Metabolic epoxidation of an alpha,beta-unsaturated oxime generates sensitizers of extreme potency. Are nitroso intermediates responsible?

Metabolic activation of inherently nonprotein-reactive compounds (prohaptens) in the skin can lead to development of contact allergy, a chronic skin disease. The prohapten hypothesis has existed for more than 20 years; yet, detailed knowledge regarding the mechanisms of activation as well as what structural moieties can be transformed to protein-reactive sensitizers is still limited. Today, the consideration of cutaneous bioactivation is important when developing nonanimal-based assays for prediction of contact allergenic activity, as only methods that include skin metabolism are able to detect prohaptens as sensitizers. We have studied the mechanism of activation of the prohapten carvoxime (1), a strongly sensitizing but in itself poorly protein-reactive alpha,beta-unsaturated oxime. alpha,beta-Unsaturated oximes represent a novel class of prohaptens, which previously have never been investigated for potential metabolic activation. To identify reactive metabolites formed from 1, liver microsomal incubations in the presence of glutathione were carried out. Putative reactive metabolites were synthesized, and their allergenic activity, chemical reactivity toward nucleophiles, and ability to elicit a contact allergenic response in animals induced with 1 were assessed. We found that 1 is metabolically activated by epoxidation of the allylic carbon-carbon double bond. The alpha,beta-epoxy oxime metabolites were found to be sensitizers of extreme potency in the local lymph node assay and highly reactive toward nucleophilic amino acids and a model peptide. One of the two diastereomeric epoxy metabolites also elicited an allergic reaction in mice sensitized to 1, in the mouse ear swelling test. Furthermore, this study presents strong indications that the basis of the high reactivity and sensitizing capacity observed for the alpha,beta-unsaturated oximes is related to their ability to form highly reactive nitroso intermediates by tautomerization. To our knowledge, the formation of nitrosoalkenes by oxidative metabolism of alpha,beta-unsaturated oximes has not been shown so far.     

Characterization by liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry of two coupled oxidative-conjugative metabolic pathways for 7-ethoxycoumarin in human liver microsomes treated with alamethicin.

The microsomal metabolism of 7-ethoxycoumarin (7-EC) was investigated using liquid chromatography (LC)-NMR and liquid chromatography-mass spectrometry (LC-MS) to characterize the coupling of oxidative-conjugative metabolism events. Within microsomes, cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs) are spatially disparate, each having surface and luminal localization, respectively. To optimize cofactor and substrate transit to UGT without compromising P450 activity, the pore-forming peptide alamethicin was used for microsomal perforation. Aqueous extracts of microsomal incubations containing NADPH and UDP-glucuronic acid were injected for LC-NMR and LC-MS analysis. The analytical complementarity of LC-NMR and LC-MS permitted the identification of four metabolites (M1 to M4). The metabolites M1 and M2 are novel microsomal metabolites for 7-EC, consistent with 3-hydroxylation and subsequent glucuronidation, respectively. Metabolites M3 and M4 were 7-hydroxycoumarin (7-HC) and 7-HC glucuronide, respectively. Viewed collectively, these results illustrate the utility of alamethicin in the examination of coupled oxidative-conjugative metabolism and the synergy of LC-NMR and LC-MS in metabolite identification.     

In vitro metabolism of tolcapone to reactive intermediates: relevance to tolcapone liver toxicity

Tolcapone is a catechol-O-methyltransferase (COMT) inhibitor used for control of motor fluctuations in Parkinson's disease (PD). Since its entry onto the market in 1998, tolcapone has been associated with numerous cases of hepatotoxicity, including three cases of fatal fulminant hepatic failure. The cause of this toxicity is not known; however, it does not occur with the use of the structurally similar drug entacapone. It is known that tolcapone is metabolized to amine (M1) and acetylamine (M2) metabolites in humans, but that the analogous metabolites were not detected in a limited human study of entacapone metabolism. We hypothesized that one or both of these tolcapone metabolites could be oxidized to reactive species and that these reactive metabolites might play a role in tolcapone-induced hepatocellular injury. To investigate this possibility, we examined the ability of M1 and M2 to undergo in vitro bioactivation by electrochemical and enzymatic methods. Electrochemical experiments revealed that M1 and M2 are more easily oxidized than the parent compound, in the order M1 > M2 > tolcapone. There was a general correlation between oxidation potential and the half-lives of the compounds in the presence of two oxidizing systems, horseradish peroxidase and myeloperoxidase. These enzymes catalyzed the oxidation of M1 and M2 to reactive species that could be trapped with glutathione (GSH) to form metabolite adducts (C1 and C2). Each metabolite was found to only form one GSH conjugate, and the structures were tentatively identified using LC-MS/MS. Following incubation of M1 and M2 with human liver microsomes in the presence of GSH, the same adducts were observed, and their structures were confirmed using LC-MS/MS and (1)H NMR. Experiments with chemical P450 inhibitors and cDNA-expressed P450 enzymes revealed that this oxidation is catalyzed by several P450s, and that P450 2E1 and 1A2 play the primary role in the formation of C1 while P450 1A2 is most important for the production of C2. Taken together, these data provide evidence that tolcapone-induced hepatotoxicity may be mediated through the oxidation of the known urinary metabolites M1 and M2 to reactive intermediates. These reactive species may form covalent adducts to hepatic proteins, resulting in damage to liver tissues, although this supposition was not investigated in this study.     

Xenobiotic-metabolizing enzymes involved in activation and detoxification of carcinogenic polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens and metabolized by a variety of xenobiotic-metabolizing enzymes such as cytochrome P450 (P450 or CYP), epoxide hydrolase, glutathione transferase, UDP-glucuronosyltransferase, sulfotransferase, NAD(P)H quinone oxidoreductase 1, and aldo-keto reductase. These enzymes mainly participate in the conversion of PAHs to more polar and water-soluble metabolites, and the resultant metabolites are readily excreted from the body. However, during the course of metabolism, a variety of unstable and reactive intermediates of PAHs are formed, and these metabolites attack DNA, causing cell toxicity and transformation. P450s and epoxide hydrolase convert PAHs to proximate carcinogenic metabolites, PAH-diols, and these products are further metabolized by P450s to ultimate carcinogenic metabolites, PAH diol-epoxides, or by aldo-keto reductase to reactive PAH o-quinones. PAHs are also activated by P450 and peroxidases to reactive radical cations that bind covalently to DNA. The oxygenated and reactive metabolites of PAHs are usually converted to more polar and detoxified products by phase II enzymes. Inter-individual differences exist in levels of expression and catalytic activities of a variety of enzymes that activate and/or detoxify PAHs in various organs of humans and these phenomena are thought to be critical in understanding the basis of individual differences in response to PAHs. Factors affecting such variations include induction and inhibition of enzymes by diverse chemicals and, more importantly, genetic polymorphisms of enzymes in humans.     

Possible involvement of singlet oxygen species as multiple oxidants in p450 catalytic reactions

Cytochrome P450 (P450) constitutes a superfamily of enzymes which activate dioxygen and carry out monooxygenation reactions of large numbers of endogenous and xenobiotic compounds. Drug metabolism is a particularly important P450 function, and, therefore, elucidating the metabolic products and pathways of drugs is essential for drug development. To explain the substrate selectivity of P450 reactions, it is necessary to understand the formation of multiple activated oxygen species to determine the type of catalyzed reactions, in addition to conducting structure analyses of P450s. Although an oxo-Fe(IV)-porphyrin-pi-cation radical is regarded as an activated oxygen species in P450 reactions, a nucleophilic Fe(III)-peroxo species has also been proposed as another oxidant. In the past decade, various studies indicated that P450-catalyzed oxygenations are complex, and that a single reaction pathway cannot explain all of the experimental results. In addition, the microsomal P450 system is known to generate reactive oxygen species (ROS). However, the contribution of ROS to P450 reactions remains unclear. We recently found that singlet oxygen (1O2) was involved in both several rat liver microsomal P450 reactions and four human CYP subfamily activities, as confirmed by the ESR spin-trapping method. In this review, we describe the studies that have been conducted on the detection and characterization of ROS in P450 reactions related to drug metabolism that involve the possibility of 1O2 in the P450 catalytic cycle. Gaining an understanding of the activated oxygen species that determine the type of drug metabolism will help us to predict the important metabolites formed.     

Use of a systems model of drug-induced liver injury (DILIsym) to elucidate the mechanistic differences between acetaminophen and its less-toxic isomer,AMAP, in mice

Acetaminophen (APAP) has been used as a probe drug to investigate drug-induced liver injury (DILI). In mice, 3′-hydroxyacetanilide (AMAP), a less-toxic isomer of APAP, has also been studied as a negative control. Various mechanisms for the divergence in toxicological response between the two isomers have been proposed. This work utilized a mechanistic, mathematical model of DILI to test the plausibility of four mechanistic hypotheses. Simulation results were compared to an array of measured endpoints in mice treated with APAP or AMAP. The four hypotheses included: (1) quantitative differences in drug metabolism profiles as a result of different affinities for the relevant enzymes; (2) differences in the amount of reactive metabolites produced due to cytochrome P450 (CYP450) inhibition by the AMAP reactive metabolites; (3) differences in the rate of conjugation between the reactive metabolites and proteins; (4) differences in the downstream effects or potencies of the reactive metabolites on vital components within hepatocytes. The simulations did not support hypotheses 3 or 4 as the most likely hypotheses underlying the difference in hepatoxic potential of APAP and AMAP. Rather, the simulations supported hypotheses 1 and 2 (less reactive metabolite produced per mole of AMAP relative to APAP). Within the simulations, the difference in reactive metabolite formation was equally likely to have occurred from differential affinities for the relevant drug metabolism enzymes or from direct CYP450 inhibition by the AMAP reactive metabolite. The demonstrated method of using simulation tools to probe the importance of possible contributors to toxicological observations is generally applicable across species.     

Conjugated dienes as prohaptens in contact allergy: in vivo and in vitro studies of structure-activity relationships, sensitizing capacity, and metabolic activation

There is a great interest in developing in vitro/in silico methods for the prediction of contact allergenic activity. However, many proposed methods do not take the activation of prohaptens to sensitizers by skin metabolism into account. As a consequence, consumer products containing potent sensitizers could be marketed. To identify prohaptens, studies regarding their structure-activity relationships and the mechanisms of their activation must be conducted. In the present investigation, we have studied the structure-activity relationships for alkene prohaptens. A series of seven alkenes (1-7), all of the same basic structure but with variation in the number and position(s) of the double bond(s), were designed and screened for sensitizing capacity using the murine local lymph node assay. Compounds 1-7 were also incubated with liver microsomes in the presence of glutathione to trap and identify reactive metabolites. The metabolic conversion of three alkenes (9-11) to epoxides (12-15) was also studied along with comparison of their sensitizing capacity. Our results show that conjugated dienes in or in conjunction with a six-membered ring are prohaptens that can be metabolically activated to epoxides and conjugated with GSH. Related alkenes containing isolated double bonds and an acyclic conjugated diene were shown to be weak or nonsensitizers. For the first time, the naturally occurring monoterpenes alpha-phellandrene, beta-phellandrene, and alpha-terpinene were demonstrated to be prohaptens able to induce contact allergy. The difference in sensitizing capacity of conjugated dienes as compared to alkenes with isolated double bonds was found to be due to the high reactivity and sensitizing capacity of the allylic epoxides metabolically formed from conjugated dienes. We recommend that these structure-activity relationship rules are incorporated into in silico predictive databases and propose that the prediction of contact allergenic activity of suspected prohaptens is based on assessment of susceptibility to metabolic activation and chemical reactivity of potential metabolites.     

Characterization of the human cytochrome P450 forms involved in metabolism of tamoxifen to its alpha-hydroxy and alpha,4-dihydroxy derivatives

Tamoxifen is a known hepatocarcinogen in rats and is associated with an increased incidence of endometrial cancer in patients. One mechanism for these actions is via bioactivation, where reactive metabolites are generated that are capable of binding to DNA or protein. Several metabolites of tamoxifen have been identified that appear to predispose to adduct formation. These include alpha-hydroxytamoxifen, alpha,4-dihydroxytamoxifen, and alpha-hydroxy-N-desmethyltamoxifen. Previous studies have shown that cytochrome P450 (P450) enzymes play an important role in the biotransformation of tamoxifen. The aim of our work was to determine which P450 enzymes were capable of producing alpha-hydroxylated metabolites from tamoxifen. When tamoxifen (18 or 250 microM) was used as the substrate, P450 3A4, and to a lesser extent, P450 2D6, P450 2B6, P450 3A5, P450 2C9, and P450 2C19 all produced a metabolite with the same HPLC retention time as alpha-hydroxytamoxifen at either substrate concentration tested. This peak was well-separated from 4-hydroxy-N-desmethyltamoxifen, which eluted substantially later under the chromatographic conditions used. No alpha,4-dihydroxytamoxifen was detected in incubations with any of the forms with tamoxifen as substrate. However, when 4-hydroxytamoxifen (100 microM) was used as the substrate, P450 2B6, P450 3A4, P450 3A5, P450 1B1, P450 1A1, and P450 2D6 all produced detectable concentrations of alpha,4-dihydroxytamoxifen. These studies demonstrate that multiple human P450s, including forms found in the endometrium, may generate reactive metabolites in women undergoing tamoxifen therapy, which could subsequently play a role in the development of endometrial cancer.     

In vitro characterization of 4′-(p-toluenesulfonylamide)-4-hydroxychalcone using human liver microsomes and recombinant cytochrome P450s

1.?4′-(p-Toluenesulfonylamide)-4-hydroxychalcone (TSAHC) is a synthetic sulfonylamino chalcone compound possessing anti-cancer properties. The aim of this study was to elucidate the metabolism of TSAHC in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of TSAHC.

2.?TSAHC was incubated with HLMs or recombinant P450 isoforms (rP450) in the presence of an nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-regenerating system. The metabolites were identified and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). P450 isoforms, responsible for TSAHC metabolite formation, were characterized by chemical inhibition and correlation studies in HLMs and enzyme kinetic studies with a panel of rP450 isoforms.

3.?Two hydroxyl metabolites, that is M1 and M2, were produced from the human liver microsomal incubations (K_m and V_max values were 2.46?µM and 85.1?pmol/min/mg protein for M1 and 9.98?µM and 32.1?pmol/min/mg protein for M2, respectively). The specific P450 isoforms responsible for two hydroxy-TSAHC formations were identified using a combination of chemical inhibition, correlation analysis and metabolism by expressed recombinant P450 isoforms. The known P450 enzyme activities and the rate of TSAHC metabolite formation in the 15 HLMs showed that TSAHC metabolism is correlated with CYP2C and CYP3A activity. The P450 isoform-selective inhibition study in HLMs and the incubation study of cDNA-expressed enzymes also showed that two hydroxyl metabolites M1 and M2 biotransformed from TSAHC are mainly mediated by CYP2C and CYP3A, respectively. These findings suggest that CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 isoforms are major enzymes contributing to TSAHC metabolism.     

Metabolism of capsaicin by cytochrome P450 produces novel dehydrogenated metabolites and decreases cytotoxicity to lung and liver cells

Capsaicin is a common dietary constituent and a popular homeopathic treatment for chronic pain. Exposure to capsaicin has been shown to cause various dose-dependent acute physiological responses including the sensation of burning and pain, respiratory depression, and death. In this study, the P450-dependent metabolism of capsaicin by recombinant P450 enzymes and hepatic and lung microsomes from various species, including humans, was determined. A combination of LC/MS, LC/MS/MS, and LC/NMR was used to identify several metabolites of capsaicin that were generated by aromatic (M5 and M7) and alkyl hydroxylation (M2 and M3), O-demethylation (M6), N- (M9) and alkyl dehydrogenation (M1 and M4), and an additional ring oxygenation of M9 (M8). Dehydrogenation of capsaicin was a novel metabolic pathway and produced unique macrocyclic, diene, and imide metabolites. Metabolism of capsaicin by microsomes was inhibited by the nonselective P450 inhibitor 1-aminobenzotriazole (1-ABT). Metabolism was catalyzed by CYP1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Addition of GSH (2 mM) to microsomal incubations stimulated the metabolism of capsaicin and trapped several reactive electrophilic intermediates as their GSH adducts. These results suggested that reactive intermediates, which inactivated certain P450 enzymes, were produced during catalytic turnover. Comparison of the rate and types of metabolites produced from capsaicin and its analogue, nonivamide, demonstrated similar pathways in the P450-dependent metabolism of these two capsaicinoids. However, production of the dehydrogenated (M4), macrocyclic (M1), and omega-1-hydroxylated (M3) metabolites was not observed for nonivamide. These differences may be reflective of the mechanism of formation of these metabolites of capsaicin. The role of metabolism in the cytotoxicity of capsaicin and nonivamide was also assessed in cultured lung and liver cells. Lung cells were markedly more sensitive to cytotoxicity by capsaicin and nonivamide. Cytotoxicity was enhanced 5 and 40% for both compounds by 1-ABT in BEAS-2B and HepG2, respectively. These data suggested that metabolism of capsaicinoids by P450 in cells represented a detoxification mechanism (in contrast to bioactivation).     

Differential selectivity in carbamazepine-induced inactivation of cytochrome P450 enzymes in rat and human liver

Oxidative metabolism of carbamazepine results in covalent binding of its reactive metabolite to liver microsomal proteins, which has been proposed as an important event in pathogenesis of the hypersensitivity reactions to this drug. Although the proposed reactive metabolites are produced by cytochrome P450 enzymes (P450 or CYP), the impact of the formation of unstable metabolites on the enzyme itself has not been elucidated. The present study examines the alteration of P450 enzyme activities during the metabolism of carbamazepine. Liver microsomes from rats and humans were preincubated with carbamazepine in the presence of NADPH, and subsequently assayed for monooxygenase activities representing several P450s. No evidence was obtained for inactivation of CYP2C11, CYP3A, CYP1A1/2 or CYP2B1/2 in rat liver microsomes during the carbamazepine metabolism, whereas the CYP2D enzyme was inactivated in a manner related to the preincubation time. Interestingly, under the same protocol human liver microsomes did not exhibit inactivation of CYP2D6, as well as there being no CYP2C8, CYP2C9 or CYP3A4 inactivation, whereas CYP1A2 was inactivated. Reduced glutathione could not protect against the observed inactivation of the P450s. These results suggest that CYP2D enzyme(s) in rats and CYP1A2 in humans biotransform carbamazepine into reactive metabolites, resulting in inactivation of the enzyme themselves, and raise the possibility that the P450 isoforms participate in toxicity induced by the drug in both animal species.     

In vitro metabolism of simazine,atrazine and propazine by hepatic cytochrome P450 enzymes of rat,mouse and guinea pig,and oestrogenic activity of chlorotriazines and their main metabolites

1. The in vitro metabolism of chlorotriazines, simazine (SIZ), atrazine (ATZ) and propazine (PRZ) in liver microsomes from rat, mouse and guinea pig and the oestrogenic activity of chlorotriazines and their main metabolites have been studied. 2. The formation rates of products in chlorotriazine metabolism were determined by HPLC. The principal reactions catalysed by the cytochrome P450 (P450) system were N- monodealkylation and isopropylhydroxylation in all liver microsomes. As a result, 2- chloro-4-ethylamino-6-amino-1,3,5-triazine (M1) (SIZ-M1 for SIZ and ATZ-M1 for ATZ) and 2-chloro-4-amino-6-isopropylamino-1,3,5-triazine (M2) (ATZ-M2 for ATZ and PRZ-M2 for PRZ), and 2-chloro-4-ethylamino-6-(1-hydroxyisopropylamino)-1,3,5- triazine (M3)(ATZ-M3for ATZ) and 2-chloro-4-isopropylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazine (M4) (PRZ-M4 for PRZ) were detected as the metabolites. N- bidealkylation was not found in this system. 3. The formation rates of N-deethylated metabolites (SIZ-M1 and ATZ-M2) were generally higher in mouse than in rat and guinea pig. The formation rates of N- deisopropylated metabolites (ATZ-M1 and PRZ-M2) in guinea pig were the lowest among the three animal species. The formation rates of isopropylhydroxylated metabolites (ATZM3 and PRZ-M4) were remarkably low in mouse compared with rat and guinea pig. 4. The enzyme kinetics of chlorotriazine metabolism were examined by Eadie-Hofstee analyses. Some species differences in Michaelis-Menten parameters for each metabolite were observed, and the ranking orders were varied among the metabolites. 5. The binding affinity of chlorotriazines (SIZ, ATZ and PRZ) and their metabolites (M1-4) for recombinant human oestrogen receptor-alpha was assayed using the fluorescence polarization method. The binding a nity of M2 was significantly higher than those of parent compounds and other metabolites, although the oestrogenic activity was remarkably low compared with that of 17 beta-oestradiol (E2). 6. These results suggest that the pattern of metabolism of SIZ, ATZ and PRZ by the P450 system differs extensively among rat, mouse and guinea pig, and that M2 may be an activated metabolite of chlorotriazines.     

In vitro metabolism of simazine, atrazine and propazine by hepatic cytochrome P450 enzymes of rat, mouse and guinea pig, and oestrogenic activity of chlorotriazines and their main metabolites

1. The in vitro metabolism of chlorotriazines, simazine (SIZ), atrazine (ATZ) and propazine (PRZ) in liver microsomes from rat, mouse and guinea pig and the oestrogenic activity of chlorotriazines and their main metabolites have been studied. 2. The formation rates of products in chlorotriazine metabolism were determined by HPLC. The principal reactions catalysed by the cytochrome P450 (P450) system were N-monodealkylation and isopropylhydroxylation in all liver microsomes. As a result, 2-chloro-4-ethylamino-6-amino-1,3,5-triazine (M1) (SIZ-M1 for SIZ and ATZ-M1 for ATZ) and 2-chloro-4-amino-6-isopropylamino-1,3,5-triazine (M2) (ATZ-M2 for ATZ and PRZ-M2 for PRZ), and 2-chloro-4-ethylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazine (M3) (ATZ-M3 for ATZ) and 2-chloro-4-isopropylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazi ne (M4) (PRZ-M4 for PRZ) were detected as the metabolites. N-bidealkylation was not found in this system. 3. The formation rates of N-deethylated metabolites (SIZ-M1 and ATZ-M2) were generally higher in mouse than in rat and guinea pig. The formation rates of N-deisopropylated metabolites (ATZ-M1 and PRZ-M2) in guinea pig were the lowest among the three animal species. The formation rates of isopropylhydroxylated metabolites (ATZ-M3 and PRZ-M4) were remarkably low in mouse compared with rat and guinea pig. 4. The enzyme kinetics of chlorotriazine metabolism were examined by Eadie-Hofstee analyses. Some species differences in Michaelis-Menten parameters for each metabolite were observed, and the ranking orders were varied among the metabolites. 5. The binding affinity of chlorotriazines (SIZ, ATZ and PRZ) and their metabolites (M1-4) for recombinant human oestrogen receptor-alpha was assayed using the fluorescence polarization method. The binding affinity of M2 was significantly higher than those of parent compounds and other metabolites, although the oestrogenic activity was remarkably low compared with that of 17beta-oestradiol (E2). 6. These results suggest that the pattern of metabolism of SIZ, ATZ and PRZ by the P450 system differs extensively among rat, mouse and guinea pig, and that M2 may be an activated metabolite of chlorotriazines.     

Metabolic detoxication pathways for sterigmatocystin in primary tracheal epithelial cells

Human health effects of inhaled mycotoxins remain poorly documented, despite the large amounts present in bioaerosols. Among these mycotoxins, sterigmatocystin is one of the most prevalent. Our aim was to study the metabolism and cellular consequences of sterigmatocystin once it is in contact with the airway epithelium. Metabolites were analyzed first in vitro, using recombinant P450 1A1, 1A2, 2A6, 2A13, and 3A4 enzymes, and subsequently in porcine tracheal epithelial cell (PTEC) primary cultures at an air-liquid interface. Expressed enzymes and PTECs were exposed to sterigmatocystin, uniformly enriched with (13)C to confirm the relationship between sterigmatocystin and metabolites. Induction of the expression of xenobiotic-metabolizing enzymes upon sterigmatocystin exposure was examined by real-time quantitative real-time polymerase chain reaction. Incubation of 50 μM sterigmatocystin with recombinant P450 1A1 led to the formation of three metabolites: monohydroxy-sterigmatocystin (M1), dihydroxy-sterigmatocystin (M2), and one glutathione adduct (M3), the latter after the formation of a transient epoxide. Recombinant P450 1A2 also led to M1 and M3. P450 3A4 led to only M3. In PTEC, 1 μM sterigmatocystin metabolism resulted in a glucuro conjugate (M4) mainly excreted at the basal side of cells. If PTEC were treated with β-naphthoflavone prior to sterigmatocystin incubation, two other products were detected, i.e., a sulfo conjugate (M5) and a glucoro conjugate (M6) of hydroxy-sterigmatocystin. Exposure of PTEC for 24 h to 1 μM sterigmatocystin induced an 18-fold increase in the mRNA levels of P450 1A1, without significantly induced 7-ethoxyresorufin O-deethylation activity. These data suggest that sterigmatocystin is mainly detoxified and is unable to produce significant amounts of reactive epoxide metabolites in respiratory cells. However, sterigmatocystin increases the P450 1A1 mRNA levels with unknown long-term consequences. These in vitro results obtained in the porcine pulmonary tract need to be confirmed in human epithelial cells.     

Phenytoin metabolism by human cytochrome P450: involvement of P450 3A and 2C forms in secondary metabolism and drug-protein adduct formation.

The anticonvulsant phenytoin (5,5-diphenylhydantoin) provokes a skin rash in 5 to 10% of patients, which heralds the start of an idiosyncratic reaction that may result from covalent modification of normal self proteins by reactive drug metabolites. Phenytoin is metabolized by cytochrome P450 (P450) enzymes primarily to 5-(p-hydroxyphenyl-),5-phenylhydantoin (HPPH), which may be further metabolized to a catechol that spontaneously oxidizes to semiquinone and quinone species that covalently modify proteins. The aim of this study was to determine which P450s catalyze HPPH metabolism to the catechol, proposed to be the final enzymatic step in phenytoin bioactivation. Recombinant human P450s were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Novel bicistronic expression vectors were constructed for P450 2C19 and the three major variants of P450 2C9, i.e., 2C9*1, 2C9*2, and 2C9*3. HPPH metabolism and covalent adduct formation were assessed in parallel. P450 2C19 was the most effective catalyst of HPPH oxidation to the catechol metabolite and was also associated with the highest levels of covalent adduct formation. P450 3A4, 3A5, 3A7, 2C9*1, and 2C9*2 also catalyzed bioactivation of HPPH, but to a lesser extent. Fluorographic analysis showed that the major targets of adduct formation in bacterial membranes were the catalytic P450 forms, as suggested from experiments with human liver microsomes. These results suggest that P450 2C19 and other forms from the 2C and 3A subfamilies may be targets as well as catalysts of drug-protein adduct formation from phenytoin.     

Trimethoprim: novel reactive intermediates and bioactivation pathways by cytochrome p450s

Trimethoprim (TMP) is a widely used antibacterial agent that is usually considered as a safe drug. TMP has, however, been implicated in rare adverse drug reactions (ADRs) in humans. Bioactivation to a reactive iminoquinone methide intermediate has been proposed as a possible cause for the toxicity of the drug. However, little is known about the cytochrome P450s (P450s) involved in this bioactivation and in the metabolism of TMP in general. In this study, we have investigated the metabolism and bioactivation of TMP by human liver microsomes (HLM) and rat liver microsomes, by recombinant human cytochrome P450s, and by the bacterial P450 BM3 mutant M11(his). In addition to non GSH-dependent metabolites, five GSH adducts were identified in the HLM incubations. Next to two major GSH adducts probably originating from the iminoquinone methide intermediate described previously, three minor GSH adducts were also identified, indicating that other types of reactive intermediates are formed by HLM, such as ortho-quinones and para-quinone methide intermediates. The major GSH adducts were produced by P450 1A2 and P450 3A4, while the minor GSH adducts were mainly formed by P450 1A2, P450 3A4, and P450 2D6. Although preliminary, these results might implicate that genetic polymorphisms in P450 enzymes could play a role in the onset of TMP-related ADRs in humans.     

Role of cytochrome P450 2E1 in the metabolism of acrylamide and acrylonitrile in mice

Acrylonitrile (AN) and acrylamide (AM) are commonly used in the synthesis of plastics and polymers. In rodents, AM and AN are metabolized to the epoxides glycidamide and cyanoethylene oxide, respectively. The aim of this study was to determine the role of cytochrome P450 in the metabolism of AM and AN in vivo. Wild-type (WT) mice, WT mice pretreated with aminobenzotriazole (ABT, 50 mg/kg ip, 2 h pre-exposure), and mice devoid of cytochrome P450 2E1 (P450 2E1-null) were treated with 50 mg/kg [(13)C]AM po. WT mice and P450 2E1-null mice were treated with 2.5 or 10 mg/kg [(13)C]AN po. Urine was collected for 24 h, and metabolites were characterized using (13)C NMR. WT mice excreted metabolites derived from the epoxides and from direct GSH conjugation with AM or AN. Only metabolites derived from direct GSH conjugation with AM or AN were observed in the urine from ABT-pretreated WT mice and P450 2E1-null mice. On the basis of evaluation of urinary metabolites at these doses, these data suggest that P450 2E1 is possibly the only cytochrome P450 enzyme involved in the metabolism of AM and AN in mice, that inhibiting total P450 activity does not result in new pathways of non-P450 metabolism of AM, and that mice devoid of P450 2E1 do not excrete metabolites of AM or AN that would be produced by oxidation by other cytochrome P450s. P450 2E1-null mice may be an appropriate model for the investigation of the role of oxidative metabolism in the toxicity or carcinogenicity of these compounds.     

Identification of 17-alpha-ethynylestradiol-modified active site peptides and glutathione conjugates formed during metabolism and inactivation of P450s 2B1 and 2B6

The oral contraceptive 17-alpha-ethynylestradiol (17EE) is a mechanism-based inactivator of cytochrome P450s (P450s) 2B1 and 2B6. Inactivation of P450s 2B1 and 2B6 in the reconstituted system by [3H]17EE resulted in labeling of the P450 apoprotein. Mass spectral analysis of 17EE-inactivated P450 2B1 showed an increase in the mass of the apoprotein by 313 Da, consistent with the mass of 17EE plus one oxygen atom. P450s 2B1 and 2B6 were inactivated with [3H]17EE and digested with CNBr. Separation of these peptides resulted in the identification of one major labeled peptide for each enzyme. N-Terminal sequencing of these peptides yielded the amino acid sequences PYTDAVIHEI (for P450 2B1) and PYTEAV (for P450 2B6) that corresponded to amino acids P347-M376 and P347-M365 in P450s 2B1 and 2B6, respectively. Electrospray ionization (ESI)-liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization (MALDI)-MS analysis of the P450 2B1-derived peptide resulted in a mass of 3654 Da consistent with the mass of the P347-M376 peptide (3385 Da) plus a 268 Da 17EE adduct. Chemically reactive intermediates of 17EE that were generated during the metabolism of 17EE by P450s 2B1 and 2B6 were trapped with gluthathione (GSH). ESI-LC-MS/MS analysis of 17EE-GSH conjugates from the incubation mixtures indicated that P450s 2B1 and 2B6 generated different reactive 17EE intermediates that were responsible for the inactivation and protein modification or the formation of GSH conjugates by these two enzymes.     

Identification of N-(hydroxymethyl) norcotinine as a major product of cytochrome P450 2A6, but not cytochrome P450 2A13-catalyzed cotinine metabolism

Cotinine formation is the major pathway of nicotine metabolism in smokers, and the primary pathway of cotinine metabolism is trans-3'-hydroxylation. trans-3'-Hydroxycotinine and its glucuronide conjugate account for up to 50% of the nicotine metabolites excreted by smokers. Minor metabolites of cotinine excreted by smokers include norcotinine and cotinine N-oxide, each of which account for <5% of the nicotine dose. It has been reported that P450 2A6 is the catalyst of cotinine metabolism. However, we report here that the major product of P450 2A6-catalyzed cotinine metabolism is N-(hydroxymethyl)norcotinine, a previously unknown human metabolite of cotinine. N-(Hydroxymethyl)norcotinine was chemically synthesized, and its stability under the conditions of the enzyme reactions was confirmed. The products of P450 2A6-catalyzed [5-3H]cotinine metabolism were quantified by radioflow HPLC. The identification of N-(hydroxymethyl)norcotinine as the major metabolite was based on HPLC analysis on three unique systems and coelution with N-(hydroxymethyl)norcotinine standard. 5'-Hydroxycotinine and trans-3'-hydroxycotinine were minor products of P450 2A6-catalyzed cotinine metabolism, accounting for 14 and 8% of the total cotinine metabolites, respectively. N-(Hydroxymethyl)norcotinine was a product of cotinine metabolism by the extrahepatic P450, 2A13, but it was a minor one. The major product of P450 2A13-catalyzed cotinine metabolism was 5'-hydroxycotinine, which was formed at twice the rate of trans-3'-hydroxycotinine. The identification of all cotinine metabolites formed by both enzymes was confirmed by LC/MS/MS analysis. Kinetic parameters for cotinine metabolism were determined for P450 2A6 and P450 2A13. This work has confirmed that the major metabolite of cotinine in smokers, trans-3'-hydroxycotinine, is only a minor metabolite of P450 2A6-catalyzed cotinine metabolism.     

Enhancement of doxorubicin toxicity following activation by NADPH cytochrome P450 reductase.

Treatment of MCF-7 cells with doxorubicin in the presence of purified rat NADPH cytochrome P450 reductase (P450red) and NADPH resulted in a marked enhancement of drug cytotoxicity. No potentiation of cell killing was observed when the drug was incubated with P450red and NADPH prior to addition to the cells, implicating the involvement of short-lived species. The increase in doxorubicin toxicity was clearly related to its metabolism by P450red. Of a variety of free radical scavenging agents tested only glutathione was effective in protecting against the toxic metabolites produced by reductive activation. Preliminary experiments also showed that doxorubicin binding to cellular proteins and DNA occurred to a greater extent when cells were treated with the drug and P450red. Our data indicate that oxygen radicals do not play a primary role in cytochrome P450 reductase-mediated doxorubicin cytotoxicity and suggest that, in this case, the mechanism of toxicity involves a reactive species which binds to cellular nucleophiles.