Metabolism of 2-nitrofluorene,an environmental pollutant,by liver preparations of sea bream,Pagrus major

1. The in vitro metabolism of 2-nitrofluorene (NF), an environmental pollutant, was examined in fish, focusing on nitro-reduction followed by N -acylation and hydroxylation. 2. When NF was incubated with liver microsomes or cytosol of sea bream, Pagrus major, in the presence of NADPH or 2-hydroxypyrimidine, 2-aminofluorene (AF) was formed. 3. When AF was incubated with liver cytosol in the presence of acetyl-CoA or N -formyl-L-kynurenine, 2-acetylaminofluorene (AAF) or 2-formylaminofluorene (FAF) was formed, respectively. AAF and FAF thus formed were deacylated to the parent AF by the liver preparations. 4. AF, AAF and FAF were oxidized to 7-hydroxy or 5-hydroxy derivatives by the liver microsomes. 5. Nitro-reduction, N -acylation and ring-hydroxylation of NF and the metabolites were also observed in rat liver preparations. These activities in sea bream livers were lower than those of rat liver. However, the order of magnitude of these activities in fish was the same as in rat. 6. It is suggested that NF is effectively reduced to AF by the cytochrome P450 system or aldehyde oxidase, and the acylated metabolites, AAF and FAF, generated by arylamine acetyltransferase and formamidase were hydroxylated by the cytochrome P450 system in fish in the same way as in rat. Further, the acetylamino and formylamino derivatives were interconverted via amino derivatives in the fish.     

Metabolism of 2-nitrofluorene,an environmental pollutant,by liver preparations of sea bream,Pagrus major

1. The in vitro metabolism of 2-nitrofluorene (NF), an environmental pollutant, was examined in fish, focusing on nitro-reduction followed by N-acylation and hydroxylation. 2. When NF was incubated with liver microsomes or cytosol of sea bream, Pagrus major, in the presence of NADPH or 2-hydroxypyrimidine, 2-aminofluorene (AF) was formed. 3. When AF was incubated with liver cytosol in the presence of acetyl-CoA or N-formyl-L-kynurenine, 2-acetylaminofluorene (AAF) or 2-formylaminofluorene (FAF) was formed, respectively. AAF and FAF thus formed were deacylated to the parent AF by the liver preparations. 4. AF, AAF and FAF were oxidized to 7-hydroxy or 5-hydroxy derivatives by the liver microsomes. 5. Nitro-reduction, N-acylation and ring-hydroxylation of NF and the metabolites were also observed in rat liver preparations. These activities in sea bream livers were lower than those of rat liver. However, the order of magnitude of these activities in fish was the same as in rat. 6. It is suggested that NF is effectively reduced to AF by the cytochrome P450 system or aldehyde oxidase, and the acylated metabolites, AAF and FAF, generated by arylamine acetyltransferase and formamidase were hydroxylated by the cytochrome P450 system in fish in the same way as in rat. Further, the acetylamino and formylamino derivatives were interconverted via amino derivatives in the fish.     

Mechanisms of fenthion activation in rainbow trout (Oncorhynchus mykiss) acclimated to hypersaline environments

Previous studies in rainbow trout have shown that acclimation to hypersaline environments enhances the toxicity to thioether organophosphate and carbamate pesticides. In order to determine the role of biotransformation in this process, the metabolism of the thioether organophosphate biocide, fenthion was evaluated in microsomes from gills, liver and olfactory tissues in rainbow trout (Oncorhynchus mykiss) acclimated to freshwater and 17‰ salinity. Hypersalinity acclimation increased the formation of fenoxon and fenoxon sulfoxide from fenthion in liver microsomes from rainbow trout, but not in gills or in olfactory tissues. NADPH-dependent and independent hydrolysis was observed in all tissues, but only NADPH-dependent fenthion cleavage was differentially modulated by hypersalinity in liver (inhibited) and gills (induced). Enantiomers of fenthion sulfoxide (65% and 35% R- and S-fenthion sulfoxide, respectively) were formed in liver and gills. The predominant pathway of fenthion activation in freshwater appears to be initiated through initial formation of fenoxon which may be subsequently converted to the most toxic metabolite fenoxon R-sulfoxide. However, in hypersaline conditions both fenoxon and fenthion sulfoxide formation may precede fenoxon sulfoxide formation. Stereochemical evaluation of sulfoxide formation, cytochrome P450 inhibition studies with ketoconazole and immunoblots indicated that CYP3A27 was primarily involved in the enhancement of fenthion activation in hypersaline-acclimated fish with limited contribution of FMO to initial sulfoxidation.     

Extrahepatic metabolism of carbamate and organophosphate thioether compounds by the flavin-containing monooxygenase and cytochrome P450 systems.

The cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) enzymes are the major oxidative enzymes in phase I metabolism. Many organophosphate and carbamate thioether compounds are excellent substrates for these enzymes. Stereoselective sulfoxidation of fenthion and methiocarb by human liver, kidney, and microsomes was investigated. A high level of stereoselectivity in the formation of fenthion +-sulfoxide was observed in kidney and intestinal microsomes. This activity was not inhibited by the P450 inhibitor 1-aminobenzotriazole but was dramatically reduced following mild heat treatment. In liver, fenthion was metabolized to its sulfoxide in a nonstereoselective manner, and the activity was sensitive to both 1-aminobenzotriazole and heat treatment. The carbamate pesticide methiocarb also was sulfoxidated with a high degree of stereoselectivity in human kidney microsomes. Human liver microsomes formed both stereoisomers in equal amounts. Sulfoxide formation in kidney was not inhibited by 1-aminobenzotriazole but was abolished in liver microsomes. Formation of methiocarb sulfoxides was not observed in intestinal microsomes. The relative contribution of FMO1 and FMO3 to the sulfoxidation of carbophenothion, demeton-O, ethiofencarb, fonofos, and methiocarb also was investigated by using baculovirus-expressed recombinant proteins. FMO1 showed the highest catalytic activity for all pesticides. This study indicates that FMO1 may have a bigger role in extrahepatic metabolism than previously thought.     

The participation of human hepatic P450 isoforms, flavin-containing monooxygenases and aldehyde oxidase in the biotransformation of the insecticide fenthion

Although fenthion (FEN) is widely used as a broad spectrum insecticide on various crops in many countries, very scant data are available on its biotransformation in humans. In this study the in vitro human hepatic FEN biotransformation was characterized, identifying the relative contributions of cytochrome P450 (CYPs) and/or flavin-containing monooxygenase (FMOs) by using single c-DNA expressed human enzymes, human liver microsomes and cytosol and CYP/FMO-specific inhibitors.Two major metabolites, FEN-sulfoxide and FEN-oxon (FOX), are formed by some CYPs although at very different levels, depending on the relative CYP hepatic content. Formation of further oxidation products and the reduction of FEN-sulfoxide back to FEN by the cytosolic aldehyde oxidase enzyme were ruled out. Comparing intrinsic clearance values, FOX formation seemed to be favored and at low FEN concentrations CYP2B6 and 1A2 are mainly involved in its formation. At higher levels, a more widespread CYP involvement was evident, as in the case of FEN-sulfoxide, although a higher efficiency of CYP2C family was suggested.Hepatic FMOs were able to catalyze only sulfoxide formation, but at low FEN concentrations hepatic FEN sulfoxidation is predominantly P450-driven. Indeed, the contribution of the hepatic isoforms FMO₃ and FMO₅ was generally negligible, although at high FEN concentrations FMO's showed activities comparable to the active CYPs, accounting for up to 30% of total sulfoxidation. Recombinant FMO₁ showed the highest efficiency with respect to CYPs and the other FMOs, but it is not expressed in the adult human liver. This suggests that FMO₁-catalysed sulfoxidation may represent the major extra-hepatic pathway of FEN biotransformation.     

Stereoselective sulfoxidation by human flavin-containing monooxygenase. Evidence for catalytic diversity between hepatic, renal, and fetal forms.

The stereoselective formation of p-tolyl methyl sulfoxide from the corresponding sulfide has been examined in detergent-solubilized human adult liver, adult kidney, and fetal liver microsomes, in order to compare the functional activities of human flavin-containing monooxygenase(s). Solubilization with detergent was performed to eradicate the contribution that cytochrome P-450 would make to the net stereochemistry. Consistent with studies in experimental animal livers, solubilized human fetal liver and adult kidney microsomes formed (R)-p-tolyl methyl sulfoxide in greater than 86% enantiomeric excess. These enzyme activities were sensitive to methimazole inhibition and were markedly thermolabile in the absence of NADPH, attributes that are consistent with a flavin-containing monooxygenase-mediated process. However, solubilized adult human liver microsomes displayed little stereoselectivity (0-40% enantiomeric excess) for the formation of (R)-p-tolyl methyl sulfoxide, although this reaction also displayed several of the characteristics of a flavin-containing monooxygenase-dependent process, including sensitivity to methimazole inhibition and NADPH protection against heat inactivation. Furthermore, this lack of stereoselectivity was not attenuated by the inclusion of activated oxygen scavengers in reaction mixtures. Human tissue metabolite profiling was further studied by using the ethyl, propyl, and isopropyl p-tolyl sulfides. Parallel changes in product stereochemistry as a function of increasing steric bulk were observed with the fetal liver and adult kidney tissue, whereas an anomalous profile was again observed with adult human liver. These data are consistent with the presence of functionally discrete complements of the flavin-containing monooxygenase in detergent-solubilized adult and fetal human liver microsomes.     

In vitro metabolism of methiocarb and carbaryl in rats,and its effect on their estrogenic and antiandrogenic activities

In this work, we examined the metabolism of the carbamate insecticides methiocarb and carbaryl by rat liver microsomes and plasma, and its effect on their endocrine-disrupting activities. Methiocarb and carbaryl were not enzymatically hydrolyzed by rat liver microsomes, but were hydrolyzed by rat plasma, mainly to methylthio-3,5-xylenol (MX) and 1-naphthol, respectively. When methiocarb was incubated with rat liver microsomes in the presence of NADPH, methiocarb sulfoxide was formed. The hydrolysis product, MX, was also oxidized to the sulfoxide, 3,5-dimethyl-4-(methylsulfinyl)phenol (SP), by rat liver microsomes in the presence of NADPH. These oxidase activities were catalyzed by cytochrome P450 and flavin-containing monooxygenase. Methiocarb and carbaryl both exhibited estrogen receptor α (ERα) and ERβ agonistic activity. MX and 1-naphthol showed similar activities, but methiocarb sulfoxide and SP showed markedly decreased activities. On the other hand, methiocarb and carbaryl exhibited potent antiandrogenic activity in the concentration range of 1 × 10⁻⁶–3 × 10⁻⁵ M. Their hydrolysis products, MX, and 1-naphthol also showed high activity, equivalent to that of flutamide. However, methiocarb sulfoxide and SP showed relatively low activity. Thus, hydrolysis of methiocarb and carbaryl and oxidation of methiocarb to the sulfoxide markedly modified the estrogenic and antiandrogenic activities of methiocarb and carbaryl.     

Mixed function oxidase in the mammary gland and liver microsomes of lactating rats. Effects of 3-methylcholanthrene and beta-naphthoflavone

Mammary gland and liver microsomes of lactating rats were examined for the components of mixed function oxidase and related enzyme activities. Cytochrome b5, NADH- and NADPH- dependent cytochrome c reductase activities were 15-, 6- and 10-fold lower, respectively, in the mammary gland than in the liver microsomes. The determination of cytochrome P-450 (P-448) in the mammary gland microsomes required elimination of the spectral interferences by hemoglobin and cytochrome aa3. The presence of the latter in this fraction was also shown by cytochrome c oxidase activity. Cytochrome aa3 was reduced by anaerobic incubation of mammary gland microsomes, in the presence of antimycin A, with sodium succinate, phenazine ethosulfate, and sodium ascorbate for 30 min at room temperature. Spectral resolution of the dithionite-reduced cytochrome P-450 (P-488) carbon monoxide complex occurred 30 min after gassing. The basal level of cytochrome P-450 was about 500-fold greater in the liver than in the mammary gland microsomes. Pretreatment of lactating rats with the inducers of hepatic cytochrome P-448, 3-methylcholanthrene and beta-naphthoflavone, increased the cytochrome content 3- to 10-fold, in the mammary gland and liver microsomes, respectively. The induction of cytochrome P-448 in microsomes of both tissues was also shown by type I binding spectra obtained with N-2-fluorenylacetamide. Using hydroxylation of benzo[a]pyrene and N-2-fluorenylacetamide as a measure of mixed function oxidase activity, we found that the basal activities, which were 4- to 8-fold greater in the liver microsomes, were increased in both tissues after treatment of rats with the inducers. The induced activities were inhibited by 0.1 micrometers alpha-napthoflavone in vitro, indicating a dependence on cytochrome P-448. The data suggest that the mammary gland, an extrahepatic target for carcinogens, is capable of their metabolism.     

Synthesis of fenthion sulfoxide and fenoxon sulfoxide enantiomers: effect of sulfur chirality on acetylcholinesterase activity

Earlier reports have demonstrated that recombinant flavin-containing monooxygenase 1 (FMO1) catalyzes the oxidation of the organophosphate pesticide fenthion to (+)-fenthion sulfoxide in a stereoselective fashion. In order to elucidate the absolute configuration of the sulfoxide metabolite produced, we established an efficient synthesis of both enantiomers of fenthion sulfoxide, which were transformed into chiral fenoxon sulfoxides using a two-step protocol. The use of chiral oxidants, namely, N-(phenylsulfonyl)(3,3-dichlorocamphoryl) oxaziridines, afforded enantioenriched fenthion sulfoxides with high ee (>82%) from the parent sulfide. Single recrystallizations afforded chiral fenthion sulfoxides with >99% ee, measured by chiral HPLC analysis. The absolute configuration of the (+)-sulfoxide generated from fenthion metabolism by FMO1 was determined to be (R)-(+)-fenthion sulfoxide, confirmed by X-ray crystallographic analysis of the (S)-(-)-antipode. Inhibition of human recombinant (hrAChE) and electric eel (eeAChE) acetylcholinesterase were assayed with fenthion, fenoxon, and the racemates and enantiomers of fenthion sulfoxide and fenoxon sulfoxide. Results revealed stereoselective inhibition with (R)-(+)-fenoxon sulfoxide when compared with that of (S)-(-)-fenoxon sulfoxide (IC50 of 6.9 and 6.5 microM vs 230 and 111 microM in hrAChE and eeAChE, respectively). Fenthion sulfoxide (R or S enantiomers) did not present anti-AChE properties. Although the stereoselective sulfoxidation of fenthion to (R)-(+)-fenthion sulfoxide by FMO represents a detoxification pathway, the results of this study support the notion that subsequent oxidative desulfuration of (R)-(+)-fenthion sulfoxide (in vivo) may represent a critical bioactivation pathway, resulting in the production of (R)-(+)-fenoxon sulfoxide, a potent AChE inhibitor.     

Studies on N-demethylation of methamphetamine by liver microsomes of guinea-pigs and rats: the role of flavin-containing mono-oxygenase and cytochrome P-450 systems

Relative participation of flavin-containing mono-oxygenase and cytochrome P-450 systems in N-hydroxylation of and formaldehyde release from methamphetamine were studied in vitro using liver microsomes of guinea-pigs and rats. In guinea pigs, only methimazole, an inhibitor of flavin-containing mono-oxygenase, significantly suppressed the above reactions. Formaldehyde release from methamphetamine was significantly inhibited not only by methimazole but also by inhibitors of the cytochrome P-450 system in liver microsomes from rats, but not guinea-pigs. Pretreatment of guinea-pigs with phenobarbital and 3-methylcholanthrene did not enhance the metabolism of methamphetamine. Pretreatment of rats with phenobarbital but not 3-methylcholanthrene increased slightly the N-demethylation of methamphetamine by liver microsomes. The results indicate that a marked species difference exists in the enzymes concerned with N-demethylation of methamphetamine. N-Oxidation predominates in guinea-pigs, whereas in rats, N-oxidation and C-oxidation of the methyl group participate equally as the initial reaction of the N-demethylation pathway.     

In vitro metabolism of clindamycin in human liver and intestinal microsomes.

Incubations with human liver and gut microsomes revealed that the antibiotic, clindamycin, is primarily oxidized to form clindamycin sulfoxide. In this report, evidence is presented that the S-oxidation of clindamycin is primarily mediated by CYP3A. This conclusion is based upon several lines of in vitro evidence, including the following. 1) Incubations with clindamycin in hepatic microsomes from a panel of human donors showed that clindamycin sulfoxide formation correlated with CYP3A-catalyzed testosterone 6beta-hydroxylase activity; 2) coincubation with ketaconazole, a CYP3A4-specific inhibitor, markedly inhibited clindamycin S-oxidase activity; and 3) when clindamycin was incubated across a battery of recombinant heterologously expressed human cytochrome P450 (P450) enzymes, CYP3A4 possessed the highest clindamycin S-oxidase activity. A potential role for flavin-containing monooxygenases (FMOs) in clindamycin S-oxidation in human liver was also evaluated. Formation of clindamycin sulfoxide in human liver microsomes was unaffected either by heat pretreatment or by chemical inhibition (e.g., methimazole). Furthermore, incubations with recombinant FMO isoforms revealed no detectable activity toward the formation of clindamycin sulfoxide. Beyond identifying the drug-metabolizing enzyme responsible for clindamycin S-oxidation, the ability of clindamycin to inhibit six human P450 enzymes was also evaluated. Of the P450 enzymes examined, only the activity of CYP3A4 was inhibited (approximately 26%) by coincubation with clindamycin (100 microM). Thus, it is concluded that CYP3A4 appears to account for the largest proportion of the observed P450 catalytic clindamycin S-oxidase activity in vitro, and this activity may be extrapolated to the in vivo condition.     

Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)

The liver microsomal ethanol-inducible cytochrome P-450 (P-450IIE1) form is known to exhibit a high rate of oxidase activity in the absence of substrate and it was therefore of interest to evaluate whether this form of P-450 could contribute to microsomal and liposomal NADPH-dependent oxidase activity and lipid peroxidation. The rate of microsomal NADPH-consumption, O2--formation, H2O2-production and generation of thiobarbituric acid (TBA) reactive substances correlated to the amount of P-450IIE1 in 28 microsomal samples from variously treated rats. Anti-P-450IIE1 IgG inhibited, compared to control IgG, microsomal H2O2-formation by 45% in microsomes from acetone-treated rats and by 22% in control microsomes. NADPH-dependent generation of TBA-reactive products was completely inhibited by these antibodies, whereas preimmune IgG was essentially without effect. Liposomes containing reductase and P-450IIE1 were peroxidized in a superoxide dismutase (SOD) sensitive reaction at a 5-10-fold higher rate than membranes containing 3 other forms of cytochrome P-450. Lipid peroxidation in reconstituted vesicles dependent on the presence of P-450IIB1 was by contrast not inhibited by SOD. Microsomal peroxidase activities, using 15-(S)-hydroperoxy-5-cis-8,11,13-trans-eicosatetraenoic acid as a substrate were high in microsomes from phenobarbital- or ethanol-treated rats but low in membranes from isoniazid-treated rats, having the highest relative level of P-450IIE1. It is suggested that the oxidase activity of P-450IIE1 contributes to microsomal NADPH-dependent lipid peroxidation. The combined action of the oxidase activity by P-450IIE1 and the peroxidase activities by P-450IIB1 and other forms of P-450 may be important for the high rate of lipid peroxidation observed in e.g. microsomes from ethanol- or acetone-treated rats. The possible importance of cytochrome P-450IIE1-dependent lipid peroxidation in vivo after ethanol abuse is discussed.     

Metabolism of the alpha,beta-unsaturated ketones, chalcone and trans-4-phenyl-3-buten-2-one, by rat liver microsomes and estrogenic activity of the metabolites.

When chalcone and trans-4-phenyl-3-buten-2-one (PBO) were incubated with liver microsomes of untreated rats in the presence of NADPH, 4-hydroxychalcone and trans-4-(4-hydroxyphenyl)-3-buten-2-one (4-OH-PBO), respectively, were formed as major metabolites. Two minor metabolites of chalcone, 4'-hydroxychalcone and 2-hydroxychalcone, were also observed. The oxidase activity affording 4-hydroxychalcone was inhibited by SKF 525-A, disulfiram, ketoconazole, and alpha-naphthoflavone. The oxidase activities leading to 4-hydroxychalcone and 4'-hydroxychalcone were enhanced in liver microsomes of 3-methylcholanthrene- and phenobarbital-treated rats, respectively. The activity generating 2-hydroxychalcone was enhanced in liver microsomes of 3-methylcholanthrene- and dexamethasone-treated rats. The oxidation of PBO to 4-OH-PBO was inhibited by SKF 525-A, ketoconazole, disulfiram, and sulfaphenazole. This activity was enhanced in liver microsomes of 3-methylcholanthrene-, acetone- and phenobarbital-treated rats. 4-Hydroxylation, 4'-hydroxylation, and 2-hydroxylation of chalcone were catalyzed by rat recombinant cytochrome P450 1A1, 1A2, and 2C6; by 1A1 and 2C6; and by 1A1 and 3A1, respectively. PBO was oxidized by cytochrome P450 1A1, 1A2, 2C6, and 2E1. Chalcone and PBO were negative in an estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7. However, 4-hydroxychalcone, 2-hydroxychalcone, 4'-hydroxychalcone, and 4-OH-PBO exhibited estrogenic activity.     

Extremely high drug-reductase activity based on aldehyde oxidase in monkey liver.

Drug-reducing ability of monkey liver cytosol was examined in this study. Monkey liver cytosol exhibited significant reductase activities toward zonisamide, sulindac and imipramine N-oxide in the presence of 2-hydroxypyrimidine or benzaldehyde, an electron donor to aldehyde oxidase. These activities were abolished by inhibitors of aldehyde oxidase, such as menadione. These reductase activities in monkeys were extremely high compared to those in other animals. The zonisamide reductase activity of monkey liver cytosol was about 40-fold higher than that of the liver microsomes. It appears that the high levels of aldehyde oxidase exists in monkey liver, and zonisamide, sulindac and imipramine N-oxide are mainly reduced by this enzyme, not by cytochrome P450.     

Tissue-specific effects of fenthion on glutathione metabolism modulated by NAC and BSO in Oreochromis niloticus

Introduction:?The present study was designed to understand the effects of organophosphate (OP) insecticide and avicide fenthion on cellular redox status and the role of reduced glutathione (GSH) on fenthion toxicity in the liver and kidney of Oreochromis niloticus as a model organism. N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) were injected intraperitoneally to fenthion-exposed fish as modulators of GSH metabolism. GSH redox status, GSH-related enzyme activities, and thiobarbituric acid reactive substances (TBARS) contents were then measured spectrophotometrically at 24, 48, and 96 hours. To assess recovery from fenthion exposure, similar analyses were performed on fish transferred to non-treated water for 24, 48, and 96 hours.

Results:?Fenthion increased glutathione S-transferase (GST; EC 2.5.1.18) activity and caused changes in total GSH (tGSH), GSH and oxidized glutathione (GSSG) contents and glutathione peroxidase (GPx; EC 1.11.1.9) specific activity in the liver tissue over time. Increases observed in tGSH and GSSG contents at 24 hours were decreased by fenthion treatment at 96 hours. BSO caused a sharp decline in liver tGSH, GSH, and GSSG contents and an elevation in GST and γ-glutamyl transpeptidase (γ-GT; EC 2.3.2.2) enzyme activities. A significant decrease was observed in tGSH and GSH contents and, also, GST enzyme activities in the kidney at 48-hour fenthion treatment. On the contrary to the liver, a significant increase was observed in tGSH and GSH contents in the kidney by BSO injection. NAC application eliminated the decreasing effects of fenthion on GST activity in this tissue. NAC injection caused decreases in lipid peroxidation (LPO) levels. Decline in tGSH and GSH contents were maintained in the liver during the recovery period, and elevations in LPO levels in the kidney were observed during the same period.

Conclusions:?In conclusion, tissue-specific and time-dependent GSH redox status disturbance of fenthion were observed. BSO revealed the significance of GST-mediated GSH conjugation on the detoxification process of fenthion. NAC seemed useful to avoid the fenthion-related oxidative toxicity.     

Behavioral and immunohistological assessment of painful neuropathy induced by a single oxaliplatin injection in the rat

The in vitro metabolism of permethrin and its hydrolysis products in rats was investigated. Cis- and trans-permethrin were mainly hydrolyzed by liver microsomes, and also by small-intestinal microsomes of rats. trans-Permethrin was much more effectively hydrolyzed than the cis-isomer. When NADPH was added to the incubation mixture of the liver microsomes, three metabolites, 3-phenoxybenzyl alcohol (PBAlc), 3-phenoxybenzaldehyde (PBAld) and 3-phenoxybenzoic acid (PBAcid), were formed. However, only PBAlc was formed by rat liver microsomes in the absence of cofactors. The microsomal activities of rat liver and small intestine were inhibited by bis-p-nitrophenyl phosphate, an inhibitor of carboxylesterase (CES). ES-3 and ES-10, isoforms of the CES 1 family, exhibited significant hydrolytic activities toward trans-permethrin. When PBAlc was incubated with rat liver microsomes in the presence of NADPH, PBAld and PBAcid were formed. The NADPH-linked oxidizing activity was inhibited by SKF 525-A. Rat recombinant cytochrome P450, CYP 2C6 and 3A1, exhibited significant oxidase activities with NADPH. When PBAld was incubated with the microsomes in the presence of NADPH, PBAcid was formed. CYP 1A2, 2B1, 2C6, 2D1 and 3A1 exhibited significant oxidase activities in this reaction. Thus, permethrin was hydrolyzed by CES, and PBAlc formed was oxidized to PBAld and PBAcid by the cytochrome P450 system in rats.     

Cytochrome P450 1A1/2 mediated metabolism of trans-stilbene in rats and humans

It was demonstrated that trans-stilbene was metabolically activated to the estrogenic compound by rat liver microsomes (Sugihara et al., Toxicol. Appl. Pharmacol., 167, 46-54 (2000)). In this study, determination of the isoforms of cytochrome P450 involved in the oxidation of the proestrogen, trans-stilbene, to its hydroxylated metabolites was examined. When trans-stilbene was incubated with rat liver microsomes in the presence of NADPH, estrogenic compounds, trans4-hydroxystilbene and trans-4,4'-dihydroxystilbene were formed. Comparison of the oxidase activity among liver microsomes of untreated, 3-methylcholanthrene-treated, acetone-treated, clofibrate-treated, dexamethasone-treated and phenobarbital-treated rats toward trans-stilbene showed that those from 3-methylcholanthrene-treated rats exhibited the highest activity. Human liver microsomes also catalyzed the oxidation in varying degrees. Variation in trans-stilbene oxidase activity was closely correlated to that of phenacetin O-deethylase activity. The oxidase activity was inhibited by alpha-naphthoflavone; however, in this case trans-4,4'-dihydroxystilbene was not detected. The oxidase activity toward trans-stilbene was exhibited by recombinant human cytochrome P450 1A1 and 1A2 expressed in a human B lymphoblastoid cell line.     

Immunochemical detection of cytochrome P450 isozymes induced in rat liver byn-hexane, 2-hexanone and acetonyl acetone

Cytochrome P450 isozymes induced in rat liver by treatment withn-hexane, 2-hexanone and acetonyl acetone (given intraperitoneally 5 mmol/kg for 4 days) were investigated using enzyme assays (benzene, toluene, 7-ethoxyresorufin and 7-pentoxyresorufin metabolism) and monoclonal antibodies (anti-P450IA1/2, anti-P450IIB1/2, anti-P450IIC11/6, anti-P450IIE1(91) and anti-P450IIE1(98)).n-Hexane treatment enhanced the activities of low-K _m benzene aromatic hydroxylase and toluene side-chain oxidase, but not 7-ethoxyresorufin O-deethylase or 7-pentoxyresorufin O-depentylase. 2-Hexanone or acetonyl acetone treatment enhanced the activities of low-and high-K _m benzene aromatic hydroxylases, toluene side-chain oxidase and 7-pentoxyresorufin O-depentylase, but not of 7-ethoxyresorufin O-deethylase. Immunoblot analysis showed that anti-P450IA1/2 did not bind liver microsomal protein from either control and treated rats in the region of cytochrome P450s, whereas with anti-P450IIE1(98) a clear-cut band was seen in liver microsomes from control and treated rats, with intensities in the following order: 2-hexanone=acetonyl acetone n-hexane > control > phenobarbital. With anti-P450IIB1/2, a band was detected in microsomes from phenobarbital-treated rats, and to a lesser extent, in microsomes from 2-hexanone-and acetonyl acetone-treated rats. Like the immunoblot analysis, anti-P450IIE1(91) inhibited toluene side-chain hydroxylase activity in all microsomes, except in preparations from phenobarbital-treated rats and anti-P450IIB1 in microsomes from phenobarbital-, 2-hexanone- and acetonyl acetone-treated rats. Anti-P450IIC11/6 also inhibited toluene side-chain hydroxylase activity: the inhibited activity in the five different microsome preparations was as follows:n-hexane=control > acetonyl acetone=2-hexanone=phenobarbital. These results indicate thatn-hexane induces only quantitative alterations in the constitutive cytochrome P450 isozyme (P450IIE1), whereas its metabolites 2-hexanone and acetonyl acetone induce not only quantitative changes in constitutive cytochrome P450 (P450IIE1 and P450IIC11/6) but also a different type of isozyme (P450IIB1/2).     

Enhanced aryl hydrocarbon hydroxylase activity after interaction between solubilized cytochrome P-448 and microsomes low in endogenous cytochrome P-450

The effects of cumene hydroperoxide on microsomal mixed-function oxidase components and enzyme activities were determined. In vitro cumene hydroperoxide treatment decreased cytochrome P-450 content, benzphetamine N-demethylase activity and aryl hydrocarbon hydroxylase activity of hepatic and renal microsomes from adult male and female rats, and of hepatic microsomes from fetal rats. Cumene hydroperoxide-treated microsomes, as well as fetal liver and adult renal microsomes, which are naturally low in cytochrome P-450 and mixed-function oxidase activity, were used to incorporate partially purified hepatic cytochrome P-448 isolated from 2,3,7,8-tetrachlorodibenzo-p-dioxin-pretreated immature male rats. This resulted in an enhanced rate of benzo[a]pyrene hydroxylation. Aryl hydrocarbon hydroxylase activity was increased 12-, 26-. 31- and 53-fold when 1.0 nmole of partially purified cytochrome P-448 was incubated with fetal liver microsomes, microsomes from kidney cortex of female rats, and cumene hydroperoxide-pretreated hepatic microsomes from female and male rats, respectively. The increased rate of benzo[a]pyrene hydroxylation was linear with cytochrome P-448 over the range 0.25 to 1.0 nmole. Because cumene hydroperoxide-pretreated microsomes from male rat liver and the hepatic and renal microsomes from female rats have a combination of high NADPH-cytochrome c reductase activity and low mixed-function oxidase activity, they are an attractive choice for catalytic studies of the interaction between cytochrome P-448 and microsomes.     

Metabolism of diallyl disulfide by human liver microsomal cytochromes P-450 and flavin-containing monooxygenases.

The metabolism of diallyl disulfide (DADS), a garlic sulfur compound, was investigated in human liver microsomes. Diallyl thiosulfinate (allicin) was the only metabolite observed and its formation followed Michaelis-Menten kinetics with a Km = 0.61 +/- 0.2 mM and a Vmax = 18.5 +/- 4.2 nmol/min/mg protein, respectively (mean +/- S.E. M., n = 4). Both flavin-containing monooxygenase and the cytochrome P-450 monooxygenases (CYP) were involved in DADS oxidation, but the contribution of CYP was predominant. The cytochrome P-450 isoforms involved in this metabolism were investigated using selective chemical inhibitors, microsomes from cells expressing recombinant CYP isoenzymes, and studying the correlation of the rate of DADS oxidation with specific monooxygenase activities of human liver microsomes. Diethyldithiocarbamate and tranylcypromine inhibited allicin formation, whereas other specific inhibitors had low or no effect. Most of the different human microsomes from cells expressing CYP were able to catalyze this reaction, but CYP2E1 showed the highest affinity with a substantial activity. Furthermore, allicin formation by human liver microsomes was correlated with p-nitrophenol hydroxylase activity, a marker of CYP2E1, and tolbutamide hydroxylase activity, a marker of CYP2C9. Among these approaches only CYP2E1 was identified in each case, which suggested that DADS is preferentially metabolized to allicin by CYP2E1 in human liver. However the minor participation of other CYP forms and flavin-containing monooxygenases is likely.