Purification of liver serine protease which activates microsomal glutathione S-transferase: possible involvement of hepsin

Rat liver microsomal glutathione S-transferase (MGST1) is known to be activated by trypsin, however, it has not been clarified whether MGST1 is activated by a protease present in liver. In the present study we purified the MGST1 activating protease from liver microsomes and finally identified that the protease is hepsin, a type II transmembrane serine protease. When the protease was incubated with the purified MGST1 or liposomal MGST1 at 4 degrees C, MGST1 activity was increased 3-4.5 fold after 3-6 d. In electrophoretic and immunoblot analyses after the incubation of MGST1 with the protease MGST1 dimer and its degraded fragment were detected. These results suggest that the rat liver microsomal hepsin functions as MGST1 activating/degrading enzyme.     

Mechanisms for epigallocatechin gallate induced inhibition of drug metabolizing enzymes in rat liver microsomes

Epigallocatechin gallate (EGCG) inhibits drug metabolizing enzymes by unknown mechanisms. Here we examined if the inhibition is due to covalent-binding of EGCG to the enzymes or formation of protein aggregates. EGCG was incubated with rat liver microsomes at 1–100 μM for 30 min. The EGCG-binding proteins were affinity purified using m-aminophenylboronic acid agarose and probed with antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin, cytochrome P450 (CYP) 1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A, catechol-O-methyltransferase (COMT) and microsomal glutathione transferase 1 (MGST1). All but actin and soluble COMT were positively detected at ≥1 μM EGCG, indicating EGCG selectively bound to a subset of proteins including membrane-bound COMT. The binding correlated well with inhibition of CYP activities, except for CYP2E1 whose activity was unaffected despite evident binding. The antioxidant enzyme MGST1, but not cytosolic GSTs, was remarkably inhibited, providing novel evidence supporting the pro-oxidative effects of EGCG. When microsomes incubated with EGCG were probed on Western blots, all but the actin and CYP2E1 antibodies showed a significant reduction in binding at ≥1 μM EGCG, suggesting that a fraction of the indicated proteins formed aggregates that likely contributed to the inhibitory effects of EGCG but were not recognizable by antibodies against the intact proteins. This raised the possibility that previous reports on EGCG regulating protein expression using GAPDH as a reference should be revisited for accuracy. Remarkable protein aggregate formation in EGCG-treated microsomes was also observed by analyzing Coomassie Blue-stained SDS-PAGE gels. EGCG effects were partially abolished in the presence of 1 mM glutathione, suggesting they are particularly relevant to the in vivo conditions when glutathione is depleted by toxicant insults.     

Sodium nitroprusside regulates mRNA expressions of LTC4 synthesis enzymes in hepatic ischemia/reperfusion injury rats via NF-kappaB signaling pathway

Leukotriene (LT) C4 (LTC4) synthesis enzymes including LTC4 synthase (LTC4S), microsomal glutathione S-transferase (MGST) 2 and MGST3 can all conjugate LTA4 and reduced glutathione (GSH) to form LTC4, which is related to hepatic ischemia/reperfusion (I/R) injury. The relationship between nitric oxide (NO) and cysteinyl LTs has been shown in previous studies. However, the mechanisms of NO action on gene expression of LTC4 synthesis enzymes are still largely unclear during hepatic I/R. Adult male Sprague-Dawley rats were divided into 5 groups: a sham group (control), an I/R group, and sodium nitroprusside (SNP, 2.5, 5 and 10 microg/kg/min)+I/R groups. Livers were subjected to 60 min of partial hepatic ischemia followed by 5 h of reperfusion, saline or SNP (2.5, 5 and 10 microg/kg/min) administered intravenously. The mRNA levels of LTC4 synthesis enzymes, inducible NO synthase (iNOS) and endothelial No synthase (eNOS) in rat liver tissue were examined by RT-PCR; the protein expressions of NF-kappaB p65, p50 and IkappaBalpha in liver cell lysates and nuclear extracts were detected by Western blot analysis, and serum NO2. levels were also evaluated. Serum NO2. levels, the protein expressions of NF-kappaB p65 and p50 in the nucleus extract, and hepatic mRNA expressions of LTC4S and iNOS were decreased while hepatic mRNA of eNOS was increased in the SNP (5 and 10 microg/kg/min)+I/R groups when compared with those in the I/R group. SNP (2.5 microg/kg/min) promoted the mRNA expressions of both MGST2 and MGST3, whereas SNP (10 microg/kg/min) increased MGST2 mRNA but decreased MGST3 mRNA compared to those in I/R group. Compared with control, the mRNA expression of MGST2 and MGST3 were elevated in SNP (2.5 microg/kg/min)+I/R group, MGST3 mRNA was significantly declined in the SNP (5 and 10 microg/kg/min)+I/R groups. Immunohistochemistry staining revealed that I/R liver exhibited strong cytoplasmic and nuclear staining for NF-kappaB p65, but the livers of the SNP (2.5 microg/kg/min)+I/R group presented slight cytoplasmic and nuclear staining. But IkappaBalpha protein in all groups remains unchanged. It was concluded that SNP downregulated LTC4S mRNA expression by inhibiting NF-kappaB activation independent of IkappaBalpha, but appeared to have a dual influence on the mRNA expressions of MGST2 and MGST3 by other signaling pathways during hepatic I/R injury.     

Differential hormonal regulation of carbonyl reductase activities in liver and kidney microsomes of rat

1. In the male rat, hepatic microsomal carbonyl reductase (CR) activity decreased by testectomy (Tx) was restored to the control level by the treatment with testosterone propionate (TP), even though the enzyme activity decreased by hypophysectomy (Hx) was not increased by the treatment with TP. On the other hand, renal microsomal CR activities decreased by Tx and Hx were markedly increased by the treatment with TP. 2. The treatment with TP had no effect on the CR activity in liver microsomes of the ovariectomized or hypophysectomized female rat. On the other hand, the CR activities in kidney microsomes of the ovariectomized and hypophysectomized female rat were significantly increased by the treatment with TP. 3. The results indicate that in rat programmed by neonatal androgens, the hepatic microsomal CR activity is regulated indirectly by androgens through the hypothalamus-pituitary system, whereas the hormonal regulation of the renal microsomal CR activity is not via the pituitary. We conclude that the regulatory mechanism of the CR activity in liver microsomes is distinguishable from that in kidney microsomes.     

Irreversible protein binding of acrylonitrile

1. After i.p. injection of [2,3-14C]acrylonitrile to rats, a significant portion of radioactivity becomes irreversibly attached to proteins of liver, lung, spleen and other tissues. 2. When rat liver microsomes were incubated with [2,3-14C]acrylonitrile, a time-dependent irreversible binding of radioactivity occurred to microsomal proteins. This binding was not dependent on NADPH. A high extent of binding to heat-inactivated microsomes indicated that no enzymic metabolic step was involved. 3. The irreversible binding of [2,3-14C]acrylonitrile to rat liver microsomal protein in vitro was inhibited by thiols (cysteine, glutathione, mercaptoethanol). The greatest inhibitory potency was displayed by dithiocarb (diethyl dithiocarbamate).     

Differential hormonal regulation of carbonyl reductase activities in liver and kidney microsomes of rat

1. In the male rat, hepatic microsomal carbonyl reductase (CR) activity decreased by testectomy (Tx) was restored to the control level by the treatment with testosterone propionate (TP), even though the enzyme activity decreased by hypophysectomy (Hx) was not increased by the treatment with TP. On the other hand, renal microsomal CR activities decreased by Tx and Hx were markedly increased by the treatment with TP. 2. The treatment with TP had no effect on the CR activity in liver microsomes of the ovariectomized or hypophysectomized female rat. On the other hand, the CR activities in kidney microsomes of the ovariectomized and hypophysectomized female rat were significantly increased by the treatment with TP. 3. The results indicate that in rat programmed by neonatal androgens, the hepatic microsomal CR activity is regulated indirectly by androgens through the hypothalamus-pituitary system, whereas the hormonal regulation of the renal microsomal CR activity is not via the pituitary. We conclude that the regulatory mechanism of the CR activity in liver microsomes is distinguishable from that in kidney microsomes.     

In vitro covalent binding of new brain tracer, para-125I-amphetamine, to rat liver and lung microsomes

p-125I-amphetamine (I-Amp) is retained significantly in liver and lung during brain tomoscintigraphy. To attempt to explain this clinical observation, we have investigated the interaction of I-Amp with rat liver and lung microsomal proteins. Studies using spectral shift technique indicate that low concentration of I-Amp gives a type I complex and high concentration appears very stable type II complex with cytochrome P-450 Fe III. In the presence of NADPH, I-Amp gives rise to a 455 nm absorbing complex with similar properties to the Fe-RNO complexes. This complex formation was greatly enhanced with phenobarbital treated liver microsomes. The in vitro binding study shows that I-Amp and/or its metabolites was covalently bound to macromolecules in the presence of the molecular oxygen and NADPH-generating system. Incubation in the presence of glutathione, cystein and radical scavengers decreases binding. Mixed function oxydase (MFO) inhibitors diminish the amount of covalent binding and alter the extent of metabolite formation. The total covalent binding level increased with liver microsomes from PB pretreated rats as it was observed with the 455nm complex formation. The radioactivity distribution on microsomal proteins was examinated with SDS polyacrylamide gel electrophoresis and autoradiography. This experiment proves that the radiolabelled compounds are bound on the cytochrome P-450. The radioactivity bound increased when the PB induced rat liver microsomes were used. All these results indicate that I-Amp was activated by an oxydative process dependent on the MFO system which suggests a N-oxydation of I-Amp and the formation of reactive entities which covalently bind to proteins.     

In vitro identification of metabolites of verapamil in rat liver microsomes

AIM: To investigate the metabolism of verapamil at low concentrations in rat liver microsomes. METHODS: Liver microsomes of Wistar rats were prepared using ultracentrifuge method. The in vitro metabolism of verapamil was studied with the rat liver microsomal incubation at concentration of 1.0 μmol/L and 5.0 μmol/L. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MS^n), and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. RESULTS: Eightmetabolites, including two novel metabolites (M4 and MS), were found in rat liver microsomal incubates. They were identified as O-demethyl-verapamil isomers (M1 - M4), N-dealkylated derivatives of verapamil (MS-MT), and N, O-didemethyl-verapamil (MS). CONCLUSION: O-Demethylation and N-dealkylation were the main metabolic pathways of verapamil at low concentrations in rat liver microsomes, and the relative proportion of them in verapamil metabolism changed with different substrate concentrations.     

Regioselective and stereoselective metabolisms of pyrene and 1-bromopyrene by rat liver microsomes and effects of enzyme inducers

Due to the symmetrical property of pyrene (Py), trans-dihydrodiols formed at 4,5- and 9,10-positions are identical, as are the monohydroxylated products (phenols) formed at C1, C3, C6, and C8 positions. With a bromo substituent at C1 position of Py, 1-bromopyrene (1-BrPy) trans-4,5-dihydrodiol and 1-BrPy trans-9,10-dihydrodiol are distinctly different products, as are the phenolic products formed at C3, C6, and C8 positions. Products formed in the oxidative metabolism of 1-BrPy by rat liver microsomes were characterized by retention times on reversed-phase high performance liquid chromatography (HPLC), and by ultraviolet-visible absorption and mass spectral analyses. We have compared regioselective and stereoselective metabolisms at the K- and non-K-regions of Py and 1-BrPy by liver microsomes from untreated (control), phenobarbital (PB)-treated, 3-methylcholanthrene (MC)-treated, and polychlorinated biphenyls (PCB, Aroclor 1254)-treated rats. The effects of inducers on the relative amounts of non-K-region phenols formed in the metabolisms of Py and 1-BrPy by rat liver microsomes were: MC greater than PCB greater than PB greater than control. The relative order was PB greater than PCB greater than MC greater than control for the formation of both 1-BrPy trans-4,5-dihydrodiol and 1-BrPy trans-9,10-dihydrodiol in the metabolism of 1-BrPy. The ratios between metabolically formed 1-BrPy trans-4,5-dihydrodiol to Py trans-4,5-dihydrodiol, using 0.5 mg of microsomal protein per ml of incubation mixture, were between 0.4 and 0.6 in the presence of liver microsomes from untreated, PB-treated, and PCB-treated rats. However, the ratio was approximately 1.5 using liver microsomes from MC-treated rats. The ratios between the sum of 1-BrPy trans-9,10-dihydrodiol and 1-BrPy 9,10-epoxide to Py trans-9,10-dihydrodiol, and to 1-BrPy trans-4,5-dihydrodiol were in the range of 0.1 to 0.5 using four rat liver microsomal preparations. These data revealed the effects of a bromo substituent at C1 of Py on the regioselectivity of various rat liver microsomal enzymes toward the oxidative metabolism at various positions of 1-BrPy. The enantiomeric compositions of K-region dihydrodiols formed by four rat liver microsomal preparations were determined by chiral stationary phase HPLC and circular dichroism spectral analyses; the percentage of R,R-enantiomers were: Py trans-4,5-dihydrodiol, 78-79%; 1-BrPy trans-4,5-dihydrodiol, 74-77%; 1-BrPy trans-9,10-dihydrodiol, 86-97%.     

Role of microsomal glutathione transferase 1 in the mechanism-based biotransformation of glyceryl trinitrate in LLC-PK1 cells

Although glyceryl trinitrate (GTN) has been used in the treatment of angina for many years, details of its conversion to the proximal activator (presumed to be NO or an NO congener) of soluble guanylyl cyclase (sGC) are still unclear. We reported previously that purified microsomal glutathione transferase 1 (MGST1) mediates the denitration of GTN. In the current study, we investigated in intact cells whether this enzyme also converts GTN to species that activate sGC (mechanism-based biotransformation). We utilized LLC-PK1 cells, a cell line with an intact NO/sGC/cGMP system, and generated a stable cell line that overexpressed MGST1. MGST1 in the stably transfected cells was localized to the endoplasmic reticulum, and microsomes from these cells exhibited markedly increased GST activity. Although incubation of these cells with GTN resulted in a 3-4-fold increase in GTN biotransformation, attributed primarily to an increase in formation of the 1,3-glyceryl dinitrate metabolite, GTN-induced cGMP accumulation in cells overexpressing MGST1 was not different than that observed in wild type cells or in cells stably transfected with empty vector. To determine whether overexpression of NADPH cytochrome P450 reductase might act in concert with MGST1 to generate activators of sGC, we assessed GTN-induced cGMP accumulation in MGST1-overexpressing cells that had been transiently transfected with CPR. In this case, GTN-induced cGMP accumulation was also not different than that observed in wild type cells. We conclude that although MGST1 mediates the biotransformation of GTN in intact cells, this biotransformation does not contribute to the formation of activators of sGC.     

Metabolism of benzimidazoline-2-thiones by rat hepatic microsomes and hog liver flavin-containing monooxygenase.

The metabolism of benzimidazoline-2-thione (I) and the 1-methyl (II) and 1,3-dimethyl (III) derivatives was studied to elucidate the mechanisms of hepatic oxidation for this class of thionosulfur-containing xenobiotics. NADPH-dependent metabolism of I, II, and III to the corresponding benzimidazoles Ia, IIa, and IIIa, respectively, was observed in dexamethasone-pretreated rat hepatic microsomes. III was the only thiocarbamide converted to an amide metabolite (IIIb). The effects of heat and 1-aminobenzotriazole pretreatment suggested that rat hepatic microsomal metabolism of I was catalyzed by the flavin-containing monoxygenase (FMO) only and that of II and III by both FMO and cytochrome P450 isozymes (P450). Addition of 5.0 mM glutathione (GSH) blocked formation of all metabolites from I, II, and III. Highly purified hog liver FMO catalyzed formation of all metabolites observed in rat hepatic microsomal systems. Incubation of III with either rat liver microsomes or with highly purified hog liver FMO in the presence of [18O]water led to ca. 50% incorporation of [18O] into IIIb. When [18O] molecular oxygen was used, ca. 8% incorporation of [18O] into IIIb was observed. Highly purified hog liver FMO also converted I-III to chemically reactive species that covalently bound to protein thiols. In the presence of hog liver FMO, the covalent binding pattern of radiolabeled I-III to bovine serum albumin was essentially identical to that observed for rat hepatic microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of membrane-bound glutathione transferase activity by phospholipids including cardiolipin

Membrane-bound glutathione transferases (MGST1) distributed mostly in liver microsomal and mitochondrial membranes are activated by the thiol modification. In the present study, the effect of phospholipids on MGST1 activity was investigated using purified enzyme. When MGST1 was mixed with liposomes of cardiolipin (CL), phosphatidylcholine (PC), phosphatidylserine (PC), or phosphatidylethanolamine (PE), its activity was increased in a magnitude which was dependent on the anionic property of lipids in the order of CL>PS>PE>PC, indicating that MGST1 activity is enhanced by surrounding anionic lipids. Although MGST1 was activated by the thiol alkylation with N-ethylmaleimide (NEM), the activation was suppressed in the presence of anionic phospholipids as clearly observed in the presence of CL. Similarly, the activation of MGST1 by diamide or diamide plus glutathione through disulfide-bond formation was also disturbed in the presence of CL. Suppression of NEM-derived MGST1 activation by CL was lost when MGST1 was incubated with CL in the presence of the detergent Triton X-100. These results indicate that reactivity (stability) of the thiol in MGST1 is affected by surrounding lipids, namely CL which prevents MGST1 activation by thiol modification. Since CL is a mitochondria specific lipid located in the inner membrane, it was suggested that function of mitochondrial MGST1 could be regulated by interaction with CL.     

Metabolism of lovastatin by rat and human liver microsomes in vitro

The metabolism of lovastatin (Mevacor) was examined using isolated microsomes derived from the livers of normal and phenobarbital-treated rats and from human liver samples. Incubation of lovastatin with rat liver microsomes resulted in the formation of several polar metabolites of lovastatin. The metabolites were isolated by HPLC and identified by NMR and mass spectrometry. One fraction consisted of a 2:1 mixture of 6-hydroxy-lovastatin and the rearrangement product delta 4,5-3-hydroxy lovastatin. Addition of a trace of acid to this mixture resulted in the formation of a single aromatized product, the desacyl-delta 4a,6,8-dehydro analog of lovastatin. Another microsomal metabolite was determined to be the delta 4,8a,1-3-hydroxy-lovastatin derivative. The chromatographic pattern of metabolites produced from lovastatin by human liver microsomes was similar to that obtained with rat liver microsomes. Metabolism of lovastatin by rat liver microsomes was both time and concentration dependent; optimal microsomal metabolism occurred with 0.1 mM lovastatin, whereas higher lovastatin concentrations inhibited the reaction. The open acid form of lovastatin was poorly metabolized by both the rat and the human liver microsomes.     

Regiochemistry and enantioselectivity in the oxidative N-dealkylation of verapamil

The oxidative N-dealkylation of verapamil (1), a calcium channel antagonist, was examined in the presence of rat and human liver microsomes by using GC-MS methodology and synthesized regio-isomeric standards. All three possible secondary amine metabolites, N-methyl-4-(3,4-dimethoxyphenyl)-4-cyano-5-methylhexylamine (5), norverapamil (4), and N-methyl-2-(3,4-dimethoxyphenyl)ethylamine (3), were formed as microsomal metabolites. Compound 5 and norverapamil (4) were major products. Substrate stereoselectivity for the N-dealkylation process was determined when pseudoracemic verapamil[equimolar (S)-(-)-verapamil-d6 and (R)-(+):verapamil-d0] was used as substrate. In the presence of rat liver microsomes, a slight enantiomeric preference for the metabolism of (R)-verapamil to secondary amines 3 and 5 (S/R ratio = 0.88 and 0.78, respectively) was observed. In contrast, (S)-verapamil was preferentially metabolized to norverapamil (4) and primary amine 9 (S/R ratio = 1.20 for both). The enantioselectivity for the N-dealkylation process in the presence of human liver microsomes was slight and variable (six samples). Quantitatively, the major N-dealkylation routes in both microsomal systems yielded norverapamil (4) and secondary amine 5. Greater substrate enantioselectivity was observed for the N-dealkylation process in rat liver microsomes than in human liver microsomes. In rat liver microsomal studies, two aliphatic aldehydes (2 and 6) were successfully trapped as their O-methyloximes (7 and 11, respectively) by using methoxylamine. In addition, the alcohols formed from reduction of these aldehydes were observed, due in part to a direct reduction by NADPH.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro metabolism of amiodarone by rabbit and rat liver and small intestine

The present studies were designed to investigate whether amiodarone (Am) is metabolized in the major organs and tissues of the rat and rabbit. Incubations using Am and tissue homogenates (600 g supernatant) of rabbit and rat lung, liver, kidney, and gut revealed formation of desethylamiodarone (DEA) by the liver and gut. Subsequent experiments using the post-mitochondrial, cytosolic, and microsomal fractions of these tissues indicated that metabolism of Am was greatest in the microsomal fractions. In both species, greater DEA formation was detected for microsomes of hepatic origin. The hepatic microsomal mediated production of DEA was altered by protein concentration in both the rabbit and rat preparations with protein concentrations of 5 mg providing the greatest DEA production. DEA formation by gut microsomes was greatest at 3 mg of protein for the rabbit but exhibited no significant change from 1 mg to 10 mg of protein for the rat. In vitro metabolism of Am by rabbit and rat hepatic microsomal preparations was significantly reduced by 1 mM piperonyl butoxide, SKF 525-A, n-octylamine, and carbon monoxide. Effects of these inhibitors on rabbit and rat gut microsomal incubations were inconclusive. HPLC analysis of incubation samples revealed a species difference in the metabolism of Am as demonstrated by the detection of three metabolites in addition to DEA. The unidentified metabolites (I, II, III) were detected in rabbit hepatic microsomal incubations. Metabolite II was also detected in incubations using rabbit duodenal tissue microsomes. No metabolites other than DEA were found in incubations using rat tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

3-aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone, forms DNA adducts after metabolic activation by human and rat liver microsomes: evidence for activation by cytochrome P450 1A1 and P450 1A2

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen found in diesel exhaust and ambient air pollution. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was recently detected in the urine of salt mining workers occupationally exposed to diesel emissions. Determining the capability of humans to metabolize 3-ABA and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-ABA. Using the (32)P-postlabeling method, we found that all hepatic microsomes were competent to activate 3-ABA. DNA adduct patterns with multiple adducts, qualitatively similar to those formed in vivo in rats treated with 3-ABA, were observed. These patterns were also similar to those formed by the nitroaromatic counterpart 3-NBA and which derive from reductive metabolites of 3-NBA bound to purine bases in DNA. The role of specific cytochrome P450s (P450s) in the human hepatic microsomal samples in 3-ABA activation was investigated by correlating the P450-linked catalytic activities in each microsomal sample with the level of DNA adducts formed by the same microsomes. On the basis of this analysis, most of the hepatic microsomal activation of 3-ABA was attributable to P450 1A1 and 1A2 enzyme activity. Inhibition of DNA adduct formation in human liver microsomes by alpha-naphthoflavone and furafylline, inhibitors of P450 1A1 and 1A2, and P450 1A2 alone, respectively, supported this finding. Using recombinant human P450 1A1 and 1A2 expressed in Chinese hamster V79 cells and microsomes of baculovirus-transfected insect cells (Supersomes), we confirmed the participation of these enzymes in the formation of 3-ABA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells expressing P450 1A1 and 1A2. Using rat hepatic microsomes, we showed that both human and rat microsomes lead to the same 3-ABA-derived DNA adducts. Pretreatment of rats with beta-naphthoflavone or Sudan I, inducers of P450 1A1 and 1A2, and P450 1A1 alone, respectively, significantly stimulated the levels of 3-ABA-derived DNA adducts formed by rat liver microsomes. Utilizing purified rat recombinant P450 1A1, the participation of this enzyme in DNA adduct formation by 3-ABA was corroborated. In summary, our results strongly suggest a genotoxic potential of 3-ABA for humans. Moreover, 3-ABA is not only a suitable biomarker of exposure to 3-NBA but may also directly contribute to the high genotoxic potential of 3-NBA.     

Inhibition of mono-oxygenase activities by 1,1,1-trichloropropene 2,3-oxide, an inhibitor of epoxide hydrase, in rat liver microsomes.

Addition of 1,1,1-trichloropropene 2,3-oxide (TCPO), an inhibitor of microsomal epoxide hydrase, to rat liver microsomes caused a type I spectral change, and its magnitude was increased by pretreatment of animals with phenobarbital (PB) but not with 3-methylcholanthrene and polychlorinated biphenyls. TCPO inhibited aminopyrine N-demethylation competitively and prevented covalent binding of 2,4,2',4'-tetrachlorobiphenyl to macromolecules catalyzed by liver microsomes, although it stimulated benzolalpyrene hydroxylation significantly. It is suggested that TCPO interacts with cytochrome P-450, especially a species of the cytochrome which is inducible by PB administration, and thus inhibits mono-oxygenase activities of liver microsomes.     

UDP-glucuronosyltransferase activity toward digitoxigenin-monodigitoxoside. Differences in activation and induction properties in rat and mouse liver

Glucuronidation of digitoxigenin-monodigitoxoside (DT1), a metabolite of the cardiac glycoside digitoxin, is mediated by the microsomal isozymes, UDP-glucuronosyltransferase(s) (UDP-GT). The present studies examined the activation and induction properties of UDP-GT activity toward DT1 in hepatic microsomes of rats and mice. When compared to enzyme activity present in native (latent) microsomes of the rat (0.104 +/- 0.010 nmol/min/mg of protein), the activity toward digitoxigenin-monodigitoxoside in mouse native microsomes was 3.5-fold higher (0.379 + 0.44 mumol/min/mg of protein). After treatment with ionic (sodium cholate), zwitterionic [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)], or nonionic (Emulgen 911, Triton X-100) detergents, or with UDP-N-acetylglucosamine, enzyme activity in rat microsomes remained unchanged. In contrast, UDP-GT activity (DT1) in mouse liver microsomes treated with detergents or with the nucleotide was increased 2-3-fold above native enzyme activity. Pretreatment of rats with the microsomal enzyme inducers, 3-methylcholanthrene and phenobarbital, had no effect on this enzyme activity, whereas pretreatment with pregnenolone-16 alpha-carbonitrile (PCN) and dexamethasone (DEX) increased enzyme activity toward DT1 800 and 380%, respectively. These findings support the hypothesis that PCN and DEX induce a unique form of UDP-GT in the rat that selectively glucuronidates DT1. In marked contrast, the activity of this enzyme in mouse liver was not affected by pretreatment with any of the microsomal inducers, including PCN and DEX. In both rat and mouse, the P-450p-dependent N-ethylmorphine demethylase activity was increased 10-15-fold in PCN-pretreated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro metabolic products of RWJ-34130, an antiarrythmic agent, in rat liver preparations

The in vitro metabolism of RWJ-34130, an antiarrhythmic agent, was conducted using rat hepatic 9000 x g supernatant (S9) and microsomes in an NADPH-generating system, and the rat liver perfusion. The 100 and 20 microg ml(-1) concentrations of RWJ-34130 aqueous solution were used for microsomal incubation and liver perfusion, respectively. Unchanged RWJ-34130 (approximately 77-78% of the sample in both S9 and microsomes) plus a major metabolite, RWJ-34130 sulfoxide (20% of the sample in both S9 and microsomes) were profiled, isolated and identified from both hepatic S9 and microsomal incubates (60 min) using HPLC and mass spectrometry (MS), and by comparison to a synthetic RWJ-34130 sulfoxide, which was synthesized by reacting RWJ-34130 with MCPBA (meta-chloroperoxy benzoic acid). No unchanged RWJ-34130 was detected in the 3 h liver perfusate, however, 1-phenyl-2-oxo-pyrrolidine was profiled, isolated and identified as a major hydrolyzed metabolite of liver perfusate. RWJ-34130 is not extensively metabolized in vitro in rat hepatic S9 and microsomes. All HPLC metabolic profiles of hepatic S9 and microsomal samples (30 min, 60 min) were qualitatively and nearly quantitatively identical.