Metabolism of acrylonitrile to 2-cyanoethylene oxide in F-344 rat liver microsomes, lung microsomes, and lung cells

The metabolism of acrylonitrile to the epoxide, 2-cyanoethylene oxide (ANO) was examined in rat liver microsomes, lung microsomes, and isolated enriched lung cell preparations. GC/high resolution MS was used to quantitate ANO in microsomal and cellular extracts by monitoring the fragment ion C2H3N (m/z 41.0265). The limit of detection was 0.05 pmol of ANO/0.5 microliter of standard solution, microsomal extract, or cellular extract injected onto the column, and the linear range of analysis was 0.05 to 12.5 pmol of ANO. Kinetic parameters of Vmax, V/K, and Km were calculated for microsomal ANO formation. Liver microsomes were quantitatively more active than lung microsomes on a mg of protein basis. The Vmax (pmol of ANO formed/min/mg of protein) was 666.61 for liver and 45.07 for lung microsomes. The V/K (pmol of ANO/min/mg of protein/microM) was 12.83 for liver and 0.02 for lung microsomes. The apparent Km was 51.93 microM and 1853.83 microM for liver and lung microsomes, respectively. When calculated as nmol of ANO formed/min/nmol of microsomal P-450, the Vmax for lung was equivalent to the Vmax for liver. ANO formation in the rat lung was cell specific. The rates of metabolism in the Clara cell-enriched fraction, the alveolar type II cell-enriched fraction, and the cell suspension were 2.55, 0.38, and 0.67 pmol of ANO formed/min/mg of protein, respectively. No metabolism was observed in the endothelial (small) cell-enriched fraction or in the alveolar macrophages. The results suggest that the lung contributes to the metabolism and disposition of inhaled acrylonitrile.     

Heterologous expression of human microsomal epoxide hydrolase in Saccharomyces cerevisiae. Study of the valpromide-carbamazepine epoxide interaction

A cDNA of human microsomal epoxide hydrolase (hmEH) was constitutively and inducibly expressed in Saccharomyces cerevisiae. The heterologous enzyme was located mainly in the microsomal fraction of yeast cells. Yeast microsomes containing hmEH exerted styrene oxide hydrolase activity (Km = 300 microM; Vmax = 22 nmol/mg min) as well as carbamazepine epoxide hydrolase activity. The hmEH catalysed exclusively the formation of carbamazepine-10,11-transdihydrodiol, since no carbamazepine-10,11-cisdihydrodiol was detected. Inhibition studies using these microsomes revealed unequivocally hmEH as the target for inhibition by the antiepileptic drug valpromide. A Ki value of 27 microM was determined for the inhibitor valpromide with styrene oxide as substrate. For carbamazepine epoxide, a Ki value of 8.6 microM was obtained, which is well in line with data published for hmEH determined with human liver microsomes. Our results demonstrate the potential of heterologous gene expression in S. cerevisiae and its application to the in vitro study of pharmacological and toxicological problems.     

Metabolic activation of the tricyclic antidepressant amineptine--I. Cytochrome P-450-mediated in vitro covalent binding

Incubation of [14C]amineptine (1 mM) with hamster liver microsomes resulted in the irreversible binding of an amineptine metabolite to microsomal proteins. Covalent binding measured in the presence of various concentrations of amineptine (0.0625-1 mM) followed Michaelis-Menten kinetics. Pretreatment with phenobarbital increased not only the Vmax, but also the Km, for this binding. Covalent binding required NADPH and molecular oxygen and was decreased when the incubation was made in the presence of inhibitors of cytochrome P-450 such as piperonyl butoxide (4 mM), SKF 525-A (4 mM) or carbon monoxide (80:20 CO-O2 atmosphere). In contrast, binding was increased when microsomes from untreated hamsters were incubated in the presence of 0.5 mM 1,1,1-trichloropropene 2,3-oxide, an inhibitor of epoxide hydrolase. Metabolic activation also occurred in kidney microsomes. In vitro covalent binding to kidney microsomal proteins required NADPH and was decreased by piperonyl butoxide (4 mM) but was not increased by pretreatment with phenobarbital. We conclude that amineptine is activated by hamster liver and kidney microsomes into a chemically reactive metabolite that covalently binds to microsomal proteins.     

Comparative metabolism of carbon tetrachloride in rats, mice, and hamsters using gas uptake and PBPK modeling

No study has comprehensively compared the rate of metabolism of carbon tetrachloride (CCl4) across species. Therefore, the in vivo metabolism of CCl4 was evaluated using groups of male animals (F344 rats, B6C3F1 mice, and Syrian hamsters) exposed to 40-1800 ppm CCl4 in a closed, recirculating gas-uptake system. For each species, an optimal fit of the family of uptake curves was obtained by adjusting Michaelis-Menten metabolic constants Km (affinity) and Vmax (capacity) using a physiologically based pharmacokinetic (PBPK) model. The results show that the mouse has a slightly higher capacity and lower affinity for metabolizing CCl4 compared to the rat, while the hamster has a higher capacity and lower affinity than either rat or mouse. A comparison of the Vmax to Km ratio, normalized for milligrams of liver protein (L/h/mg) across species, indicates that hamsters metabolize more CCl4 than either rats or mice, and should be more susceptible to CCl4-induced hepatotoxicity. These species comparisons were evaluated against toxicokinetic studies conducted in animals exposed by nose-only inhalation to 20 ppm 14C-labeled CCl4 for 4 h. The toxicokinetic study results are consistent with the in vivo rates of metabolism, with rats eliminating less radioactivity associated with metabolism (14CO2 and urine/feces) and more radioactivity associated with the parent compound (radioactivity trapped on charcoal) compared to either hamsters or mice. The in vivo metabolic constants determined here, together with in vitro constants determined using rat, mouse, hamster, and human liver microsomes, were used to estimate human in vivo metabolic rates of 1.49 mg/h/kg body weight and 0.25 mg/L for Vmax and Km, respectively. Normalizing the rate of metabolism (Vmax/Km) by milligrams liver protein, the rate of metabolism of CCl4 differs across species, with hamster > mouse > rat > human.     

Induction of liver microsomal epoxide hydrolase, UDP-glucuronyl transferase and cytosolic glutathione transferase in different rodent species by 2-acetylaminofluorene or 3-methylcholanthrene

Control activities vary 12-fold for microsomal epoxide hydrolase, two-fold for UDP-glucuronyl transferase and five-fold for cytosolic glutathione (GSH) transferase among the different rodents (rat, hamster, guinea-pig, mouse) examined. For all three enzymes the activities in rat liver are towards the lower values. In these rodents, except for a 100% increase in microsomal epoxide hydrolase in guinea-pig liver, 2-acetylaminofluorene induces the three phase 2 enzymes only in rat. Treatment with 3-methylcholanthrene also produces the largest effects on these three enzyme activities in rat liver; exceptions are its failure to induce microsomal epoxide hydrolase in female rat and the large induction of cytosolic GSH transferase in hamster liver. Quantitatively, hepatic microsomal epoxide hydrolase, UDP-glucuronyl transferase and cytosolic GSH transferase activities, and their inducibility by 2-acetylaminofluorene or 3-methylcholanthrene, in male Sprague-Dawley rats are not representative for other rodent species or even, in all cases, for female rat.     

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.     

Organ distribution of epoxide hydrolases in cytosolic and microsomal fractions of normal and nafenopin-treated male DBA/2 mice

Using trans-stilbene oxide and styrene oxide as substrates, epoxide hydrolase activities were measured in cytosolic and microsomal fractions from liver, kidney, heart, lung and testis of male DBA/2 mice. The activities towards these two substrates are remarkably organ specific: trans-stilbene oxide was most effectively hydrolyzed in subcellular fractions from liver, kidney and heart, whereas styrene oxide was predominantly hydrolyzed in those from liver, lung and testis. Immunoblotting experiments were performed with two polyclonal antibodies isolated from goat antisera. Using an anti-mouse liver cytosolic epoxide hydrolase antibody, the corresponding antigen protein was predominantly detected in both cytosolic and microsomal fractions from liver, kidney and heart. An anti-rat liver microsomal epoxide hydrolase antibody proved to be cross-reactive with the mouse enzyme and stained SDS-gels run with microsomal fractions from liver, lung and testis. The anti-mouse liver cytosolic epoxide hydrolase antibody precipitated cytosolic epoxide hydrolase activities from liver, kidney and heart cytosolic fractions. Dietary exposure to the hypolipidemic agent nafenopin (2000 ppm/10 days) caused an induction of trans-stilbene oxide hydrolase and styrene oxide hydrolase activities in cytosolic and microsomal liver fractions whereas, in the other organs, the same activities were unaffected by this treatment. This finding was in accordance with the increased amounts of antigen protein as detected with the antibodies in liver fractions from treated animals. The anti-mouse liver cytosolic epoxide hydrolase antibody was found to precipitate the whole trans-stilbene oxide hydrolase activity also from liver cytosol of nafenopin-treated mice, which indicates the presence of a single cytosolic epoxide hydrolase following induction.     

Epoxide hydrolase in human fetal liver

Epoxide hydrolase activity towards styrene oxide was measured in the microsomal fraction of 20 human fetal livers. The enzymatic activity was 5.60 +/- 0.52 nmol/min/mg (mean +/- SE) which is about 40% of the previously reported value in human adult liver microsomes. No relation between enzymatic activity and fetal age was observed. The kinetics of the enzyme were studied in 6 different livers and found to obey Michaelis-Menten kinetics. The Km ranged between 0.25 and 0.54 mmol/l and Vmax between 7.2 and 16.7 nmol/min/mg microsomal protein. The enzyme was inhibited both by 1,1,1-trichloropropene-2,3-oxide (TCPO; 0.25 mmol/l) and benzo(a)pyrene-4,5-oxide (BPO; 0.2 mmol/l). Those substances inhibited the epoxide hydrolase by 61 and 14%, respectively, at 1 mmol/l styrene oxide. Thus TCPO was considerably more potent as an inhibitor of the fetal liver styrene oxide hydrolase. Lineweaver-Burk plots of the inhibition data revealed that TCPO exerts an uncompetitive mixed type of inhibition.     

The formation and toxicity of catechol metabolites of acetaminophen in mice

Acetaminophen is metabolized to a catechol, 3'-hydroxy-4'-hydroxyacetanilide (3-hydroxyacetaminophen), by mouse liver microsomes, and to both catechol and methylated catechol metabolites by the mouse in vivo. Although 3-hydroxyacetaminophen is less hepatotoxic in mice than acetaminophen itself, 3-methoxyacetaminophen is as hepatotoxic as acetaminophen and is subject to a glutathione threshold effect. However, neither metabolite is formed in sufficient amounts to account for the hepatotoxicity caused by acetaminophen in the mouse. Mouse liver microsomes catalyze the oxidation of acetaminophen to the catechol in an apparent cytochrome P-450-mediated reaction that is induced by phenobarbital and inhibited by piperonyl butoxide, but is surprisingly not altered by cobaltous chloride. Lineweaver-Burk analysis of the oxidation carried out by liver microsomes from control animals gave curvilinear plots that may indicate catalysis by two enzyme sites with apparent Km values of 0.011 and 0.271 mM, and apparent Vmax values of 87 and 162 pmol/mg/min, respectively. Neither an isotope effect nor an NIH shift were measurable in the microsomal metabolism of selectively deuterated analogs of acetaminophen to 3-hydroxyacetaminophen. These results, coupled with results of previous investigations with 18O2 and epoxide hydrolase, indicate that a mechanism different from either direct insertion or epoxidation is involved in the formation of the catechol metabolite of acetaminophen.     

Activation of 8-methoxypsoralen by cytochrome P-450. Enzyme kinetics of covalent binding and influence of inhibitors and inducers of drug metabolism

The kinetics of covalent binding of reactive metabolites of 8-methoxypsoralen (8-MOP) to protein were measured in incubations of liver microsomes of rats pretreated for 3 days with i.p. injections of 80 mg/kg/day of beta-naphthoflavone (BNF), phenobarbital (PB), 8-MOP, or vehicle. Covalent binding of radioactivity derived from [14C]8-MOP (labeled at the metabolically stable 4-position in the coumarin ring) required NADPH, obeyed classical Michaelis-Menten kinetics, and was inducible by both PB and BNF. Plots of V versus V/[S] were linear in liver microsomes of rats pretreated with vehicle, PB, or 8-MOP; respective values for Km were 26, 24 and 13 microM and for Vmax were 0.61, 1.70 and 0.50 nmol bound/min/mg protein. In microsomes of rats pretreated with BNF, high- and low-affinity components of covalent binding were observed with respective values for Km of 4.7 and 117 microM and for Vmax of 0.77 and 1.71 nmol bound/min/mg protein. Addition of glutathione and cysteine to the incubations decreased covalent binding by 33 and 67%, respectively, presumably by trapping reactive electrophilic metabolites. Inhibition of epoxide hydrolase with 1,1,1-trichloropropene-2,3-oxide did not affect covalent binding of reactive metabolites of 8-MOP. SKF-525A was a potent inhibitor of both the metabolism of 8-MOP and covalent binding in microsomes from rats pretreated with PB, but had only a slight effect in microsomes from rats pretreated with BNF. In contrast, alpha-naphthoflavone almost completely inhibited metabolism of 8-MOP and covalent binding in BNF-induced microsomes but had no effect in PB-induced microsomes. Apparent covalent binding was reduced by 39% in incubations with 8-MOP labeled with tritium in the metabolically labile methoxy group. Collectively, these results indicate that 8-MOP is biotransformed by two or more isozymes of cytochrome P-450 to reactive electrophiles capable of binding to tissue macromolecules.     

Properties of the microsomal and cytosolic glutathione transferases involved in hexachloro-1:3-butadiene conjugation

Hexachloro-1,3-butadiene (HCBD) is a substrate for the hepatic microsomal glutathione transferases and is metabolised at higher rates by these enzymes than their cytosolic counterparts. Conjugation reactions catalysed by the microsomal and cytosolic transferases have been studied and characterized using this substrate and 1-chloro-2,4-dinitrobenzene (CDNB). In rat liver microsomes the Km values for HCBD and CDNB were 0.91 and 0.012 mM and in cytosol 0.51 and 0.10 mM respectively. Vmax values for HCBD were 1.39 and 0.35 nmol conjugate formed/min/mg protein for microsomes and cytosol respectively. In microsomal systems HCBD was a potent competitive inhibitor of the metabolism of CDNB with a Ki value of approximately 10 microM. However, CDNB did not inhibit HCBD metabolism significantly. These data suggest that more than one microsomal enzyme is involved in HCBD metabolism. The microsomal membrane could be solubilized without significant inhibition of HCBD activity; however, some detergents did inhibit the conjugation reaction. Activity was also lost on treatment of microsomal membranes with trypsin indicating the enzyme is localized on the cytoplasmic surface of the endoplasmic reticulum. Pretreatment of the rats with Aroclor 1254, 3-methylcholanthrene or phenobarbital did not change the microsomal conjugation of HCBD or CDNB with glutathione. Of seven species investigated, a human liver sample showed the highest ratio of microsomal to cytosolic glutathione transferase activity for HCBD (in microsomes 40-fold higher specific activity than in cytosol). Glutathione conjugation appears to play a critical role in the toxicity and carcinogenicity of some halogenated hydrocarbons. These data substantiate the potentially important role for the microsomal glutathione transferase in catalysing these reactions.     

Roles of human CYP2A6 and rat CYP2B1 in the oxidation of (+)-fenchol by liver microsomes

The metabolism of (+)-fenchol was investigated in vitro using liver microsomes of rats and humans and recombinant cytochrome P450 (P450 or CYP) enzymes in insect cells in which human/rat P450 and NADPH-P450 reductase cDNAs had been introduced. The biotransformation of (+)-fenchol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Fenchol was oxidized to fenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on GC. Several lines of evidence suggested that CYP2A6 is a major enzyme involved in the oxidation of (+)-fenchol by human liver microsomes. (+)-Fenchol oxidation activities by liver microsomes were very significantly inhibited by (+)-menthofuran, a CYP2A6 inhibitor, and anti-CYP2A6. There was a good correlation between CYP2A6 contents and (+)-fenchol oxidation activities in liver microsomes of ten human samples. Kinetic analysis showed that the Vmax/Km values for (+)-fenchol catalysed by liver microsomes of human sample HG03 were 7.25 nM-1 min-1. Human recombinant CYP2A6-catalyzed (+)-fenchol oxidation with a Vmax value of 6.96 nmol min-1 nmol-1 P450 and apparent Km value of 0.09 mM. In contrast, rat CYP2A1 did not catalyse (+)-fenchol oxidation. In the rat (+)-fenchol was oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol by liver microsomes of phenobarbital-treated rats. Recombinant rat CYP2B1 catalysed (+)-fenchol oxidation. Kinetic analysis showed that the Km values for the formation of fenchone, 6-exo- hydroxyfenchol and 10-hydroxyfenchol in rats treated with phenobarbital were 0.06, 0.03 and 0.03 mM, and Vmax values were 2.94, 6.1 and 13.8 nmol min-1 nmol-1 P450, respectively. Taken collectively, the results suggest that human CYP2A6 and rat CYP2B1 are the major enzymes involved in the metabolism of (+)-fenchol by liver microsomes and that there are species-related differences in the human and rat CYP2A enzymes.     

Determination of the human cytochrome P450s involved in the metabolism of 2n-propylquinoline

1 2n-Propylquinoline (2nPQ) is a newly developed drug for visceral antileishmaniasis and its activity has been previously evaluated in mice following oral administration. The study was carried out to investigate the kinetic formation of 2nPQ metabolites and to characterize the human liver CYP forms involved in its oxidative metabolism. 2. The inhibition of 2nPQ metabolite formation by specific substrates or inhibitors of CYP forms and correlation studies were performed in human liver microsomes. 2nPQ biotransformation was then studied in human lymphoblasts expressing specific CYPs and microsomal epoxide hydrolase. 3. Three major metabolites were produced by human liver microsomes and their structures were identified by ESI-LC/MS: dihydroxy-2n-propylquinoline, 3'-hydroxy-2n-propylquinoline and 1'-hydroxy-2n-propylquinoline. An intermediary metabolite, epoxy-2n-propylquinoline, formed by CYP was also biotransformed by microsomal epoxide hydrolase into dihydroxy-2n-propylquinoline. 4. 2nPQ oxidation follows Michaelis-Menten kinetics. In human liver microsomes, its metabolism was extremely inhibited by pilocarpine, coumarin and diethyldithiocarbamate. From a panel of 12 human liver microsome samples, the rate of 2nPQ oxidation was highly correlated with the activities of CYP2A6 and CYP2E1. Human lymphoblasts expressing specific CYPs showed the involvement of CYP2A6, CYP2E1 and CYP2C19. 5. The results indicate that 2nPQ metabolites are 3'- and 1'-hydroxylated by human liver microsomes and an epoxy-2n-propylquinoline is biotransformed into a dihydroxy-2n-propylquinoline by microsomal epoxide hydrolase.     

Proliferation of peroxisomes and induction of cytosolic and microsomal epoxide hydrolases in different strains of mice and rats after dietary treatment with clofibrate

1. The effects of dietary clofibrate (0.5%, w/w, for 10 days) on seven inbred strains of mice—C57BL/6, C57BL/B10A(5R), ATL/OLA, C3H/HE/OLA, BALB/C, CBA/CA and A/J/OLA—and three strains of rats—Sprague-Dawley, Wistar and LOU/OLA—have been investigated. Liver weight, peroxisome proliferation, catalase activity, cytosolic, microsomal and mitochondrial epoxide hydrolase activities, cytochrome oxidase activity, microsomal cytochrome P-450 content and cytosolic glutathione transferase activity in liver were determined, together with cytosolic and microsomal epoxide hydrolase and cytosolic glutathione transferase activities in the kidneys.

2. In all cases peroxisome proliferation and induction of cytosolic epoxide hydrolase were observed in livers of rodents exposed to clofibrate. Thus, no non-responsive strains were found and further evidence for a coupling between these two phenomena was provided. In many cases significant increases in the liver microsomal cytochrome P-450 content and decreases in the hepatic cytosolic glutathione transferase activity were also seen.

3. High levels of cytosolic epoxide hydrolase were found in the rat kidney. In several strains of mice and rats renal cytosolic epoxide hydrolase activity was increased by clofibrate.

4. There were often considerable strain differences. However, in general mice had higher cytosolic epoxide hydrolase and glutathione transferase activities, whereas rats had higher microsomal epoxide hydrolase activities.     

Proliferation of peroxisomes and induction of cytosolic and microsomal epoxide hydrolases in different strains of mice and rats after dietary treatment with clofibrate

1. The effects of dietary clofibrate (0.5%, w/w, for 10 days) on seven inbred strains of mice--C57BL/6, C57BL/B10A(5R), ATL/OLA, C3H/HE/OLA, BALB/C, CBA/CA and A/J/OLA--and three strains of rats--Sprague-Dawley, Wistar and LOU/OLA--have been investigated. Liver weight, peroxisome proliferation, catalase activity, cytosolic, microsomal and mitochondrial epoxide hydrolase activities, cytochrome oxidase activity, microsomal cytochrome P-450 content and cytosolic glutathione transferase activity in liver were determined, together with cytosolic and microsomal epoxide hydrolase and cytosolic glutathione transferase activities in the kidneys. 2. In all cases peroxisome proliferation and induction of cytosolic epoxide hydrolase were observed in livers of rodents exposed to clofibrate. Thus, no non-responsive strains were found and further evidence for a coupling between these two phenomena was provided. In many cases significant increases in the liver microsomal cytochrome P-450 content and decreases in the hepatic cytosolic glutathione transferase activity were also seen. 3. High levels of cytosolic epoxide hydrolase were found in the rat kidney. In several strains of mice and rats renal cytosolic epoxide hydrolase activity was increased by clofibrate. 4. There were often considerable strain differences. However, in general mice had higher cytosolic epoxide hydrolase and glutathione transferase activities, whereas rats had higher microsomal epoxide hydrolase activities.     

Rat liver metabolism and toxicity of 2,2,2-trifluoroethanol.

2,2,2-Trifluoroethanol (TFE) is a metabolite of anesthetic agents and chlorofluorocarbon alternatives. Its toxicity in rats is a consequence of its metabolism to 2,2,2-trifluoroacetaldehyde (TFAld) and then to trifluoroacetic acid (TFAA). The enzymes involved in the toxic metabolic pathway have been investigated in this study. For the reaction of TFE to TFAld, the major hepatic metabolism associated with toxicity (as assessed by pyrazole-inhibitability) was NADPH dependent and occurred in the microsomes, whereas for TFAld conversion to TFAA, NADPH-dependent microsomal metabolism was significant, but mitochondrial and cytosolic metabolism in the presence of NADPH were also major contributors. NADPH-dependent hepatic microsomal metabolism of TFE to TFAld and TFAld to TFAA was inhibited by carbon monoxide, 2-allyl-2-isopropylacetamide, SKF-525A, metyrapone, imidazole, and pyrazole, and both reactions were oxygen dependent. The metabolism of TFE to TFAld was inhibited by diethyldithiocarbamate, a specific inhibitor of cytochrome P450E1, and by a monoclonal antibody to P4502E1, whereas the metabolism of TFAld was inhibited by neither. Ethanol pretreatment of rats enhanced the Vmax for hepatic microsomal metabolism of TFE to TFAld from 5.3 to 9.7 nmol/mg protein/min, while for TFAld to TFAA the Vmax was increased from 4.3 to 6.5 and the Km was unaffected for both reactions. Phenobarbital pretreatment of the rats did not affect any of these kinetic parameters. Coadministration of ethanol and a lethal dose of TFE very markedly decreased the lethality. Both the lethality (LD50 0.21 to 0.44 g/kg) and the metabolic kinetic parameters [(Vmax/Km)H(Vmax/Km)D = 4.2] were affected markedly when deuterated TFE replaced TFE. In contrast, deuteration of TFAld did not affect its lethality or rates of metabolism, but did affect its Km. Taken together these results indicate that P4502E1 catalyzed toxicity-associated hepatic metabolism of TFE to TFAld, while TFAld metabolism was catalyzed by a P450 which was not P4502E1. The hepatic metabolism of TFAld was not associated with its toxicity, which has been determined previously to be associated with its intestinal metabolism.     

The effect of tridiphane (2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane) on hepatic epoxide-metabolizing enzymes: indications of peroxisome proliferation

Administration of tridiphane (Tandem, DOWCO 356, 2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane) to male Swiss-Webster mice for 3 days at 100, 250, and 500 mg/kg (ip) resulted in increases in liver weight accompanied by an increase in mitotic index and increases in large particle and microsomal protein. Epoxide hydrolase (EH) activity towards cis-stilbene oxide (CSO, microsomal EH) was elevated in microsomes and cytosol, a decrease in microsomal cholesterol EH was found, and hydrolysis of trans-stilbene oxide (TSO, cytosolic EH) was elevated in the cytosol but not in the microsomes. Glutathione S-transferase (GST) activity was elevated in cytosol for CSO, TSO, and 1,2-dichloro-4-nitrobenzene (DCNB), with inconsistent responses found with 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP). Microsomal GST was not consistently effected by tridiphane. Clofibrate (500 mg/kg, 3 daily ip injections) treatment resulted in similar responses in liver size, microsomal protein, and the EHs. The increase in cytosolic EH activity previously has been noted only in animals treated with peroxisome proliferators. Examination of livers from mice treated with 250 mg/kg tridiphane revealed that an increase in hepatic peroxisomes was apparent after 3 days of treatment. This was accompanied by decreases in serum cholesterol and triglyceride levels and increases in liver carnitine acetyl transferase and cyanide-insensitive oxidation of palmitoyl-CoA. This study demonstrates that tridiphane does have in vivo effects on mammalian epoxide-metabolizing enzymes and extends the association of increased cytosolic epoxide hydrolase activity with peroxisome proliferation.     

Determination of the human cytochrome P450s involved in the metabolism of 2n -propylquinoline

1 2 n -Propylquinoline (2 n PQ) is a newly developed drug for visceral antileishmaniasis and its activity has been previously evaluated in mice following oral administration. The study was carried out to investigate the kinetic formation of 2 n PQ metabolites and to characterize the human liver CYP forms involved in its oxidative metabolism. 2. The inhibition of 2 n PQ metabolite formation by specific substrates or inhibitors of CYP forms and correlation studies were performed in human liver microsomes. 2 n PQ biotransformation was then studied in human lymphoblasts expressing specific CYPs and microsomal epoxide hydrolase. 3. Three major metabolites were produced by human liver microsomes and their structures were identified by ESI-LC/MS: dihydroxy-2 n -propylquinoline, 3'-hydroxy-2 n -propylquinoline and 1'-hydroxy-2 n -propylquinoline. An intermediary metabolite, epoxy-2 n -propylquinoline, formed by CYP was also biotransformed by microsomal epoxide hydrolase into dihydroxy-2 n -propylquinoline. 4. 2 n PQ oxidation follows Michaelis-Menten kinetics. In human liver microsomes, its metabolism was extremely inhibited by pilocarpine, coumarin and diethyldithiocarbamate. From a panel of 12 human liver microsome samples, the rate of 2 n PQ oxidation was highly correlated with the activities of CYP2A6 and CYP2E1. Human lymphoblasts expressing specific CYPs showed the involvement of CYP2A6, CYP2E1 and CYP2C19. 5. The results indicate that 2 n PQ metabolites are 3'- and 1'-hydroxylated by human liver microsomes and an epoxy-2 n -propylquinoline is biotransformed into a dihydroxy-2 n -propylquinoline by microsomal epoxide hydrolase.     

Slow oxidation of acetoxime and methylethyl ketoxime to the corresponding nitronates and hydroxy nitronates by liver microsomes from rats, mice, and humans.

Acetoxime and methylethyl ketoxime (MEKO) are tumorigenic in rodents, inducing liver tumors in male animals. The mechanisms of tumorigenicity for these compounds are not well defined. Oxidation of the oximes to nitronates of secondary-nitroalkanes, which are mutagenic and tumorigenic in rodents, has been postulated to play a role in the bioactivation of ketoximes. In these experiments, we have compared the oxidation of acetoxime and methylethyl ketoxime to corresponding nitronates in liver microsomes from different species. The oximes were incubated with liver microsomes from mice, rats, and several human liver samples. After tautomeric equilibration and extraction with n-hexane, 2-nitropropane and 2-nitrobutane were quantitated by GC/MS-NCI (limit of detection of 250 fmol/injection volume). In liver microsomes, nitronate formation from MEKO and acetoxime was dependent on time, enzymatically active proteins, and the presence of NADPH. Nitronate formation was increased in liver microsomes of rats pretreated with inducers of cytochrome P450 and reduced in the presence of inhibitors (n-octylamine and diethyldithiocarbamate). Rates of oxidation of MEKO (Vmax) were 1.1 nmol/min/mg (mice), 0.5 nmol/min/mg (humans), and 0.1 nmol/min/mg (rats). In addition to nitronates, several minor metabolites were also enzymatically formed (two diastereoisomers of 3-nitro-2-butanol, 2-hydroxy-3-butanone oxime and 2-nitro-1-butanol). Acetoxime was also metabolized to the corresponding nitronate at rates approximately 50% of those observed with MEKO oxidation in the three species examined. 2-Nitro-1-propanol was identified as a minor product formed from acetoxime. No sex differences in the capacity to oxidize acetoxime and MEKO were observed in the species examined. The observed results show that formation of sec-nitronates from ketoximes occurs slowly, but is not the only pathway involved in the oxidative biotransformation of these compounds. Due to the lack of sex-specific oxidative metabolism, other metabolic pathways or mechanisms of tumorigenicity not involving bioactivation may be involved in the sex-specific tumorigenicity of ketoximes in rodents.     

Characterization of distinct forms of cytochromes P-450, epoxide metabolizing enzymes and UDP-glucuronosyltransferases in rat skin

Study of drug metabolizing enzyme activity was undertaken in skin microsomal and cytosolic fractions of male and female rats. The presence of several isoforms was revealed from their activities towards selected substrates and from their cross immunoreactivity using antibodies raised against purified hepatic or renal cytochromes P-450, epoxide hydrolase and UDP-glucuronosyltransferases. Cytochrome P-450 content was precisely quantified by second derivative spectrophotometry, 23.1 and 16.5 pmol/mg protein in males and females, respectively. The monooxygenase activity associated to cytochromes P-450IIB1 and P-450IA1 was determined through O-dealkylation of ethoxy-; pentoxy- and benzoxyresorufin. The activity ranged between 4 and 2 nmol/min/mg protein for male and female rats, respectively. These results and Western blot analysis indicated that rat skin microsomes contain both monooxygenase systems associated with cytochromes P-450IIB1 and P-450IA1. By contrast lauric acid hydroxylation, supported by cytochrome P-450IVA1, was not detectable. Activities of epoxide metabolizing enzymes (microsomal and cytosolic epoxide hydrolases; glutathione S-transferase) were also characterized in skin. Microsomes catalysed the hydratation of benzo(a)pyrene-4,5-oxide and cis-stilbene oxide at the same extent, whatever the sex, although the specific activity was 10 times lower than in liver. The hydratation of trans-stilbene oxide by soluble epoxide hydrolase was four times lower than in the liver. Conjugation of cis-stilbene oxide with glutathione in skin and liver proceeded at essentially similar rates, as the specific activity of glutathione S-transferase in skin was only two times less than that measured in hepatic cytosol. Glucuronidation of 1-naphthol, bilirubin but not of testosterone could be followed in the microsomal fraction. Revelation by Western blot indicated that both the isoforms involved in conjugation of phenols and bilirubin were present in skin microsomes. By contrast, the isoform catalysing the conjugation of testosterone was apparently missing. When immunoblotting was carried out using specific antibodies raised against the renal isoforms, the same result was obtained. In addition, an intense staining corresponding to a 57 kD-protein was observed.