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PubMed1980

Cholesterol alpha- and beta-epoxides as obligatory intermediates in the hepatic microsomal metabolism of cholesterol to cholestanetriol.

A high performance liquid chromatographic method for the good separation and direct determination of cholesterol alpha-epoxide (5,6 alpha-epoxy-5 alpha-cholestan-3 beta-ol) and beta-epoxide (5,6 beta-epoxy-5 beta-cholestan-3 beta-ol) was introduced to the study of microsomal lipid peroxidation-mediated oxygenation of the cholesterol double bond. In the presence of NADPH, FeSO4, and ADP, bovine liver microsomes converted [4-14C] cholesterol to the alpha-epoxide, beta-epoxide, and cholestanetriol (5 alpha-cholestane-3 beta,5,6 beta-triol) in the ratio 1.0:4.3:0.7. Obligatory intermidiacy of both cholesterol alpha- and beta-epoxides and essential role of microsomal cholesterol epoxide hydratease in the conversion of cholesterol to cholestanetriol were established by using the isotope trapping method as well as the cholesterol epoxide hydratase inhibitor, 5,6 alpha-imino-5 alpha-cholestan-3 beta-ol. Hepatic microsomal P-450 played no appreciable role in the epoxidation of cholesterol. Microsomal cholesterol epoxide hydratase was with no doubt found to differ in nature from microsomal xenobiotic epoxide hydratase. Microsomal hydrolysis of styrene oxide and safrole oxide (0.1 mM each) was almost completely inhibited by 3,3,3-trichloro-1-propene oxide (1 mM) but not by 5,6 alpha-imino-5 alpha-cholestan-3 beta-ol (1 mM). However, microsomal hydrolysis of both cholesterol alpha- and beta-epoxides was remarkably accelerated by 3,3,3-trichloro-1-propene oxide and inhibited by 5,6 alpha-imino-5 alpha-cholestan-3 beta-ol.

Animals↗
PubMed1983

Esterification of cholesterol and 25-hydroxycholesterol by rat liver microsomes.

The properties of an enzyme in rat liver microsomes was described that catalyzed the formation of 25-hydroxycholesteryl ester in the presence of labeled sterol and oleoyl-CoA. The reaction was similar in several respects to that of cholesteryl ester formation by acyl-CoA: cholesterol acyltransferase. Trypsin pretreatment of microsomes inhibited the esterification of both sterols and a similar dose-dependent inhibition was produced by addition of progesterone and several androgens. Microsomes with an enhanced cholesterol content resulting from in vivo treatment with ethinyl estradiol showed increased esterifying activity towards both cholesterol and 25-hydroxycholesterol. Esterification of endogenous microsomal cholesterol was increased by the addition of 25-hydroxycholesterol, concomitant with 25-hydroxycholesteryl ester formation. To assess the relationship between the association of sterols with membranes and sterol ester formation, microsomes were preincubated with either sterol, reisolated by ultracentrifugation in a density gradient and then analyzed chemically or enzymatically. Cholesterol and 25-hydroxycholesterol both associated with microsomes and the added sterol was subsequently esterified. Maximal esterification was only partially dependent on the amount bound. Progesterone, which inhibited sterol esterification, did not bind to microsomes and no inhibition was observed in reisolated microsomes, indicating that the inhibition produced by progesterone was reversible.

Animals↗
PubMed1983

Phospholipid fatty acid modification of rat liver microsomes affects acylcoenzyme A:cholesterol acyltransferase activity.

The effect of phospholipid fatty acyl composition on the activity of acylcoenzyme A:cholesterol acyltransferase was investigated in rat liver microsomes. Specific phosphatidylcholine replacements were produced by incubating the microsomes with liposomes and bovine liver phospholipid-exchange protein. Although the fatty acid composition of the microsomes was modified appreciably, there was no change in the microsomal phospholipid or cholesterol content. As compared to microsomes enriched for 2 h with dioleoylphosphatidylcholine, those enriched with dipalmitoylphosphatidylcholine exhibited 30-45% less acyl-CoA:cholesterol acyltransferase activity. Enrichment with 1-palmitoyl-2-linoleoylphosphatidylcholine increased acyl-CoA:cholesterol acyltransferase activity by 20%. By contrast, dilinoleoylphosphatidylcholine abolished microsomal acyl-CoA:cholesterol acyltransferase activity almost completely. Addition of cofactors that stimulated microsomal lipid peroxidation inhibited acyl-CoA:cholesterol acyltransferase activity by only 10%, however, and did not increase the inhibition produced by submaximal amounts of dilinoleoylphosphatidylcholine. Certain of the phosphatidylcholine replacements produced changes in palmitoyl-CoA hydrolase, NADPH-dependent lipid peroxidase, glucose-6-phosphatase and UDPglucuronyl transferase activities, but they did not closely correlate with the alterations in acyl-CoA:cholesterol acyltransferase activity. Electron spin resonance measurements with the 5-nitroxystearate probe indicated that microsomal lipid ordering was reduced to a roughly similar extent by dioleoyl- or by dilinoleoylphosphatidylcholine enrichment. Since these enrichments produce widely different effects on acyl-CoA:cholesterol acyltransferase activity, changes in bulk membrane lipid fluidity cannot be the only factor responsible for phospholipid fatty acid compositional effect on acyl-CoA:cholesterol acyltransferase. The present results are more consistent with a modulation resulting from either changes in the lipid microenvironment of acyl-CoA:cholesterol acyltransferase or a direct interaction between specific phosphatidylcholine fatty acyl groups and acyl-CoA:cholesterol acyltransferase.

Acyl Coenzyme A↗
PubMed1986

Studies on the mechanism of formation of 4-hydroxynonenal during microsomal lipid peroxidation.

The mechanism of the formation of 4-hydroxynonenal through the NADPH-linked microsomal lipid peroxidation was investigated. The results were as follows: 4-hydroxynonenal arises exclusively from arachidonic acid contained in the polar phospholipids, neither arachidonic acid of the neutral lipids nor linoleic acid of the polar or neutral lipids are substrates for 4-hydroxynonenal generation. This finding results from the estimation of the specific radioactivity of 4-hydroxynonenal produced by microsomes prelabelled in vivo with [U-14C]arachidonic acid. Phospholipid-bound 15-hydroperoxyarachidonic acid would have the structural requirements needed for 4-hydroxynonenal (CH3-(CH2)4-CH(OH)-CH=CH-CHO). Microsomes supplemented with 15-hydroperoxyarachidonic acid and NADPH, ADP/iron converted only minimal amounts (0.6 mol%) of 15-hydroperoxyarachidonic acid into 4-hydroxynonenal; similarly, 15-hydroperoxyarachidonic acid incubated at pH 7.4 in the presence of ascorbate/iron yielded only small amounts of 4-hydroxynonenal with a rate orders of magnitude below that observed with microsomes. Phospholipid-bound 15-hydroperoxyarachidonic acid is therefore not a likely intermediate in the reaction pathway leading to 4-hydroxynonenal. The rate of 4-hydroxynonenal formation is highest during the very initial phase of its formation and the onset does not show a lag phase, suggesting a transient intermediate predominantly formed during the early phase of microsomal lipid peroxidation. After 60 min of incubation, 204 nmol polyunsaturated fatty acids (20 nmol 18:2, 143 nmol 20:4, 41 nmol 22:6) were lost per mg microsomal protein and the incubation mixture contained 206 nmol lipid peroxides, 71.6 nmol malonic dialdehyde and 4.6 nmol 4-hydroxynonenal per mg protein. Under artificial conditions (pH 1.0, ascorbate/iron, 20 h of incubation) not comparable to the microsomal peroxidation system, 15-hydroperoxyarachidonic acid can be decomposed in good yields (15 mol%) into 4-hydroxynonenal. Autoxidation of arachidonic acid in the presence of ascorbate/iron gave after 25 h of incubation 2.8 mol% (pH 7.4) and 1.5 mol% (pH 1.0) 4-hydroxynonenal. The most remarkable difference between the non-enzymic system and the enzymic microsomal system is that the latter forms 4-hydroxynonenal at a much higher rate.

Aldehydes↗
PubMed1982

Role of catalase and hydroxyl radicals in the oxidation of methanol by rat liver microsomes.

In view of the presence of adventitious catalase in isolated microsomes, and the requirement for H2O2, it has been suggested that NADPH-dependent oxidation of methanol by rat liver microsomes was mediated exclusively by the peroxidatic activity of catalase. However, H2O2 may also serve as a precursor of the hydroxyl radical, and methanol reacts with hydroxyl radicals to produce formaldehyde. Inhibition of H2O2 production should therefore decrease methanol oxidation by either a hydroxyl radical-dependent pathway or a catalase-dependent pathway. To attempt to clarify some of the controversies concerning the roles of H2O2 and catalase in the microsomal pathway of oxidation of short chain alcohols, studies were carried out to determine the nature of the pathway responsible for methanol oxidation by isolated microsomes. In the absence of the catalase inhibitor azide, methanol may be oxidized primarily by the peroxidatic activity of catalase since there was little effect on methanol oxidation by competing hydroxyl radical scavengers. Azide, which inhibited catalase activity greater than 95%, inhibited NADPH-dependent oxidation of methanol by 30-50%. The azide-insensitive (catalase-independent) pathway of methanol oxidation was inhibited by scavengers of hydroxyl radicals. The inhibition of the scavengers reflected the rate constant for interaction with hydroxyl radicals and was greater at lower concentrations of methanol than at higher concentrations, suggesting competition between the scavengers and methanol. The addition of H2O2 stimulated the oxidation of methanol in the presence of azide; H2O2 may serve as a precursor of the hydroxyl radical. Iron-EDTA, which is known to increase hydroxyl radical production, stimulated the oxidation of methanol in the absence and presence of azide. The stimulation by iron-EDTA was blocked by the competing hydroxyl radical scavengers even in the absence of azide, suggesting that the added iron-EDTA favorably with microsomal catalase for H2O2 to produce hydroxyl radicals (or a species with the oxidizing power of the hydroxyl radical). These results suggest that in microsomes, depending on the absence or presence of azide, methanol may be oxidized by two primary pathways, one involving the peroxidatic activity of catalase, and the other in which hydroxyl radicals, generated from microsomal electron transfer, play a role. In view of the crucial role played by H2O2 in both pathways, inhibition of H2O2 formation should not be interpreted solely as evidence for a role for catalase in the microsomal oxidation of alcohols.

Animals↗
PubMed1982

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.

2-Acetylaminofluorene↗
PubMed1985

Cimetidine interaction with liver microsomes in vitro and in vivo. Involvement of an activated complex with cytochrome P-450.

The O-deethylation of 7-ethoxycoumarin was inhibited in a mixed type manner by cimetidine in vitro and in microsomes isolated from rats treated with cimetidine in vivo. It was found that the inhibition was even greater if cimetidine was preincubated with the microsomal suspension in the presence of an NADPH-generating system prior to the addition of substrate. In vitro the decrease in activity was accompanied by a decrease in cytochrome P-450 content. This decrease was unaffected by the addition of EDTA to the microsomal suspensions, eliminating the possibility that free radical production was responsible for the decrease in cytochrome P-450. The decrease in activity and cytochrome P-450 content following preincubation of microsomal suspensions with cimetidine could be attenuated if potassium ferricyanide was added to the suspensions. The deethylation activity and cytochrome P-450 content of liver microsomes prepared from cimetidine-treated rats was decreased compared to control animals. The activity and cytochrome P-450 content of microsomes from cimetidine-treated rats could also be restored if microsomes were washed with potassium ferricyanide prior to incubation with substrate. It is proposed that an intermediate complex of cimetidine and cytochrome P-450 could be involved in the inhibition of microsomal metabolism by cimetidine.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1986

Incorporation of cytochrome b5 into rat liver microsomal membranes. Impairment of cytochrome P-450-dependent mixed function oxidase activity.

Cytochrome b5 was purified to electrophoretic homogeneity from the liver microsomes of untreated rats and reincorporated into liver microsomes from phenobarbital-treated rats, resulting in an approximate three-fold enrichment of the cytochrome b5 specific content (1.5 nmol haemoprotein X mg-1 protein). Our results have shown that the N-demethylation of benzphetamine was progressively inhibited in cytochrome b5-fortified microsomal preparations. Using stopped flow, visible difference spectrophotometry, the NADPH-driven reduction kinetics of cytochrome P-450 were examined in the modified microsomes over the first few seconds of reaction. Increasing the amount of incorporated cytochrome b5 resulted in a progressive inhibition of the initial, fast phase reduction rate constant of microsomal cytochrome P-450, both in the absence and presence of the type I substrate benzphetamine. Although the initial rate of NADPH-driven cytochrome b5 reduction was the same for both native and cytochrome b5-fortified microsomes, the extent of cytochrome b5 reduction was greater in the fortified microsomes. If cytochrome b5 has a positive role to play in cytochrome P-450-dependent mixed function oxidase activity either as an effector or in electron transfer or both, the former haemoprotein must be already present in sufficient concentrations in the native microsomes.

Animals↗
PubMed1988

The mechanism of the suicidal, reductive inactivation of microsomal cytochrome P-450 by carbon tetrachloride.

1. Stoichiometric losses of microsomal haem and cytochrome P-450 were observed when carbon tetrachloride (CCl4) was incubated anaerobically with rat liver microsomes using NADPH or sodium dithionite as a reducing agent. A rapid destruction of haem was also observed during the non-enzymatic reductive incubation of CCl4 with soluble haem preparations (methaemalbumin) in presence of sodium dithionite. The results indicate that haem is both the site and the target of the suicidal activation of CCl4 by cytochrome P-450. 2. When an additional, fluorimetric assay for haem determination was used, an equimolar loss of protoporphyrin IX fluorescence was also observed in both the enzymatic and non-enzymatic system, indicating that the haem moiety of cytochrome P-450 has undergone a structural change, involving either loss or labilization of the porphyrin tetrapyrrolic structure. In both systems the loss of porphyrin was prevented by carbon monoxide (CO). 3. A dichlorocarbene-cytochrome P-450 ligand complex is partially responsible for the difference spectrum obtained on addition of CCl4 to anaerobically reduced rat liver microsomes. A molar extinction coefficient for this complex has been calculated. The carbene trapping agent 2,3-dimethyl-2-butene (DMB) strongly inhibited (greater than 95%) the formation of this spectrum but did not modify the loss of haem in reduced CCl4-supplemented microsomal incubations. The results suggest that dichlorocarbene (:CCl2) is not significantly involved in CCl4-dependent haem destruction. 4. Pretreatment of rats with different microsomal enzyme inducers was responsible for similar but not identical patterns of :CCl2 and CO formation and haem loss during incubation of CCl4 with reduced microsomes. This indicates a critical role of CCl4 metabolism in the suicidal destruction of cytochrome P-450 haem and suggests that the apoprotein of cytochrome P-450 is capable of modulating not only the metabolism of CCl4 to :CCl2 but also the hydrolysis of :CCl2 to CO. 5. Inactivation of cytochrome P-450 by CCl4 with reduced microsomes from Aroclor-pretreated rats was saturable and followed pseudo first-order kinetics. This provides further evidence to conclude that CCl4 activation is a suicidal process where the reactive metabolite(s) formed bind to haem, we predict, in a one to one stoichiometry. 6. The partition ratio between loss of cytochrome P-450 haem and CCl4 metabolism by liver microsomes from Aroclor pretreated rats has been investigated using limiting concentrations of CCl4. It was calculated that approximately 26 molecules of CCl4 had to be metabolised to achieve the loss of one molecule of haem.

Animals↗
PubMed1988

Evidence for metabolism of 3-methylindole by prostaglandin H synthase and mixed-function oxidases in goat lung and liver microsomes.

3-Methylindole (3MI) is the causative agent of a naturally occurring lung disease in cattle. The effects of 3MI on the prostaglandin H synthase (PHS) and mixed-function oxidase (MFO) systems in lung and liver microsomes were investigated. Addition of 3MI in goat lung microsomes resulted in a pronounced increase in PHS activity as indicated by both the initial rate and total oxygen consumption. The effect of 3MI on PHS activity was dependent on arachidonic acid and inhibited by indomethacin. PHS was capable of activating [14C]3MI to a reactive intermediate as indicated by the covalent binding of 3MI to microsomal protein. [14C]3MI was converted to water-soluble 3MI metabolites in the PHS system. Biosynthesis of total prostaglandins was enhanced 69% with 0.5 mM 3MI. PHS-catalyzed co-oxidation of 3MI was shown to be independent of the MFO-catalyzed metabolism of 3MI. PHS and MFO enzyme activities were found to exist in both goat lung and liver microsomes. PHS activity was significantly higher in lung microsomes than in liver microsomes. Pulmonary MFO activity, unlike hepatic MFO activity, was enhanced with addition of 3MI. This indicates a tissue selectivity for the metabolism of 3MI. Lung microsomal PHS and MFO systems were capable of activating 3MI to a greater extent than liver microsomes. Thus, the combined effects of PHS and MFO systems in the activation of 3MI, the selectivity of pulmonary MFO to metabolize 3MI, and the capacity for altered prostaglandin biosynthesis in lung tissue may explain the tissue specificity of 3MI-induced toxicity.

Animals↗
PubMed1989

Ability of 1-methyltetrazole-5-thiol with microsomal activation to inhibit aldehyde dehydrogenase.

Antibiotics that contain the 1-methyltetrazole-5-thiol (MTT) leaving group are associated with an adverse effect when alcohol is ingested after their administration. Therefore, the ability of MTT to inhibit an enzyme in alcohol metabolism, aldehyde dehydrogenase (ALDH), was examined. In the absence of microsomes, MTT did not inhibit ALDH obtained from either yeast or rat liver. In the presence of rat hepatic microsomes, MTT was able to inhibit the enzyme from both sources. The characteristics of the inhibition were studied, using the yeast enzyme, and found to be dependent upon the length of incubation with the hepatic microsomes and upon the concentration of MTT. Inhibition required the presence of NADH and was not detected if the microsomes were heat treated. Dilution did not reverse the inhibition. Intact antibiotics which contain the MTT moiety did not cause an inhibition of yeast ALDH unless the antibiotics were first treated with potassium hydroxide and then incubated with microsomes. Inhibition of ALDH activity measured in the mitochondrial plus microsomal fractions of rat liver also required NADH and was prevented by glutathione and heat treatment of the microsomes. These results indicate that microsomal activation of MTT is necessary for inhibition of aldehyde dehydrogenase. The behavior of MTT described here may explain the adverse effect observed if alcohol is ingested following administration of MTT-containing antibiotics.

Aldehyde Dehydrogenase↗
PubMed1990

Effects of ethanol and inhibitors on the binding and metabolism of acetaminophen and N-acetyl-p-benzoquinone imine by hepatic microsomes from control and ethanol-treated rats.

Acetaminophen is metabolized by cytochrome P450 to N-acetyl-p-benzoquinone imine (NABQI). This metabolite reacts with critical cellular macromolecules to give toxicity. The administration of 10% ethanol in the drinking water to 100 g male rats for 6 weeks markedly increases the toxicity of acetaminophen. This increase was associated with a 71% increase in microsomal protein binding of acetaminophen [4.8 pmol/min/mg protein in control microsomes versus 8.2 pmol/min/mg protein in ethanol microsomes (P less than 0.01)] and a 131% increase in aniline hydroxylase [0.52 nmol/min/mg protein in control microsomes versus 1.20 nmol/min/mg protein in ethanol microsomes (P less than 0.001)]. On the other hand, cysteine conjugation of acetaminophen showed an increase of only 12% [2.8 nmol/min/mg protein in control microsomes versus 3.1 nmol/min/mg protein in ethanol microsomes (P less than 0.05)]. Ethylmorphine- and benzphetamine N-demethylases did not increase. In microsomes from both control and ethanol animals, imidazole (1 mM) inhibited the two N-demethylases, aniline hydroxylation and acetaminophen binding by 85-95% but inhibited the cysteine conjugation by only 50%. For control and ethanol animals, both 80% CO/20% O2 and SKF-525A (1 mM) totally inhibited cysteine conjugation but only inhibited the other activities by about 36-60%. KCN (1 mM) had no effect on any of the activities except protein binding (60-67% inhibition). Scavengers of reactive oxygen [mannitol (1 mM), dimethyl sulfoxide (1 mM), superoxide dismutase (15 micrograms/mL) and catalase (65 micrograms/mL)] had no effect on any of the reactions. Of all these treatments only CO/O2 decreased the protein binding and cysteine conjugation of NABQI in the presence of either NADP+ or NADPH. The data from the inhibitor studies and the effect of ethanol on acetaminophen and NABQI metabolism would suggest that protein binding and cysteine conjugation are catalyzed by different isozymes of cytochrome P450. Finally, the current results indicate that the increased toxicity of acetaminophen observed with ethanol more closely parallels the increase in protein binding activity rather than cysteine conjugation.

Acetaminophen↗
PubMed1990

Biotransformation of lovastatin--III. Effect of cimetidine and famotidine on in vitro metabolism of lovastatin by rat and human liver microsomes.

The effects of the H2-receptor antagonists, cimetidine and famotidine, on the microsomal metabolism of [14C]lovastatin were investigated. Liver microsomes were prepared from control, phenobarbital- and 3-methylcholanthrene-pretreated rats and humans (male and female). Concentration-dependent inhibition of the metabolism of lovastatin (0.1 mM) was observed with cimetidine (0.1 to 1.0 mM). In contrast, famotidine at a similar concentration was a very weak inhibitor. The formation of 6'beta-hydroxy-lovastatin, the major microsomal metabolite of lovastatin, was similarly inhibited. The results suggest that in vivo metabolic interaction with concomitantly administered lovastatin is less likely with famotidine than with cimetidine. Phenobarbital pretreatment produced 58% stimulation in overall metabolism, whereas 3-methylcholanthrene pretreatment had no effect relative to control rats (5.4 nmol/mg protein/min). Liver microsomes from phenobarbital-pretreated rats produced 67% more of the 6'beta-hydroxy-lovastatin but 63-66% less of the 3''-hydroxy and 6'-exomethylene metabolites. Liver microsomes from 3-methylcholanthrene-treated rats also produced less 3"-hydroxy-lovastatin (49%) but similar quantities of the other two metabolites. 6'beta-Hydroxy-lovastatin was a major metabolite with human liver microsomes. Interestingly with these microsomes, hydroxylation at the 3''-position of the molecule was a negligible pathway and hydrolysis to the hydroxy acid form was not observed. The formation of 6'-exomethylene-lovastatin was also catalyzed by human liver microsomes (0.5 to 0.8 nmol/mg protein/min).

Animals↗
PubMed1992

Covalent binding of 14C- and 35S-labeled thiocarbamides in rat hepatic microsomes.

The covalent binding of a series of 14C- or 35S-labeled benzimidazole-2-thione (MBI) derivatives to rat liver microsomal proteins was studied to determine the mechanisms of hepatic monooxygenase oxidation of model anti-hyperthyroid compounds. All thiocarbamides tested (including methimazole) produced an NADPH-dependent loss of cytochrome P450 (P450) chromophore which could be prevented by the addition of glutathione (GSH). The covalent binding of MBI to liver microsomal proteins from dexamethasone (DEX)-pretreated rats was enhanced 10-fold with NADPH, unaffected by P450 inactivation with 1-aminobenzotriazole (ABT) and attenuated by GSH addition. Heat treatment of microsomes to inactivate the flavin-containing monooxygenase (FMO) decreased the observed binding. Equivalent amounts of [35S]- and [14C]MBI were covalently bound to hepatic microsomal proteins, suggesting retention of both the carbon and sulfur portions of the molecule in the MBI/protein adduct. Thiophilic reagents effected release of covalently bound [14C]- and [35S]MBI in equal amounts suggesting the presence of disulfide bonds between an MBI-derived sulfenic acid and microsomal protein thiols. Coincubation with bovine serum albumin (BSA) resulted in NADPH-dependent binding of [14C]-MBI to BSA sulfhydryls which was blocked by prior treatment of BSA with iodoacetamide. 1-Methyl-benzimidazole-2-thione (MMBI) also covalently bound to microsomal proteins and BSA but at levels lower than with MBI. P450, however, appeared to be more important than FMO in the metabolism of MMBI based on the effects of microsome heat pretreatment or ABT addition. In addition, ca. 1.5-fold more 35S- than 14C-label became bound. The covalent binding of [35S]1,3-dimethyl-benzimidazole-2-thione (DMMBI) to microsomal proteins was ca. six times greater than that of [14C]DMMBI. ABT, catalase and superoxide dismutase had a minimal effect on [35S]DMMBI binding, while FMO inactivation decreased binding by ca. 30%. These findings suggest that both monooxygenases contribute significantly to the hepatic metabolism of thiocarbamides. However, FMO activates thiocarbamides primarily to sulfenic acids, whereas P450 appears to produce both sulfenic acid and other reactive sulfur-derived metabolites. Thiol groups of P450 and other proteins are the molecular targets for these reactive species formed during the hepatic metabolism of anti-hyperthyroid drugs.

Animals↗
PubMed1992

The topography of trifluoroacetylated protein antigens in liver microsomal fractions from halothane treated rats.

Sera from patients with halothane hepatitis contain immunoglobulin G (IgG) antibodies to trifluoroacetylated liver microsomal proteins of 100, 76, 59, 57 and 54 kDa, which are produced as a consequence of metabolism of halothane to trifluoroacetyl halide by cytochrome(s) P450. In the present study, the membrane topographies of the various antigens in rat liver microsomal fractions were investigated. Liver microsomal fractions from rats treated with halothane in vivo, and rat liver microsomal fractions which had been incubated with halothane in vitro, were used as the source of trifluoroacetyl antigens. The antigens were detected by immunoblotting. Whereas the 100, 76, 59 and 57 kDa antigens were solubilized from the microsomal membrane by either 0.1 M sodium carbonate or 0.1% (w/v) sodium deoxycholate, the 54 kDa antigen was not solubilized by 0.1% (w/v) sodium deoxycholate. In intact microsomal fractions, the 100, 76, 59 and 57 kDa antigens were not degraded appreciably by trypsin unless detergent was added to permeabilize the microsomal membrane. These results indicate that the 54 kDa antigen is an integral membrane protein, whereas the 100, 76, 59 and 57 kDa antigens are peripheral membrane proteins situated within the lumen of microsomal vesicles, and hence presumably located within the lumen of the endoplasmic reticulum in vivo.

Animals↗
PubMed1993

Activation of the anti-cancer drug ifosphamide by rat liver microsomal P450 enzymes.

The NADPH-dependent metabolism of ifosphamide catalyzed by rat liver microsomes was investigated in order to identify individual P450 enzymes that activate this anti-cancer drug and to ascertain their relationship to the P450 enzymes that activate the isomeric drug cyclophosphamide. Pretreatment of rats with phenobarbital or clofibrate increased by up to 8-fold the activation of both ifosphamide and cyclophosphamide catalyzed by isolated liver microsomes. Studies using P450 form-selective inhibitory antibodies demonstrated that constitutively expressed P450s belonging to subfamily 2C (forms 2C11/2C6) make significant contributions to the activation of both oxazaphosphorines in uninduced male rat liver microsomes, while the phenobarbital-inducible P450 2B1 was shown to be a major catalyst of these activations in phenobarbital-induced microsomes. Pretreatment of rats with dexamethasone increased liver microsomal activation of ifosphamide approximately 6-fold without a corresponding effect on cyclophosphamide activation rates. Ifosphamide activation catalyzed by dexamethasone-induced liver microsomes was minimally inhibited by anti-P450 2B or anti-P450 2C antibodies, but was selectively inhibited by anti-P450 3A antibodies. Selective inhibition of liver microsomal ifosphamide activation was also effected by the macrolide antibiotic triacetyloleandomycin, an inhibitor of several dexamethasone-inducible 3A P450s. These studies establish that a dexamethasone-inducible family 3A P450 can make an important contribution to rat liver microsomal ifosphamide activation, and suggest that dexamethasone pretreatment might provide a useful approach for modulation of ifosphamide metabolism in order to improve its therapeutic efficacy in cancer patients.

Animals↗
PubMed1993

An investigation of the formation of cytotoxic, genotoxic, protein-reactive and stable metabolites from naphthalene by human liver microsomes.

Chemically reactive epoxide metabolites have been implicated in various forms of drug and chemical toxicity. Naphthalene, which is metabolized to a 1,2-epoxide, has been used as a model compound in this study in order to investigate the effects of perturbation of detoxication mechanisms on the in vitro toxicity of epoxides in the presence of human liver microsomes. Naphthalene (100 microM) was metabolized to cytotoxic, protein-reactive and stable, but not genotoxic, metabolites by human liver microsomes. The metabolism-dependent cytotoxicity and covalent binding to protein of naphthalene were significantly higher in the presence of phenobarbitone-induced mouse liver microsomes than with human liver microsomes. The ratio of trans-1,2-dihydrodiol to 1-naphthol was 8.6 and 0.4 with the human and the induced mouse microsomes, respectively. The metabolism-dependent toxicity of naphthalene toward human peripheral mononuclear leucocytes was not affected by the glutathione transferase mu status of the co-incubated cells. Trichloropropene oxide (TCPO; 30 microM), an epoxide hydrolase inhibitor, increased the human liver microsomal-dependent cytotoxicity (19.6 +/- 0.9% vs 28.7 +/- 1.0%; P = 0.02) and covalent binding to protein (1.4 +/- 0.3% vs 2.8 +/- 0.2%; P = 0.03) of naphthalene (100 microM), and reversed the 1,2-dihydrodiol to 1-naphthol ratio from 6.6 (without TCPO) to 2.6, 0.6 and 0.1 at TCPO concentrations of 30, 100 and 500 microM, respectively. Increasing the human liver microsomal protein concentration reduced the cytotoxicity of naphthalene, while increasing its covalent binding to protein and the formation of the 1,2-dihydrodiol metabolite. Co-incubation with glutathione (5 mM) reduced the cytotoxicity and covalent binding to protein of naphthalene by 68 and 64%, respectively. Covalent binding to protein was also inhibited by gestodene, while stable metabolite formation was reduced by gestodene (250 microM) and enoxacin (250 microM). The study demonstrates that human liver cytochrome P450 enzymes metabolize naphthalene to a cytotoxic and protein-reactive, but not genotoxic, metabolite which is probably an epoxide. This is rapidly detoxified by microsomal epoxide hydrolase, the efficiency of which can be readily determined by measurement of the ratio of the stable metabolites, naphthalene 1,2-dihydrodiol and 1-naphthol.

Adult↗
PubMed1994

Glucuronidation and isomerization of all-trans- and 13-cis-retinoic acid by liver microsomes of phenobarbital- or 3-methylcholanthrene-treated rats.

Glucuronidation and isomerization of all-trans-retinoic acid (tr-RA) and 13-cis-retinoic acid (13-cis-RA) were investigated in an in vitro system using liver microsomes of differently pretreated rats. In agreement with their thermodynamic stability, more retinoic acid was isomerized from the 13-cis form to the all-trans form than vice versa. Also some 9-cis-retinoic acid (9-cis-RA) could be found. Isomerization was reduced, but in contrast to glucuronidation was still important if boiled microsomes were used. This supports the view that isomerization can proceed as a non-enzymatic process. 3-Methylcholanthrene (MC) pretreatment of the rats increased the microsomal glucuronidation of 13-cis-RA and tr-RA and the formation of 13-cis-retinoyl-beta-glucuronide was enhanced up to 7-fold by MC-induced rat microsomes. The rates of glucuronidation by uninduced and phenobarbital-induced rat microsomes differed only slightly. In addition to glucuronides of the applied retinoic acid isomers (13-cis-RA and tr-RA), 9-cis-RA and its glucuronide were found. Induction of retinoid glucuronidation by pretreatment with MC indicates that this metabolic reaction is catalysed by a MC-inducible UGT isozyme. After two recently described pathways (conversions of retinol to retinal and of retinyl methyl ether to retinol) this is a third step of retinoid metabolism, induced by pretreatment with MC. With human microsomes no more than traces of glucuronides were detected; also, incubations with human microsomes resulted in a lower degree of isomerization than with rat microsomal fractions.

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