesis. These secondary adducts retain a reactive aldehyde with the potential to form cross-links and are likely to contribute significantly to furan's toxic and carcinogenic effects.     

Biomarkers of furan exposure by metabolic profiling of rat urine with liquid chromatography-tandem mass spectrometry and principal component analysis

Furan has been found in a number of heated food items and is carcinogenic in the liver of rats and mice. Estimates of human exposure on the basis of concentrations measured in food are not reliable because of the volatility of furan. A biomarker approach is therefore indicated. We searched for metabolites excreted in the urine of male Fischer 344 rats treated by oral gavage with 40 mg of furan per kg of body weight. A control group received the vehicle oil only. Urine collected over two 24-h periods both before and after treatment was analyzed by a column-switching LC-MS/MS method. Data were acquired by a full scan survey scan in combination with information dependent acquisition of fragmentation spectra by the use of a linear ion trap. Areas of 449 peaks were extracted from the chromatograms and used for principal component analysis (PCA). The first principal component fully separated the samples of treated rats from the controls in the first post-treatment sampling period. Thirteen potential biomarkers selected from the corresponding loadings plot were reanalyzed using specific transitions in the MRM mode. Seven peaks that increased significantly upon treatment were further investigated as biomarkers of exposure. MS/MS information indicated conjugation with glutathione on the basis of the characteristic neutral loss of 129 for mercapturates. Adducts with the side chain amino group of lysine were characterized by a neutral loss of 171 for N-acetyl- l-lysine. Analysis of products of in vitro incubations of the reactive furan metabolite cis-2-butene-1,4-dial with the respective amino acid derivatives supported five structures, including a new 3-methylthio-pyrrole metabolite probably formed by beta-lyase reaction on a glutathione conjugate, followed by methylation of the thiol group. Our results demonstrate the potential of comprehensive mass spectrometric analysis of urine combined with multivariate analyses for metabolic profiling in search of biomarkers of exposure.     

Synthesis of a 2'-deoxyguanosine adduct of cis-2-butene-1,4-dial, a reactive metabolite of furan

Furan is a liver and kidney toxicant and a hepatocarcinogen in rodents. Its reactive metabolite, cis-2-butene-1,4-dial, reacts with nucleosides to form adducts in vitro. The reaction with 2'-deoxyguanosine generates 3-(2'-deoxy-beta-D-erythropentafuranosyl)-3,5,6,7-tetrahydro-6-hydroxy-7-(ethane-2"-al)-9H-imidazo[1,2-alpha]purine-9-one as the major reaction product. A synthetic approach to this adduct is presented in this report. The key step in this synthesis is the preparation of 2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-1-(1,2,5,6-tetrahydroxyhexan-3-yl)guanosine. Treatment of this intermediate with sodium periodate gave three reaction products: a one-substituted adduct, 2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-1-(2,5-dihydroxy-tetrahydrofuran-3-yl)guanosine; a 1,N(2)-cyclic adduct, 3-[2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-beta-D-erythropentafuranosyl]-6-hydroxy-8-formyl-5,6,7,8-tetrahydropyrimidino[1,2-alpha]purin-10(3H)-one; and the 1,N(2)-bicyclic adduct, 3-[2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-beta-D-erythropentafuranosyl]-3,5,6,7-tetrahydro-6-hydroxy-7-(ethane-2"-al)-9H-imidazo[1,2-alpha]purine-9-one. The one-substituted and 1,N(2)-cyclic reaction products were unstable and rearranged over time to yield the 1,N(2)-bicyclic 2'-deoxyguanosine adducts. The desired reaction product was obtained as a mixture of four diastereomers by removing the tert-butyldimethylsilyl groups with hydrogen fluoride. This synthetic approach to the cis-2-butene-1,4-dial-derived dGuo adducts confirms our previous structural characterization of the in vitro cis-2-butene-1,4-dial-dGuo reaction product. These studies demonstrate that the observed 1,N(2) bicyclic structure is the thermodynamically stable isomer, supporting our previous observations that this adduct is the major product formed in vitro. Finally, these studies provide the necessary groundwork for the preparation of oligonucleotides with site specifically incorporated cis-2-butene-1,4-dial-derived adducts.     

Correlation of urinary furan with plasma gamma-glutamyltranspeptidase levels in healthy men and women.

Furan is a colorless, volatile compound that is found in heat-treated foods, such as canned and jarred foods, at levels up to 100 ppb. When animals ingest high doses, furan metabolites, such as cis-2-butene-1,4-dial, cause severe hepatotoxicity and carcinogenicity. However, the levels and effects of furan on humans are not known. Therefore, we measured urinary furan in 100 healthy individuals consumed normal diet (49 men, 51 women) using solid phase micro-extraction-gas chromatography-mass spectrometry (SPME-GCMS). Urinary furan was detected in 56 subjects (31 males, 25 females) and ranged up to 3.14 ppb. In individuals with detectable urinary furan, the level of gamma-glutamyltranspeptidase (gamma-GT), a marker for liver damage, was strongly correlated with the urinary furan concentration (r=0.56, p<0.0001). Linear regression analysis indicated that the urinary furan level was significantly associated with gamma-GT in both univariate (p<0.0001) and multiple (p=0.0001) models including age, sex, body weight, and blood pressure as covariates. To our knowledge, this is the first study to measure detectable levels of furan in human urine. These levels of urinary furan, which may be dietary origin, could be hepatotoxic in humans; therefore; the metabolic fates and potential toxicity of dietary furan in humans should be investigated further.     

Bioactivation of 4-ipomeanol by CYP4B1: adduct characterization and evidence for an enedial intermediate

4-Ipomeanol (IPO) is a pneumotoxin that is bioactivated to a reactive intermediate that binds to DNA and other cellular macromolecules. Despite over 30 years of research in this area, detailed structural information on the nature of the IPO reactive intermediate is still lacking. In the present study, we reacted IPO with rabbit CYP4B1 in the presence of exogenous nucleophiles and analyzed the products by liquid chromatography/electrospray ionization-mass spectrometry. Coincubation of IPO and rabbit CYP4B1 with glutathione gave rise to multiple products due likely to the presence of both sulfur and nitrogen nucleophiles in the same trapping molecule. Reaction mixtures containing equimolar N-acetyl cysteine (NAC) and N-acetyl lysine (NAL) provided a major NADPH- and CYP4B1-dependent product. A combination of high-resolution mass spectrometry and two-dimensional NMR analysis following large-scale isolation of the biologically derived material provided evidence for an N-substituted cysteinyl pyrrole derivative of IPO, analogous to that characterized previously in model chemical studies conducted with cis-2-butene-1,4-dial. Purified native rabbit lung CYP4B1 and purified recombinant rabbit CYP4B1 produced the trapped NAC/NAL-IPO pyrrole adduct at rates of 600-700 nmol/nmol P450/30 min. A panel of 14 commercially available recombinant human CYPs was also studied, and substantial rates of IPO bioactivation (>100 nmol/nmol/30 min) were observed with CYP1A2, CYP2C19, CYP2D6, and CYP3A4. These studies provide evidence for the formation of an enedial reactive intermediate during CYP-mediated IPO bioactivation, identify multiple human liver P450s capable of IPO bioactivation, and demonstrate that the same reactive intermediate is formed by both rabbit CYP4B1 and human P450s.     

Hepatic microsomal metabolism of indole to indoxyl, a precursor of indoxyl sulfate.

The aim of our study was to determine which microsomal cytochrome P450 isozyme(s) were responsible for the microsomal oxidation of indole to indoxyl, an important intermediate in the information of the uremic toxin indoxyl sulfate. Indole was incubated together with an NADPH-generating system and rat liver microsomes. Formation of indigo, an auto-oxidation product of indoxyl, was used to determine the indole-3-hydroxylation activity. Apparent Km and Vmax values of 0.85 mM and 1152 pmol min(-1) mg(-1) were calculated for the formation of indoxyl from indole using rat liver microsomes. The effects of various potential inducers and inhibitors on the metabolism of indole to indoxyl by rat liver microsomes were studied to elucidate the enzymes responsible for metabolism. Studies with general and isozyme-specific P450 inhibitors demostrated that P450 enzymes and not FMO are responsible for the formation of indoxyl. In the induction studies, rate of indoxyl formation in the microsomes from untreated vs induced rats correlated nearly exactly with the CYP2E1 activity (4-nitrophenol 2-hydroxylation). These results suggests that CYP2E1 is the major isoform for the microsomal oxidation of indole to indoxyl.     

Detection of glutathione conjugates derived from 4-ipomeanol metabolism in bile of rats by liquid chromatography-tandem mass spectrometry.

Earlier studies postulated that bioactivation of 4-ipomeanol by cytochrome P450 enzymes may occur through oxidation of its furan ring, following a mechanism similar to the bioactivation of other furan-containing compounds. This would lead to the formation of furan epoxides and alpha,beta-unsaturated di-aldehyde-reactive metabolites that can conjugate with glutathione. These metabolites are thought to be responsible for the cytotoxic and anticancer properties of 4-ipomeanol. We hypothesized that if 4-ipomeanol is metabolized following this pathway, its glutathione conjugates would be isobaric (molecular ion mass = 492 Da) and would be excreted in bile. To investigate this hypothesis, we analyzed by liquid chromatography-tandem mass spectrometry the bile of rats administered d0/d6 4-ipomeanol (1:1 ratio) intravenously. Hexadeuterated 4-ipomeanol had all deuterium atoms incorporated on its aliphatic chain. Multiple reaction monitoring scans of bile for the mass transition: MH+/(MH - 129)+, which is characteristic of glutathione conjugates, detected four glutathione conjugates. The observation of the isotope cluster (M + 1)+ (d0)/(MH + 6)+ (d6) in a 1:1 molar ratio confirmed that these conjugates were derived from 4-ipomeanol. Retention of the six deuterium atoms in the glutathione conjugates detected, (MH + 6)+, indicates that the bioactivation of 4-ipomeanol took place on the furan ring moiety. Rat hepatic microsomal incubations provided additional evidence. From this study, the mass of the reactive metabolites of 4-ipomeanol can be inferred. The inferred mass (186 Da) matches the mass postulated. A pathway of 4-ipomeanol bioactivation is proposed here. This work represents one step forward to understanding the mechanism of bioactivation of 4-ipomeanol.