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PubMed1985

Monoclonal antibodies distinguish among isozymes of the cytochrome P-450b subfamily.

Polyclonal antibody has been shown previously to react identically with cytochromes P-450b and P-450e purified from Long Evans rats and a strain variant of cytochrome P-450b purified from Holtzman rats (P-450bH). In the present study, an array of 12 different monoclonal antibodies produced against cytochrome P-450b has been used to distinguish among these closely related phenobarbital-inducible rat hepatic cytochromes P-450. In immunoblots and enzyme-linked immunosorbent assays, 10 monoclonal antibodies bind to cytochromes P-450b, P-450e, and P-450bH; one monoclonal antibody (B50) recognizes cytochromes P-450b and P-450bH but not cytochrome P-450e; and one monoclonal antibody (B51) is specific for cytochrome P-450b. In addition, one monoclonal antibody (BEF29) reacts strongly with cytochrome P-450f, and another antibody (BEA33) reacts weakly with cytochrome P-450a. No cross-reactions with cytochromes P-450c, P-450d, and P-450g-P-450j were detected with any of the monoclonal antibodies in these assays. Six spatially distinct epitopes on cytochrome P-450b were identified, and differences in antibody reactivity provided evidence for three additional overlapping epitopes. Several monoclonal antibodies are potent inhibitors of testosterone and benzphetamine metabolism supported by cytochrome P-450b in a reconstituted system. B50 and BE52 do not inhibit metabolism of the two substrates by microsomes from untreated rats, but inhibit benzphetamine N-demethylation and testosterone metabolism to 16 alpha- and 16 beta-hydroxytestosterone as well as androstenedione formation 67-94% by microsomes from phenobarbital-treated rats. No other pathways of testosterone metabolism are inhibited by these monoclonal antibodies. The differential inhibition of microsomal metabolism of benzphetamine and testosterone by these monoclonal antibodies is a reflection of the content and inducibility of cytochromes P-450b and P-450e as well as other cytochrome P-450 isozymes.

Animals↗
PubMed1984

Substrate binding site of microsomal cytochrome P-450 directly faces membrane lipids.

Cytochrome P-450, purified from liver microsomes of phenobarbital-treated rabbits, was incorporated into dimyristoylphosphatidylcholine liposomes. The binding of benzphetamine to the liposome-bound cytochrome P-450 was examined by measuring the benzphetamine-induced spectral change at various temperatures. The van't Hoff plot of the apparent spectral dissociation constant showed a distinct break at the temperature of phase transition of the synthetic lipid. On the other hand, no such break was observed for benzphetamine binding to microsomal bound cytochrome P-450. These results suggest that the substrate binding site of cytochrome P-450 is embedded in the apolar interior of phospholipid bilayer membranes.

Animals↗
PubMed1987

Reduction and catalytic properties of cytochrome P-450 LM2 in reconstituted system containing monomeric carriers.

The membrane microsomal monooxygenase system can be reconstituted in solution from NADPH-specific flavoprotein and cytochrome P-450 which exist in the monomeric state in the presence of Emulgen 913 at molar ratio of the proteins and detergent of 1:1:300. Oxidized and dithionite-reduced monomers of cytochrome P-450 were much less thermostable than its initial aggregates, while thermal stability of NADPH-specific flavoprotein did not depend on its aggregation state. Binding spectra of cytochrome P-450 monomers with benzphetamine were atypical and had an absorbance minimum at 422 nm only. The addition of benzphetamine and/or flavoprotein to cytochrome P-450 monomers did not cause the spin equilibrium shift and the low-spin form content was higher than 85% in all cases. Investigation of the dependence of the initial rates of NADPH-dependent cytochrome P-450 reduction and benzphetamine oxidation on the stoichiometry of the flavoprotein and cytochrome P-450 at their constant total concentration showed that the molar ratio of 1:1 was required for maximal activity. Thus this system works in full accordance with the mass action law.

Animals↗
PubMed1993

Identification of the human and animal hepatic cytochromes P450 involved in clonazepam metabolism.

This report characterizes the cytochrome P450 isozyme involved in clonazepam metabolism. This study was undertaken using a library of liver microsomal fractions prepared from untreated rabbits or those treated with drugs known to specifically induce various cytochrome P450 isozymes (ie P450 2B4 by phenobarbital, P450 1A1 and P450 1A2 by 3-methylcholanthrene and beta-naphthoflavone, P450 2E1 by acetone and ethyl alcohol, and P450 3A6 by erythromycin). Only microsomes obtained from phenobarbital-treated rabbits exhibited a type II binding spectrum upon addition of clonazepam (Ks(app) = 31.4 +/- 3.8 microM) and significantly metabolized clonazepam to 7-aminoclonazepam. Benzphetamine, which is a known substrate for P450 2B1 was also extensively metabolized by microsomes prepared from phenobarbital treated rabbits. This indicates that the same isozyme (P450 2B subfamily) was involved in the biotransformation of both substrates. Experiments performed on 14 human liver microsomal preparations showed a wide interindividual variability (from 1-4) and a good correlation (r = 0.70) between benzphetamine and clonazepam metabolism. Since P450 3A4 (nf25) was involved in benzphetamine metabolism, clonazepam was probably nitroreduced by the same isozyme. An oligonucleotide specific for the P450 3A4 gene subfamily was synthetized and used for hybridization on total RNA from human liver samples. Two transcripts of 2.2 and 3.0 kb were detected and the level of the 2.2 kb mRNA expression was significantly correlated (r = 0.61) with the intensity of clonazepam nitroreduction by the corresponding microsome batches.

Animals↗
PubMed2004

Metabolism of N,N',N"-triethylenethiophosphoramide by CYP2B1 and CYP2B6 results in the inactivation of both isoforms by two distinct mechanisms.

The anticancer drug N,N,"N"-triethylenethiophosphoramide (tTEPA) inactivated CYP2B6 and CYP2B1 in the reconstituted system in a time-, concentration-, and NADPH-dependent manner indicative of mechanism-based inactivation. The KI value for the inactivation of CYP2B1 was 38 microM, the kinact was 0.3 min(-1), and the t1/2 value was 2.5 min. Spectral carbon monoxide (CO) binding and high-performance liquid chromatography heme studies of the tTEPA-inactivated CYP2B1 suggest that the loss in the enzymatic activity was primarily due to the binding of a reactive tTEPA intermediate to the 2B1 apoprotein. Inactivation by tTEPA in the presence of 7-ethoxycoumarin, an alternate substrate, reduced the rate of inactivation of CYP2B1. Incubations with tTEPA and NADPH resulted in greater than 90% loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation and testosterone hydroxylation activity of CYP2B1. In contrast, benzphetamine metabolism was significantly less inhibited (47%). CYP2B6 was inactivated by tTEPA with a KI value of 50 microM, a k inact value of 0.1 min(-1), and a t1/2 value of 14 min. However, unlike CYP2B1, the tTEPA-inactivated human isoform showed losses in the cytochrome P450 (P450) CO spectrum, the pyridine hemochrome spectrum, and in the amount of native heme that were comparable with the loss in the 7-EFC and benzphetamine activity, suggesting that activity loss was brought about by a tTEPA-reactive intermediate damaging the CYP2B6 heme. CYP2B6 could only be protected from the tTEPA-dependent inactivation by the 2B6-specific substrate bupropion but not by other substrates of CYP2B such as benzphetamine, testosterone, or 7-ethoxycoumarin. The data indicate that tTEPA metabolism by these two 2B isoforms results in inactivation of the P450s by two distinct mechanisms.

Amines↗
PubMed1987

Inhibition of hepatic microsomal lipid peroxidation by endogenous glycogen in the rat.

The effect of endogenous glycogen on lipid peroxidation was examined in hepatic microsomes from rats. Microsomes were prepared to retain endogenous hepatic glycogen (Pg+) or to minimize it (Pg-). The indices of lipid peroxidation examined included the rate of NADPH-dependent formation of malondialdehyde (MDA) and the concomitant destruction of cytochrome P-450 and decline in the linearity of benzphetamine N-demethylase activity in microsomes. Cytochrome P-450 was destroyed during benzphetamine N-demethylation in microsomes with the loss being more extensive in Pg- than in Pg+. The destruction of cytochrome P-450 and the concomitant loss in linearity of benzphetamine N-demethylation in Pg- were prevented by added EDTA. Added linoleic acid hydroperoxide (LAHP) also caused a time-dependent loss of cytochrome P-450 in microsomes with the rate being greater in Pg- than in Pg+. The results show that glycogen inhibits hepatic microsomal lipid peroxidation and suggest that variations in glycogen content may contribute to disparities in in vitro oxidative activities between different microsomal samples. Such disparities may be minimized by the removal of glycogen during the preparation of microsomes and then supplementing the incubation mixtures with EDTA. The in vivo relevance of the observed antioxidant effect of glycogen is discussed in terms of the possible modulation by the polysaccharide of hepatotoxicity by agents whose effects may be mediated by lipid peroxidation.

Animals↗
PubMed1993

Role of lysine and arginine residues of cytochrome P450 in the interaction between cytochrome P4502B1 and NADPH-cytochrome P450 reductase.

Chemical modification of cytochrome P450 was used to study the involvement of lysine and arginine residues in the interaction between cytochrome P450 and NADPH-cytochrome P450 reductase. Acetylation of 2.2 and 8.5 mol of lysine/mole of P450 by acetic anhydride led to 38.7 and 95% reductions, respectively, in benzphetamine demethylation activity by NADPH-dependent reconstituted P450/reductase complex, while modification of up to 8.5 mol of lysine/mol of P450 did not inhibit cumene hydroperoxide-supported P450-dependent benzphetamine demethylation. Acetylation of lysine residues by acetic anhydride does not grossly disturb the P450 protein conformation as revealed by absolute, CO-difference and fluorescence spectral studies. Modification of P4502B1 by acetic anhydride did not affect its substrate binding ability either. Lysine residues of P4502B1 putatively involved in the interaction with reductase have been identified by radiolabeling of lysine residues with [14C]acetic anhydride followed by trypsin digestion, HPLC separation, and amino acid microsequencing. Radiolabeled lysines occur at positions 251, 384, 422, 433, and 473. Modification of arginine residues in P4502B1 with phenylglyoxal and 2,3-butanedione seemed to have no significant effect on the benzphetamine demethylation activity of P4502B1 either reconstituted with reductase and NADPH or supported by cumene hydroperoxide. Studies of incorporation of [14C]phenylglyoxal showed no concentration- or time-dependent incorporation of phenylglyoxal into the P4502B1. These results support the hypothesis of a predominant role of lysine residues of P450 in the electrostatic interaction with NADPH-cytochrome P450 reductase.

Acetic Anhydrides↗
PubMed1989

Purification and characterization of hepatic steroid hydroxylases from untreated rainbow trout.

Purification of cytochrome P450 from liver microsomes of untreated juvenile male rainbow trout yielded five fractions designated LMC1 to LMC5. All fractions, except LMC4 and LMC5, appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed minimum molecular weights of 50,000 (LMC1), 54,000 (LMC2), 56,000 (LMC3), 58,000 (LMC4), and 59,000 (LMC5). Specific contents ranged from 2.8 (LMC3) to 14.9 (LMC5) nmol heme/mg protein. The catalytic activity of LMC1, LMC2, and LMC5 toward various substrates was examined. LMC2 exhibited the highest estradiol 2-hydroxylase activity and progesterone 16 alpha-hydroxylase activity. LMC2 also was most active in the metabolic activation of aflatoxin B1 (AFB1). In contrast, LMC5 was most active in catalyzing the 6 beta- and 16 beta-hydroxylation of testosterone and the 6 beta-hydroxylation of progesterone. LMC1 showed the highest lauric acid hydroxylase activity. The three isozymes tested had low activity (for LMC2 and LMC5) or no activity (for LMC1) toward benzphetamine or benzo[a]pyrene. Polyclonal antibodies to all five isozymes were raised in rabbits and the antibodies were used to examine the contribution of the P450s to microsomal enzyme activities. The results of microsomal enzyme inhibition studies with polyclonal antibodies showed that anti-LMC2 IgG significantly inhibited the oxidative metabolism of testosterone, lauric acid, AFB1, and benzphetamine. Anti-LMC5 IgG inhibited the oxidation of progesterone, estradiol, benzo[a]pyrene, and benzphetamine. Anti-LMC1 IgG slightly inhibited the microsomal hydroxylation of lauric acid. Anti-LMC3 and anti-LMC4 IgG did not inhibit any of the measured microsomal enzyme activities. These findings suggest that individual constitutive isozymes of trout cytochrome P450 have well-defined contributions to the microsomal metabolism of steroids, fatty acids, and xenobiotics.

Animals↗
PubMed1984

The interaction between two forms of cytochrome P-450 during drug oxidation in the reconstituted system containing limited amount of NADPH-cytochrome P-450 reductase.

The activities of drug oxidation in a reconstituted system which contains two forms of cytochrome P-450 and a limiting amount of NADPH-cytochrome P-450 reductase were determined. Cytochrome P-450 (termed MC P-4481 and MC P-4482) purified from liver microsomes of 3-methyl-cholanthrene-treated rats was active in both 2- and 4-hydroxylation of biphenyl but cytochrome P-450 (termed PB P-450) purified from liver microsomes of phenobarbital-treated rats was active in 4-hydroxylation of biphenyl only. PB P-450, MC P-4481 and MC P-4482 were most active toward benzphetamine N-demethylation, aniline hydroxylation and 7-ethoxycoumarin O-deethylation, respectively. PB P-450 inhibited the activity of biphenyl 2-hydroxylation supported by MC P-4481 or MC P-4482. On the contrary, no inhibition of PB P-450 supported benzphetamine N-demethylation was observed when MC P-4481 or MC P-4482 was added to the system containing PB P-450 and limited amount of the reductase. The apparent Km of PB P-450 for the reductase obtained from double reciprocal plot of the reductase concentration and the activity of biphenyl hydroxylase or benzphetamine N-demethylation was lower than that of MC P-4481 or MC P-4482. These and other results suggest that there is a certain hierarchy among the cytochrome P-450 species for receiving electrons from reductase.

Animals↗
PubMed1993

Picrotoxin as a potent inducer of rat hepatic cytochrome P450, CYP2B1 and CYP2B2.

The induction by the central stimulant picrotoxin of hepatic drug-metabolizing enzymes was studied in rats. The hepatic content of P450 and the activity of benzphetamine N-demethylation increased gradually after administration of picrotoxin dissolved in drinking water (2 mg/mL), to three-times higher levels than the initial values at the third day of treatment. The increase in benzphetamine N-demethylase activity by picrotoxin was somewhat higher than the increase produced by phenobarbital. Supporting these results, immunoblot analysis showed that CYP2B1 and 2B2 proteins in the liver microsomes were increased by picrotoxin Picrotoxinin and picrotin, which are components of the picrotoxin molecule, had the same ability to induce the hepatic activity of benzphetamine N-demethylation. The liver microsomal activities of testosterone 16 alpha- and 16 beta-hydroxylation were enhanced significantly after treatment with picrotoxinin and picrotin. However, benzo[a]pyrene 3-hydroxylation, aniline 4-hydroxylation, and testosterone hydroxylations at the 2 alpha- and 7 alpha-positions were not increased by picrotoxinin and picrotin treatment. In addition to monooxygenase, significant induction of glutathione S-transferase activity for 1-chloro-2,4-dinitrobenzene and UDP-glucuronyltransferase activity for 4-hydroxybiphenyl and 4-nitrophenol was also observed by pretreatment of picrotoxin. These results clearly indicate that picrotoxin is an inducer of phenobarbital-inducible liver enzymes.

Animals↗
PubMed1984

Sites of action of cadmium in vitro on hepatic, adrenal, and pulmonary microsomal monooxygenases in guinea pigs.

Preincubation of hepatic, adrenal, or pulmonary microsomal preparations with cadmium produced time-dependent decreases in monooxygenase (benzphetamine demethylase, benzo(a)pyrene hydroxylase) activities. Addition of cadmium after the preincubation period had little or no effect on microsomal metabolism. As a result of preincubation with cadmium, hepatic cytochrome P-450 levels declined and the magnitude of the benzphetamine-induced type I spectral change in hepatic microsomes decreased. Cadmium also decreased hepatic NADPH-cytochrome c and NADPH-cytochrome P-450 reductase activities but had no effect on NADH-cytochrome c reductase activity. Cadmium similarly decreased cytochrome P-450 concentrations and NADPH-cytochrome c reductase activity in lung microsomes without affecting NADH-cytochrome c reductase activity. Preincubation of adrenal microsomes with cadmium had no effects on cytochrome P-450 levels, on the benzphetamine-induced type I spectrum, or on NADH-cytochrome c reductase activity. However, decreases in adrenal NADPH-cytochrome c and NADPH-cytochrome P-450 reductase activities resulted which closely paralleled the decline in adrenal monooxygenase activities. EDTA extraction of hepatic, adrenal, or pulmonary microsomes after the preincubation exposure removed about 95% of the cadmium but did not diminish the effects of the metal on microsomal monooxygenases. The results indicate that cadmium has somewhat varying sites of action on hepatic, adrenal, and pulmonary monooxygenases, but in all three tissues electron transfer to cytochrome P-450 is compromised. In addition, the effects of cadmium on microsomal metabolism persist fully even after removal of approximately 95% of the metal.

Adrenal Glands↗
PubMed1988

Acute and subacute effects of miconazole nitrate on hepatic styrene oxide hydrolase and cytochrome P-450-dependent monooxygenase activities in male and female AKR/J mice.

The imidazole-containing anti-fungal drug, miconazole nitrate, was shown to enhance hepatic microsomal styrene oxide hydrolase and inhibit several cytochrome P-450-dependent monooxygenase activities in the AKR/J mouse. Miconazole was a more potent inhibitor of cytochrome P-450-dependent monooxygenase activities in microsomes from male than female mice, and inhibitory potency also varied with substrate. When administered in vivo miconazole nitrate stimulated epoxide hydrolase activity, but had a substrate-dependent biphasic effect on cytochrome P-450-dependent monooxygenase activities. Monooxygenase activities with benzo[a]pyrene and benzphetamine were inhibited to varying degrees in liver homogenate and hepatic microsomes from mice sacrificed 45 min after miconazole administration. After repeated administration of miconazole, liver weight, microsomal protein yield and cytochrome P-450 were increased, as were specific monooxygenase activities with ethoxycoumarin and ethoxyresorufin, but benzphetamine N-demethylase activity was decreased. These results suggested that a metabolite of miconazole was responsible for the inhibition of benzphetamine N-demethylase. It was of special interest that ethoxyresorufin O-deethylase activity was induced in the AKR/J mouse by miconazole, since the AKR/J mouse is not responsive to induction by aromatic hydrocarbons.

Animals↗
PubMed1983

The influence of in vitro procedures which diminish NADPH cytochrome c reductase activity on oxidative drug metabolism in lung and liver microsomes.

Rabbit lung and liver microsomes were subjected to three procedures which decreased NADPH cytochrome c reductase activity; flavoprotein antibody, trypsin and subtilisin digestion. The effects on benzphetamine and p-nitroanisole demethylation and amine metabolic-intermediate complex formation were investigated. In general, the proteolytic digestion had a greater inhibitory effect on oxidation reactions for a given loss of NADPH cytochrome c reductase activity than did flavoprotein antibody; and of the two proteases, subtilisin, which also diminishes the cytochrome b5 reduction pathway, had a greater inhibitory effect than trypsin. Subtilisin digestion had similar effects in both liver and lung microsomes; a loss of flavoprotein without a loss of cytochrome P-450; but whereas all three oxidative reactions decreased in unison as the flavoprotein was lost in the liver, benzphetamine demethylation was less susceptible to flavoprotein depletion than the other two reactions in lung microsomes. With trypsin digestion flavoprotein was removed without loss of cytochrome P-450 only in lung microsomes; in liver microsomes the cytochrome P-450 was susceptible to tryptic degradation. In lung microsomes, benzphetamine and p-nitroanisole demethylations were less susceptible to flavoprotein loss than metabolic-intermediate complex formation.

Animals↗
PubMed1996

Immunochemical and sub-structural characterization of sheep lung cytochrome P450LgM2.

Sheep lung cytochrome P450LgM2 belonging to gene subfamily 2B, was obtained in highly purified form and antibodies against sheep lung cytochrome P450LgM2 were produced in rabbits by using the previously developed methods in our laboratory. Immunological and enzymatic studies showed that antibodies against lung cytochrome P450LgM2 inhibited benzphetamine N-demethylation, ethylmorphine N-demethylation and aniline 4-hydroxylation reactions in sheep lung microsomes about 99, 80 and 62%, respectively. Benzphetamine N-demethylation reaction in sheep lung microsomes was only catalyzed by cytochrome P450LgM2 isozyme while the other isozymes of P450 as well as P450LgM2 are also involved in the metabolism of ethylmorphine and aniline. Similar to lung microsomes, benzphetamine N-demethylase activity of the reconstituted systems containing purified sheep lung cytochrome P450LgM2 or phenobarbital (PB)-treated rabbit liver cytochrome P450LM2(2B4) was also inhibited by P450LgM2 antibodies about 95 and 82%, respectively. A 50% inhibitory effect of sheep P450LgM2 antibodies was also observed in ethylmorphine N-demethylase activity of the reconstituted system containing purified sheep lung cytochrome P450LgM2. SDS-PAGE peptide maps obtained following the partial proteolysis of purified sheep lung cytochrome P450LgM2 and PB-rabbit liver P450LM2(2B4) isozymes, using chymotrypsin and papain, were similar in general. However they showed some differences both qualitatively and quantitatively, suggesting that some differences exist among the amino acid sequences of sheep lung cytochrome P450LgM2 and rabbit liver cytochrome P4502B4.

Animals↗
PubMed1985

Differential haemin-mediated restoration of cytochrome P-450 N-demethylases after inactivation by allylisopropylacetamide.

Administration of allylisopropylacetamide (AIA) to phenobarbital-pretreated rats results in the destruction of several phenobarbital-inducible cytochrome P-450 isoenzymes and a correspondingly marked loss of benzphetamine N-demethylase and ethylmorphine N-demethylase activities. Accordingly, the ion-exchange h.p.l.c. or DEAE-cellulose-chromatographic profile of solubilized microsomal preparations from such rats revealed a marked decrease in the cytochrome P-450 content of several eluted fractions compared with that of microsomes from corresponding non-AIA-treated controls. Incubation of liver homogenates from such rats with haemin restores not only cytochrome P-450 content from 35 to 62% of original values, but also benzphetamine N-demethylase and ethylmorphine N-demethylase activities, from 23 to 67%, and from 12 to 36% of original values respectively. Moreover, the chromatographic profiles of microsomes prepared from such homogenates indicated increases of cytochrome P-450 content only in some fractions. Reconstitution of mixed-function oxidase activity of cytochrome P-450 by addition of NADPH: cytochrome P-450 reductase to these fractions indicated that incubation with haemin restored benzphetamine N-demethylase activity predominantly, but ethylmorphine N-demethylase activity only minimally. After injection of [14C]AIA, a significant amount of radiolabel was found covalently bound to protein in chromatographic fraction III, and this binding was unaffected by incubation with haemin. Furthermore, the extent of this binding is apparently equimolar to the amount of cytochrome P-450 refractory to haemin reconstitution in that particular fraction. Whether such refractoriness reflects structural inactivation of the apo-cytochrome remains to be determined. Nevertheless, the evidence presented very strongly argues for AIA-mediated inactivation of multiple phenobarbital-induced isoenzymes, only a few of which are structurally and functionally reparable by haemin.

Acetamides↗
PubMed1978

Rapid conformational changes of cytochrome P-450: effect of dimyristoyl lecithin.

Binding of benzphetamine to purified microsomal cytochrome P-450 from rat liver causes a shift in the heme spin state of the protein to favor the high-spin form. This shift is strongly temperature dependent. A rapid temperature jump of a cytochrome P-450/epsilon benzphetamine mixture, monitored by changes in the Soret absorptions of the heme, reveals two relaxation processes: one in a 50-msec time range (tau(f)) and the other in a 0.3-sec time range (tau(s)). Both relaxations reflect conformational changes of the protein after the substrate binding. No bimolecular reaction of benzphetamine and the enzyme has been resolved. This indicates that there is no absorption change of the heme associated with the initial binding. In the presence of dimyristoyl lecithin, at 25 degrees C tau(f) decreases by nearly one order of magnitude whereas tau(s) decreases to one-third. The enhancement of rates by added phospholipid is both temperature- and concentration-dependent: rates are accelerated only above the gel-liquid crystalline transition temperature, and this effect saturates near the enzyme/lipid ratio of 1:20. In contrast, the lipid does not have significant effect on the equilibrium binding curve of the substrate. These results suggest that the lipid may form an envelope around the enzyme and, depending on its crystalline state, regulates the rate of the substrate-induced conformational changes of cytochrome P-450.

Animals↗
PubMed1981

Alterations in hepatic mixed-function oxidase activity of rainbow trout after acute treatment with polybrominated biphenyl isomers and FireMaster BP-6.

The effects of FireMaster BP-6 and several pure isomers on hepatic microsomal mixed-function oxidase (MFO) activity in rainbow trout have been investigated. After one parenteral injection of these agents at 150 mg/kg and sacrifice 5 d later, there was no elevation of hepatic cytochrome P-450. The noncoplanar isomers 2,4,5,2',4',5'-hexa and 2,3,4,5,2',4',5'-heptabromobiphenyl did not increase any MFO activity generally associated with an increase in cytochrome P-450 levels, including the dealkylations of ethoxycoumarin and benzphetamine. The coplanar isomer 3,4,5,3',4',5'-hexabromobiphenyl produced an increase in O-deethylation of both ethoxycoumarin and ethoxyresorufin, suggesting enhancement of cytochrome P-448 associated activity, but produced no increase in benzphetamine N-dealkylation. The "mixed inducer" 2,4,5,3',4',5'-hexabromobiphenyl, produced a small but insignificant elevation of cytochrome P-448 associated MFO activities and no increase in cytochrome P-450 associated activities. At both 150 and 500 mg/kg, the commercial mixture of polybrominated biphenyls FireMaster BP-6 produced marked elevations of both ethoxycoumarin and ethoxy-resorufin dealkylations but had no effect on demethylation of benzphetamine. When microsomes from rainbow trout treated with FireMaster BP-6 were examined electrophoretically, A coomassie Blue-staining band at 57,000 daltons was intensified, as seen after treatment of fish with the cytochrome P-448 inducer beta-naphthoflavone. It is concluded that coplanar polybrominated isomers produce an induction of hepatic cytochrome P-448 associated activity, while noncoplanar isomers are ineffective as inducers of cytochrome P-450 related MFO activities in rainbow trout.

Animals↗
PubMed1995

Ketone potentiation of haloalkane-induced hepato- and nephrotoxicity. II. Implication of monooxygenases.

Previous results in Sprague-Dawley rats indicate that acetone (A), methyl ethyl ketone (MEK), and methyl isobutyl ketone (MiBK) pretreatment (3 d, po) at dosages of 6.8 and 13.6 mmol/kg potentiate CCl4 hepatotoxicity and CHCl3 nephrotoxicity, respectively. The potentiation potency profile observed was MiBK > A > MEK for liver and A > MEK > or = MiBK for kidney toxicity (Raymond & Plaa, 1995). In the present study, hepatic and renal microsomes from A-, MEK-, and MiBK-pretreated rats (6.8 or 13.6 mmol/kg) were examined for cytochrome P-450 content, substrate-specific monooxygenase activity (aminopyrine and benzphetamine N-demethylase, aniline hydroxylase) and in vitro covalent binding of 14CHCl3 and 14CCl4. Of the three ketones, only MiBK significantly increased P-450 content of liver and renal cortical microsomes. Similarly, 14CCl4 covalent binding under aerobic and anaerobic conditions was significantly increased by MiBK pretreatment only. 14CHCl3 covalent binding by renal cortical microsomes was significantly increased only under aerobic conditions by MiBK pretreatment. MiBK (13.6 mmol/kg) increased (threefold) aminopyrine N-demethylation in both liver and kidney, but only benzphetamine N-demethylation (two-fold, at 6.8 and 13.6 mmol/kg) in liver; A and MEK had no effect on either monooxygenase. All ketones at dosages of 6.8 and 13.6 mmol/kg increased aniline hydroxylation in liver (two-fold) and kidney (fivefold). Comparable profiles for P-450 induction, haloalkane covalent binding, and aminopyrine or benzphetamine N-demethylase activity were observed in liver and kidney microsomes. This profile was consistent with the ketone potentiation potency ranking profile observed in vivo for liver but not kidney injury. These findings affirm the importance of ketone-enhanced bioactivation for potentiation of CCl4 hepatotoxicity but suggest an alternative mechanism for CHCl3 nephrotoxicity.

Acetone↗