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PubMed1987

The effects of cigarette smoke compared to 3-methylcholanthrene and phenobarbitone on alkoxyresorufin metabolism by lung and liver microsomes from rats.

The rates of metabolism of phenoxazone and a homologous series of its ethers (alkoxyresorufins) by liver and lung microsomes of rats exposed to cigarette smoke were compared with the metabolism in rats pretreated with 3-methylcholanthrene (3MC) or phenobarbitone (PB). The rate of resorufin production was dependent on the length of the ether side chain. Liver and lung microsomes from control rats differed in their activity profiles (rate of resorufin production plotted against side-chain length), showing highest activity with ethoxy- and benzyloxyresorufin respectively. 3MC and PB selectively induced hepatic microsomal resorufin production with only certain of the substrates and the two agents differed in their selectivity, inducing most greatly with ethoxy- and benzyloxyresorufin respectively. Pulmonary microsomal resorufin production was induced by 3MC with a substrate selectivity similar to that shown for liver, but PB suppressed pulmonary metabolism with all the substrates. A single, short exposure to cigarette smoke induced ethoxyresorufin O-deethylase activity transiently in liver and lung microsomes. Three consecutive daily short exposures to cigarette smoke caused a weak 3MC-like induction of liver microsomal alkoxyresorufin metabolism, but the effect on lung microsomes was like weak 3MC and PB inductions combined. It is concluded that cigarette smoke induces selected cytochrome P-450-linked alkoxyresorufin O-dealkylase activities to a similar extent in both lung and liver and that the effects of cigarette smoke are characteristic of both 3MC-type and non-3MC-type inducers.

Animals↗
PubMed1988

Stimulation of the conjugation of lipid dienes in hepatic microsomes by 3,3'-dichlorobenzidine.

Pretreatment of male rats with 3,3'-dichlorobenzidine (DCB) resulted in the accumulation of conjugated dienes in lipids from hepatic microsomes. In vitro, these microsomes had 2-fold the NADPH-dependent malondialdehyde (MDA)-forming capacity of microsomes from untreated rats. To determine the mechanisms of the DCB-induced accumulation of diene conjugation, the effects of added DCB on NADPH- or iron + ascorbic acid- (Fe2+-ascorbate-) dependent diene conjugation, oxygen uptake and MDA formation were examined in microsomes from untreated rats in vitro. In the presence of NADPH, added DCB stimulated diene conjugation in microsomal lipids as did in vivo DCB pretreatment but inhibited the uptake of oxygen and the formation of MDA. When Fe2+-ascorbate was substituted for NADPH, the formation of diene conjugation, oxygen uptake, and MDA formation were inhibited by added DCB. The DCB-induced stimulation of diene conjugation, in addition to being strictly NADPH dependent, was carbon monoxide sensitive and was concomitant with the binding of added DCB to microsomal lipids. It is postulated that a metabolite of DCB generated by cytochrome P-450 reacts with membrane lipids both in vivo and in vitro in a manner analogous to the initiation of lipid peroxidation but at the same time prevents the autocatalytic decomposition of the lipids. The DCB-induced diene conjugation is interpreted as predisposing to deleterious changes in microsomes.

3,3'-Dichlorobenzidine↗
PubMed1988

Effects of long-term choline deficiency on hepatic microsomal cytochrome P-450-mediated steroid and xenobiotic hydroxylases in the female rat.

Total cytochrome P-450 levels decreased to about 80% of control in hepatic microsomes from female rats maintained for 30 weeks on a choline-deficient diet. Livers from these rats were fibrotic and had extensive fatty infiltration but, unlike livers of male rats on the same regimen, were not cirrhotic. Steroid hydroxylase activities were assessed in microsomes of female rats that received the choline-deficient diet and it was noted that the activity of the cytochrome P-450 UT-F-mediated steroid 7 alpha-hydroxylase was decreased to about 50% of the activity present in choline-supplemented control rat microsomes. Similar decreases were observed for microsomal androstenedione 6 beta-hydroxylase and aniline 4-hydroxylase activities. In female rat hepatic microsomes these two activities are probably mediated by the isozyme cytochrome P-450 ISF-G. In contrast to these findings, the activities of four other xenobiotic metabolising enzymes, as well as rates of microsomal steroid 16 alpha- and 16 beta-hydroxylation, were unchanged from control. Thus, in hepatic microsomes from choline-deficient female rats, it appears likely that levels of the non-sexually differentiated cytochromes P-450 UT-F and ISF-G are decreased. Unlike the situation in male rats, long term choline deficiency does not appear to influence levels of sexually-differentiated P-450 enzymes in the female rat.

Androstenedione↗
PubMed1989

Interactions between paraquat and ferric complexes in the microsomal generation of oxygen radicals.

Transition metals may play a central role in the toxicity associated with paraquat. Studies were carried out to evaluate the interaction of paraquat with several ferric complexes in the promotion of oxygen radical generation by rat liver microsomes. In the absence of added iron, paraquat produced some increase in low level chemiluminescence by microsomes; there was a synergistic increase in light emission in the presence of paraquat plus ferric-ATP or ferric-citrate, but not paraquat plus either ferric-EDTA or ferric-diethylenetriamine pentaacetic acid (ferric-DETAPAC). Synergistic interactions could be observed at a paraquat concentration of 100 microM and a ferric-ATP concentration of 3 microM. In the absence or presence of paraquat, microsomal light emission was not affected by catalase or dimethyl sulfoxide (DMSO), indicating no significant role for hydroxyl radicals. Superoxide dismutase (SOD) did not affect chemiluminescence in the absence of paraquat but produced some inhibition in the presence of paraquat; this inhibition by SOD was most prominent in the absence of added iron and less pronounced in the presence of ferric-ATP or ferric-citrate. Although microsomal chemiluminescence is closely associated with lipid peroxidation, paraquat did not increase malondialdehyde production as reflected by production of thiobarbituric acid-reactive components. However, lipid peroxidation was sensitive to inhibition by SOD in the presence, but not in the absence, of paraquat, analogous to results with chemiluminescence. Paraquat synergistically increased microsomal hydroxyl radical production as measured by the production of ethylene from 2-keto-4-thiomethylbutyrate in the presence of ferric-EDTA or ferric-citrate. The interaction of paraquat with microsomes and ferric complexes resulted in an increase in oxygen radical generation. Various ferric complexes can increase the catalytic effectiveness of paraquat in promoting microsomal generation of oxygen radicals, although, depending on the reaction being investigated, the nature of the ferric complex is important.

Animals↗
PubMed1989

Histamine inhibition of mixed function oxidase activity in rat and human liver microsomes and in the isolated perfused rat liver.

The imidazole ring is a common structural feature of some xenobiotics that inhibit cytochrome P-450-catalysed reactions. Histamine is a 4-substituted imidazole and a preliminary study has shown it to be an inhibitor of rat liver microsomal drug oxidation. This work has now been extended. Histamine appears to be a competitive inhibitor of the alpha-hydroxylation (HM) (Ki = 164 microM; IC50 at 20 microM = 308 microM) and O-demethylation (ODM) (Ki = 243 microns; IC50 at 20 microM = 400 microM) of metoprolol in rat liver microsomes. Of the metabolites of histamine only N-acetylhistamine showed comparable inhibitory potency to that of the parent compound. Histamine impaired the disappearance of lignocaine when incubated with rat liver microsomes. This was accompanied by a corresponding inhibition of 3-hydroxy-lignocaine appearance. Histamine produced a type II spectral interaction with rat liver microsomes (lambda max = 432 nm, lambda min = 408 nm; Ks = 0.11 mM). When histamine was incubated alone with rat liver microsomes no loss of substrate was observed. The oxidation of metoprolol by human liver microsomes was impaired by histamine (IC50 values for ODM appearance at 25 microM: liver HL1 greater than 10, HL3 = 3.8 and HL4 = 3.7 mM). In comparison, cimetidine had an IC50 value of 1.5 mM using microsomes from liver HL3. Addition of histamine impaired the elimination of metoprolol by the isolated perfused rat liver in a dose-dependent manner (P less than 0.001, one-way analysis of variance). These data demonstrate that histamine can enter hepatocytes, interact with cytochrome P-450 and inhibit some drug oxidation reactions. The physiological relevance of inhibition of drug metabolism by histamine remains to be determined.

Animals↗
PubMed1990

Oxidative activation of the thiophene ring by hepatic enzymes. Hydroxylation and formation of electrophilic metabolites during metabolism of tienilic acid and its isomer by rat liver microsomes.

Tienilic acid (TA) is metabolized by liver microsomes from phenobarbital-treated rats in the presence of NADPH with the major formation of 5-hydroxytienilic acid (5-OHTA) which is derived from the regioselective hydroxylation of the thiophene ring of TA. During this in vitro metabolism of TA, reactive electrophilic intermediates which bind irreversibly to microsomal proteins are formed. 5-Hydroxylation of TA and activation of TA to reactive metabolites which covalently bind to proteins both required intact microsomes, NADPH and O2 and are inhibited by metyrapone and SKF 525A, indicating that they are dependent on monooxygenases using cytochromes P-450. Microsomal oxidation of an isomer of tienilic acid (TAI) bearing the aroyl substituent on position 3 (instead of 2) of the thiophene ring also leads to reactive intermediates able to bind covalently to microsomal proteins. Covalent binding of TAI, as that of TA, depends on cytochrome P-450-dependent monooxygenases and is almost completely inhibited in the presence of sulfur containing nucleophiles such as glutathione, cysteine or cyteamine. These results show that 5-OHTA, which has been reported as the major metabolite of TA in vivo in humans, is formed by liver microsomes by a cytochrome P-450-dependent reaction. They also show that two thiophene derivatives, TA and TAI, bind to microsomal proteins after in vitro metabolic activation, TAI giving a much higher level of covalent binding than TA (about 5-fold higher) and a much higher covalent binding: stable metabolites ratio (4 instead of 0.5).

Animals↗
PubMed1992

Effects of cyclophosphamide and adriamycin on rat hepatic microsomal glucuronidation and lipid peroxidation.

The effect of cytotoxic drug administration, as a single dose i.p. to rats (six rats/treatment group), on hepatic microsomal UDP-glucuronosyltransferase (UGT) activity was investigated. Glucuronidation of morphine in microsomes from control rats apparently involved at least two enzymes. Administration of cyclophosphamide (CP; 200 mg/kg 7 days prior to killing) significantly increased the rate of morphine glucuronidation over the range 0.05-10 mM, and significantly increased the apparent Vmax for the high capacity isoenzyme from 1.25 +/- 0.12 to 1.95 +/- 0.39 nmol/mg/min. In contrast, the activity of 1-naphthol UGT was not significantly altered by administration of CP. Rats treated with the same dose of CP 1 day prior to killing showed a significant decrease in microsomal morphine-UGT activity at 0.05 and 2.5 mM morphine, but a significant increase in activity was observed following administration of CP or Adriamycin (AD; 10 mg/kg) 4 days prior to killing. The extent of microsomal lipid peroxidation was significantly increased in microsomes obtained from rats treated with CP or AD 4 days prior to killing, and was positively correlated (P less than 0.001) with the rate of glucuronidation of 0.05 and 2.5 mM morphine. Preincubation of microsomes in the presence of CP (5 mM) and AD (100 microM) significantly decreased the rate of glucuronidation of 2.5 mM morphine. In vitro NADPH-mediated lipid peroxidation significantly increased the activity of both the high and low affinity morphine-UGT isoenzymes. Administration of the cytotoxic drugs CP and AD may alter microsomal morphine-UGT activity via the process of lipid peroxidation, although other mechanisms cannot be excluded.

Animals↗
PubMed1992

Mechanism of activation of rat liver microsomal glutathione transferase by noradrenaline and xanthine oxidase.

Activation of glutathione transferase activity in rat liver microsomes under a variety of conditions producing oxidative stress was investigated. Neither hydrogen peroxide (10 mM) (added or produced endogenously by glucose + glucose oxidase) nor duroquinone together with an NADPH-regenerating system (which generates the superoxide anion radical) had any significant effect on the glutathione transferase activity towards 1-chloro-2,4-dinitrobenzene. On the other hand, incubation of microsomes with 1 mM noradrenaline (which autooxidizes and generates superoxide anion radical) gave a 160% activation, as shown earlier (Aniya and Anders, J Biol Chem 264: 1998-2002, 1989). This was taken as an indication that microsomal glutathione transferase could be activated by oxidative stress. Here, we demonstrate that activation by this compound is due to covalent binding (presumably of the quinone formed during autooxidation). The xanthine/xanthine oxidase system, which generates the superoxide anion radical and hydrogen peroxide, increases microsomal glutathione transferase activity, but this activation was not dependent on the presence of xanthine. Western blots of microsomes treated with xanthine oxidase revealed that activation was due to proteolysis (presumably by contaminating proteases in the xanthine oxidase). In conclusion, there is no firm evidence that rat liver microsomal glutathione transferase is activated directly by reduced oxygen species in the microsomal system. The possibility remains that oxidative stress triggers secondary mechanisms such as generation of reactive intermediates and/or activation of proteolysis, which can in turn increase enzyme activity.

Animals↗
PubMed1993

Effect of rociverine on P450-dependent monooxygenases and its N-deethylation metabolism in rat liver microsomes.

Rociverine [2-(diethylamino)-1-methylethyl cis-1-hydroxy [bicyclohexyl]-2-carboxylate] citrate (ROC) is an antispasmodic agent therapeutically active in humans at doses of 0.5-1 mg/kg. This study investigated the effect of acute administration of the drug on hepatic microsomal cytochrome P450 (P450)-catalysed drug metabolism. Only high doses (> or = 100 mg/kg) of ROC were able to induce in rats the hepatic microsomal pentoxyresorufin O-depenthylase (PROD) and 16 beta-testosterone hydroxylase activities both associated with P4502B1/2 and the erythromycin N-dimethylase (ErD) and 2 beta-testosterone hydroxylase activities both dependent on P4503A1/2. However, at 100 and 200 mg/kg of ROC, the 16 beta-testosterone hydroxylase and PROD were the most induced activities, suggesting that P4502B1/2 are the isoforms most sensitive to ROC induction. Accordingly, ROC treatment enhanced, in a dose-dependent manner, the amount of P4502B1/2 and 3A1/2 in microsomes as assayed by western blotting. The northern blot analysis of ROC-treated rat liver showed that the P4502B1/2 induction appears to be regulated at the mRNA level as in the induction by phenobarbital (PB). The oxidative metabolism of ROC with hepatic microsomes from control or PB- and ROC-induced rats resulted in a N-deethyl ROC derivative (major metabolite) and an unknown minor ROC derivative. The kinetic parameters for the N-deethylation of ROC were studied with purified P4502B1 and with microsomes from control or rats treated with various inducers (phenobarbital, ethanol, beta-naphthoflavone, dexamethasone and rociverine). It was found that phenobarbital-, dexamethasone- and rociverine-induced microsomes deethylated ROC with a Vmax about five times higher than that (0.9 nmol/min/mg protein) of control microsomes, although with a similar affinity (Km approximately 0.3 mM). In a reconstituted system, the purified P4502B1 metabolized ROC with a high deethylation rate (22 nmol/min/nmol P450). Moreover, the ROC deethylation was inhibited by compounds such as hexobarbital, metyrapone and triacetyloleandomicin, selective inhibitors for P4502B and/or P4503A enzymes. On the other hand ROC, when added in vitro, inhibited the 16 beta- and 2 beta-testosterone hydroxylases and the PROD and ErD activities. Taken together, these results indicate that the ROC-inducible P4502B and P4503A are involved in ROC deethylation. In conclusion, it has been demonstrated that ROC is a weak phenobarbital-like inducer of P450, probably able at high and reiterated doses to alter its own metabolism, at least in the rat liver.

Acetaldehyde↗
PubMed1993

Enhancement of hepatic microsomal esterase activity following soman pretreatment in guinea pigs.

Soman (pinacolyl methylphosphonofluoridate), a highly toxic organophosphate compound, has been found to be a strong inhibitor of hepatic microsomal carboxylesterase in vitro, but an enhancer of carboxylesterase when administered in vivo. In response to this paradoxical observation, the objective of this study was to determine if soman could cause true enhancement of the metabolism of drugs in the guinea pig and, if so, to characterize the enhanced enzyme activity. Following the pretreatment of guinea pigs with 90% LD50 soman, enhancement of microsomal esterase activity was noted 12 and 24 hr after pretreatment. Using Michaelis-Menten enzyme kinetic studies, enhancement was found to occur with liver carboxylesterase and procaine esterase, but not with aniline hydroxylase. Since the soman-enzyme complex was known to undergo aging with the release of pinacolyl alcohol and the subsequent formation of pinacolone, the effects of these metabolites on the activity of liver microsomal enzymes in vitro were explored. Pinacolone and pinacolyl alcohol produced enzyme enhancement in vitro in a manner similar to that produced by soman pretreatment. These effects were compared with those made by acetone in the same incubations, since the enhancing influence of acetone has already been well documented. Similarity was found between the in vitro effects of acetone and the effects of pinacolone and pinacolyl alcohol. Lastly, the in vivo effects of pinacolone on the activities of the same liver microsomal enzymes were studied following pretreatment of the guinea pigs with 90% LDLo (lowest published lethal dose) pinacolone. Pretreating guinea pigs with pinacolone prior to killing them enhanced liver microsomal carboxylesterase and procaine esterase activities, but had no effect on microsomal aniline hydroxylase activity. This pattern of enzyme enhancement was similar to that observed after soman pretreatment. Therefore, soman was found to enhance hepatic microsomal esterase activity in the guinea pig in a manner similar to that seen with its metabolites, as well as acetone. This information may give insight into how the efficacy and toxicity of therapeutic drugs, other xenobiotics, and endogenous materials may be altered in individuals who survive an exposure to soman.

Acetone↗
PubMed1994

Differential roles of cytochromes P450 2D1, 2C11, and 1A1/2 in the hydroxylation of bufuralol by rat liver microsomes.

Bufuralol hydroxylation activities of liver microsomal cytochrome P450 (P450) enzymes were studied in the rat; the reaction has been used widely in determining levels of liver microsomal P450 2D6, which shows debrisoquine-type genetic polymorphism in humans. Liver microsomes catalyzed the conversion of bufuralol to 1'-hydroxybufuralol and a structurally unidentified metabolite (termed here as M-1) in the presence of an NADPH-generating system and molecular oxygen. Bufuralol 1'-hydroxylation activities catalyzed by the liver microsomes were not increased in rats treated with several P450 inducers, whereas beta-naphthoflavone treatment (and to a lesser extent that of isosafrole) caused a significant induction of M-1 formation. The major role of P450 1A1/2 in M-1 formation was confirmed by catalytic inhibition with anti-P450 antibodies and alpha-naphthoflavone in liver microsomes of beta-naphthoflavone-treated rats, and by reconstitution experiments containing P450 1A1 and 1A2. Among nine forms of purified rat P450 enzymes studied in the reconstituted system, P450 2C11 displayed the highest activities for bufuralol 1'-hydroxylation, followed by P450 1A1 and P450 2D1. A female-specific form of P450 2C12 did not catalyze bufuralol 1'-hydroxylation. In liver microsomes of male rats, however, P450 2D1 was the dominant enzyme because only anti-P450 2D1 antibodies, and not anti-P450 2C11 and anti-P450 1A1, inhibited the bufuralol hydroxylation activities, and a specific P450 2D1 inhibitor, quinine, caused a dramatic decrease in the hydroxylation activities. The major contribution of P450 2D1 in the bufuralol 1'-hydroxylation activities was also supported by a kinetic analysis of the reconstituted system; P450 2D1 enzyme had a very low Km value (8.4 microM) as compared with those of P450 2C11 (Km = 83 microM) and P450 1A1 (Km = 230 microM). Thus, the present results suggested that different P450 enzymes are involved in the hydroxylation of bufuralol in rat liver microsomes, and the kinetic analysis, as well as immunoinhibition and chemical inhibition experiments, may be of great importance for determining the major roles of P450 enzymes in drug hydroxylation reactions.

Adrenergic beta-Antagonists↗
PubMed1994

Metabolism of FK506, a potent immunosuppressive agent, by cytochrome P450 3A enzymes in rat, dog and human liver microsomes.

The oxidative metabolism of FK506 by liver microsomes and purified cytochrome P450 (P450) enzymes from rats, dogs and humans was studied. The major metabolite formed by liver microsomes from all species was 13-demethylated FK506, named M-I. In adult rats, liver microsomal metabolic activity toward FK506 was higher in males than in females and was stimulated by treatment with P450 3A inducers such as dexamethasone and phenobarbital. In a reconstituted monooxygenase system containing various forms of purified P450 3A enzymes, rat P450 3A2, dog P450 DPB-1 (a form of the P450 3A family) and human P450 3A4 catalyzed FK506 oxidation efficiently in the presence of cytochrome b5, a mixture of phospholipids (dilauroylphosphatidylcholine, dioleoylphosphatidylcholine and phosphatidylserine), and sodium cholate. Rat P450 2C6 and 2D1 and human P450 2CMP also metabolized FK506, with significant lower activity than the P450 3A enzymes, and other rat P450 1A, 2A, 2B, 2C and 2E families including C11 did not show catalytic activities for FK506. Anti-P450 3A2 and anti-P450 3A4 antibodies strongly inhibited FK506 oxidation catalyzed by rat and human liver microsomes, respectively. The formation rate of M-I correlated well with testosterone 2 beta- and 6 beta-hydroxylase activities in rat liver microsomes and with immunoquantified P450 3A4 content, nifedipine oxidase activity, and testosterone 6 beta-hydroxylase activity in human liver microsomes. These in vitro findings indicate that the P450 3A enzymes in liver microsomes from various species of animals, including human, play a major role in the first step oxidation of FK506.

Animals↗
PubMed1995

Iron-stimulated ring-opening of benzene in a mouse liver microsomal system. Mechanistic studies and formation of a new metabolite.

In the present study, we investigated the mechanism(s) of ring-opening of benzene in a mouse liver microsomal system in the presence of Fe2+.HPLC analysis based on coelution with authentic standards and on-line UV spectra obtained using a diode array detector indicated that benzene is metabolized to phenol, hydroquinone (HQ), trans,trans-muconaldehyde (muconaldehyde, MUC), 6-oxo-trans,trans-2,4-hexadienoic (COOH-M-CHO), 6-hydroxy-trans,trans-2,4-hexadienal (CHO-M-OH), and 6-hydroxy-trans,trans-2,4-hexadienoic acid (COOH-M-OH). CHO-M-OH was confirmed by mass spectrometry. Muconaldehyde was also metabolized to CHO-M-OH, COOH-M-CHO and COOH-M-OH, in the same microsomal system. The inhibition of muconaldehyde metabolism by microsomes in the presence of pyrazole indicates that there is cytosolic alcohol dehydrogenase (ADH) activity in the microsomes. Metabolism by contaminating ADH of muconaldehyde formed during microsomal incubation of benzene could be involved in the formation of CHO-M-OH and COOH-M-OH. The ring-opening of benzene was stimulated by added Fe2+. Hydrogen peroxide was produced in the microsomal system and consumed in the presence of added Fe2+. Addition of catalase inhibited the formation of ring-opened products, while superoxide dismutase increased their formation in the presence of azide. Singlet oxygen scavengers, i.e. histidine, deoxyguanosine, Tris and azide (at concentrations above 1.0 mM), dramatically decreased the ring-opening of benzene. Hydroxyl radical scavengers, DMSO, mannitol and formate, but not ethanol, also decreased the ring-opening of benzene. The data indicate that Fenton chemistry plays an important role in benzene ring-opening by microsomes. An unknown peak with UV absorption maxima at 275 and 345 nm was also detected. Based on pH sensitivity of the UV spectrum, the reactivity with thiobarbituric acid (giving a chromogen with absorption maximum at 532 nm) and the molecular weight (126), this compound was identified tentatively as alpha- or beta-hydroxymuconaldehyde.

Aldehydes↗
PubMed1995

Species differences in the covalent binding of [14C]tamoxifen to liver microsomes and the forms of cytochrome P450 involved.

Species differences in the NADPH-dependent covalent binding of [14C]tamoxifen to liver microsomes have been studied using preparations from humans, female F344 rats and DBA/2 mice. Protein binding has been used as an index of metabolic activation and as a surrogate for DNA binding in order to establish which forms of cytochrome P450 are responsible for genotoxicity. A panel of 12 human liver microsomes has been characterized and immunoquantified for nine cytochrome P450 isoenzymes. Binding of tamoxifen (45 microM) (25 +/- 2.5 pmol/15 min/mg protein, mean +/- SE) correlated (P < 0.05) with CYP3A4 and CYP2B6 content. Covalent binding of [14C]tamoxifen to microsomal preparations from human breast tumour tissue could also be detected but at levels 7-fold lower than in liver. The covalent binding of tamoxifen to mice, rat or human liver microsomal preparations increased with increasing substrate concentration. Covalent binding of [14C]tamoxifen (45 microM) in rats was 3.8-fold and mice 17-fold higher than in human liver microsomal preparations. In mice, the apparent Km (9.6 +/- 1.9 microM) was very much lower than for rats (119 +/- 41 microM). Pretreatment of female rats with phenobarbitone or dexamethasone resulted in a 4- to 5-fold increase in [14C]tamoxifen binding, relative to controls, consistent with the involvement of CYP2B1 and CYP3A1 in the metabolic activation. It cannot be distinguished at present if the same reactive metabolites are involved in protein and DNA binding. The greater potential of mouse liver microsomes to activate tamoxifen, relative to rats, does not reflect DNA damage or hepatocarcinogenicity seen following dosing with tamoxifen in vivo. It is concluded that covalent binding of tamoxifen to protein in vitro cannot be directly related to the carcinogenic potential of this compound. However, in the three species investigated, results suggest that the rat is a better model than the mouse for human liver microsomal activation of tamoxifen both with respect to kinetic parameters and the pattern of metabolic products.

Animals↗
PubMed1995

beta-Naphthoflavone-inducible cytochrome P4501A1 activity in liver microsomes of the marine safi fish (Siganus canaliculatus).

The cytochrome P450-dependent metabolism of benzo(a)pyrene and other xenobiotics has been investigated in liver microsomes prepared from a local marine safi fish, Siganus canaliculatus. The safi fish was found to have a well-developed microsomal monooxygenase system consisting of cytochrome P450, cytochrome b5 and NADPH-cytochrome c reductase. The fish microsomal enzyme system was able to metabolize benzo(a)pyrene, 7-ethoxycoumarin and 7-ethoxyresorufin. Male fish were found to exhibit a higher monooxygenase activity than female fish. Treatment of fish with beta-naphthoflavone was found to induce (2- to 4-fold) the activities of aryl hydrocarbon hydroxylase, ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase. HPLC analysis of the metabolites produced by incubation of benzo(a)pyrene with the liver microsomal preparation showed a predominant formation of 3-OH and 9-OH benzo(a)pyrene. There was an increased formation of benzo(a)pyrene 7,8-diol and benzo(a)pyrene 7,8,9,10-tetrol in liver microsomes prepared from beta-naphthoflavone-treated fish. Western immunoblot analysis of liver microsomes from beta-naphthoflavone-treated fish using an antibody to rat liver cytochrome P4501A1 (CYP1A1) suggested the presence of an inducible cytochrome P450 enzyme that was comparable with that of rat liver enzyme. Our results suggest that liver microsomes from the safi fish have multiple forms of cytochrome P450 with a specific beta-naphthoflavone-inducible CYP1A1 homologous protein that can metabolize a variety of substrates.

Animals↗
PubMed1982

Microsomal opiate receptors differ from synaptic membrane receptors in proteolytic sensitivity.

We have found that opiate receptors in smooth microsomal fractions differ from synaptic membrane-associated receptors in proteolytic sensitivity. With 3 proteases of different substrate specificities (trypsin, chymotrypsin and S. griseus protease) smooth microsomal opiate receptors from rat brain were consistently less sensitive to limited proteolysis than were synaptic membrane receptors. Thiamine pyrophosphatase, a luminal Golgi membrane marker enzyme, exhibited a similar resistance to S. griseus protease in microsomal preparations, while microsomal Na+/K+-ATPase (ouabain-sensitive) was readily destroyed by trypsin. We also discovered that smooth microsomal opiate receptors co-migrate with both Golgi membrane and endoplasmic reticulum marker proteins on equilibrium density gradients under isopycnic conditions. Electron microscopic examination of the Golgi-enriched fraction showed the typical cisternae frequently associated with isolated Golgi membranes. Synaptic junctions, presynaptic membranes, myelin and mitochondria were conspicuously absent from this fraction. Since the microsomes isolated in vitro showed similar topography to those in vivo, the binding sites for opiates could be localized on the luminal surface membranes of the microsomal fractions. The exquisite sensitivity of synaptic membrane opiate receptors to proteolysis suggests that these receptors are found on the extracellular surface of the synaptic junction.

Animals↗
PubMed1979

A class of strong inhibitors of microsomal monooxygenases: the ellipticines.

Ellipticine (E) and its 9-hydroxy derivative inhibit strongly various liver monooxygenase activities mediated by microsomes from control and phenobarbital (PB), benzo[alpha]pyrene (BP) or Aroclor 1254 (Aroclor)-pretreated rats. The inhibition constants, Ki, are remarkably low, and often smaller than 1 micron, particularly in the case of microsomes containing cytochrome P-448. The inhibitory potency (I50) of 9-hydroxyellipticine (9-OHE) is larger (about ten-fold) than the one of classical inhibitors (metyrapone or 7,8-benzoflavone (7,8-BF)), whatever the activities studied and the induction of microsomes. Differences exist between the mechanisms of inhibition according to the form of cytochrome P-450 present in microsomes of differently pretreated rats; whichever the activities studied, one observes: (a) a competitive inhibition towards the activity of non-induced or PB-induced microsomes and (b) a non-competitive inhibition towards the activity of Aroclor or BP-induced microsomes, at variance with 7,8-BF. These results are in good agreement with the interaction properties of the ellipticines with microsomal cytochromes P-450.

Alkaloids↗
PubMed1982

Characteristics of microsomal reduction of benzo[a]pyrene 4,5-oxide.

NADPH-reduction of benzo[a]pyrene 4,5-oxide (BP-4,5-oxide) to BP required four components from rat liver: cytochrome P-450, NADPH cytochrome P-450 reductase, phosphatidylcholine and a soluble, heat-sensitive factor which was present in 105 000 X g supernatant and was also released from microsomes by sonication. The requirement for this factor contrasts with recently reported results from Sugiura et al. (Cancer Res., 40 (1980) 2910). Oxide-reduction was 40 times faster under anaerobic conditions, but oxygen did not affect the stimulation factor. This stimulation was highest (X15) at low concentrations of microsomal protein (less than 0.1 mg/ml) and was almost absent at high concentrations of microsomal protein (greater than 1 mg/ml). Oxide-reduction activity was proportional to microsomal protein concentration in the presence of added 105 000 X g supernatant, but for microsomes alone (greater than 0.1 mg/ml) exhibited a parallel plot with an intercept at 0.08 mg/ml microsomal protein. Stimulation was highest at high concentrations of BP-4,5-oxide and a linear plot of V-1 vs. [BP-4,5-oxide]-1 was only obtained in the presence of 105 000 X g supernatant (Km = 3 microM, Vmax = 3.3 nmol/mg/min). Microsomal hydration of BP-4,5-oxide (inhibited in reductase assays) was unaffected by 105 000 X g supernatant, suggesting that stimulation of oxide-reduction did not derive from solubilization of BP-4,5-oxide. Stimulation was observed in the initial rate of reaction and was independent of incubation time. Inhibition of lipid peroxidation, removal of peroxides and deoxygenation were all excluded as explanations of the stimulatory effect.

Anaerobiosis↗