Influence of estradiol and testosterone on cytochrome P-450 and monooxygenase activity in immature brook trout,Salvelinus fontinalis

Levels of hepatic microsomal cytochrome P-450 were depressed by administration of estradiol-17β and were elevated by administration of testosterone in both male and female juvenile brook trout (Salvelinus fontinalis). Treatment-associated changes in the levels of other microsomal electron transfer components in liver did not reflect the changes in cytochrome P-450 content and were also distinct from the changes in these components in kidney. Electrophoretic analysis of hepatic microsomes revealed that estradiol treatment reduced the amounts of several proteins including some heme-staining protein at 56,000 daltons, possibly containing cytochrome P-450. Hepatic microsomal benzo[a]pyrene hydroxylase and the response to 7,8-benzoflavone in vitro were affected little by steroid treatment, and ethoxyresorufin O-deethylase activity could not be detected in any of the samples. Hepatic microsomes metabolized testosterone to a suite of products including 6β-hydroxytestosterone (the major metabolite) and 16β-hydroxytestosterone, plus as many as eleven unknown metabolites. Estradiol-17β treatment depressed the rates of testosterone metabolism and particularly the rates of 6β-hydroxylase activity but did not affect 16β-hydroxylase activity. Both activities were largely unaffected by testosterone. The results are consistent with the idea that both androgens and estrogens regulate the levels of hepatic cytochrome P-450 in brook trout and that the effect, at least of estradiol-17β, involves regulation of forms that function in specific hydroxylation of testosterone. The significance of these effects and whether factors additional to steroids are involved in this regulation of hepatic cytochromes P-450 in fish remain to be established.     

Further investigations of the metabolism of fluroxene and the degradation of cytochromes P-450 in vitro.

The effects of inhibitors and of inducing agents for cytochromes P-450 on the fluroxene mediated destruction of cytochromes P-450 were investigated with hepatic microsomes from male rats in vitro and compared with the metabolism of fluroxene (2,2,2-trifluoroethyl vinyl ether) to 2,2,2-tri-fluoroethanol under similar conditions. The fluroxene mediated destruction of cytochromes P-450 and the metabolism of fluroxene are fully inhibited under totally anaerobic conditions. Carbon monoxide, SKF 525A and metyrapone fully inhibit the fluroxene mediated destruction of cytochromes P-450 and partially inhibit the metabolism of fluroxene to trifluoroethanol in microsomes from phenobarbital pretreated rats. The K_m values for the destruction of cytochromes P-450 by fluroxene in vitro were calculated as 0.8, 3.3 and 1.5 mM for microsomes from phenobarbital induced, 3-methylcholanthrene induced and uninduced animals, respectively. V_max values for 3-methylcholanthrene and phenobarbital induced microsomes (approximately 0.5 nmol cytochromes P-450 destroyed/mg microsomal protein/7 min) are elevated compared to uninduced microsomes (0.2 nmol cytochromes P-450 destroyed/mg microsomal protein/10 min). The K_m value for the metabolism of fluroxene to trifluoroethanol in control microsomes of approximately 1.0 mM is unchanged following induction, and V_max for the production of trifluoroethanol is increased relative to controls only in phenobarbital induced microsomes. It is concluded that the fluroxene mediated destruction of cytochromes P-450 appears to involve both cytochrome P-448 and cytochrome P-450 whereas the production of trifluoroethanol from fluroxene is catalyzed by cytochrome P-450 but not by cytochrome P-448.     

Tetrachloroethylene metabolism by the hepatic microsomal cytochrome P-450 system

The interaction of tetrachloroethylene with hepatic microsomal cytochromes P-450 has been investigated using male Long-Evans rats. The spectral binding of tetrachloroethylene to cytochromes P-450 in hepatic microsomes from uninduced rats was characterized by a K_s of 0.4 mM. The K_s was not affected by phenobarbital induction, but was increased following pregnenolone-16α-carbonitrile induction. The K_M of 1.1 mM, calculated for the conversion of tetrachloroethylene to total chlorinated metabolites by the hepatic microsomal cytochrome P-450 system, was decreased by phenobarbital induction and increased by pregnenolone-16α-carbonitrile induction. The maximum extents of binding (ΔA_max) and metabolism (V_max) of tetrachloroethylene were increased by both phenobarbital and pregnenolone-16α-carbonitrile induction. Induction with β-naphthoflavone was without effect on any of the above parameters. The effects of the inducing agents on tetrachloroethylene-stimulated CO-inhibitable hepatic microsomal NADPH oxidation followed the same trend as their effects on V_max for the metabolism of tetrachloroethylene, although in all cases the extent of NADPH oxidation was 5- to 25-fold greater than the extent of metabolite production. The inhibitors of cytochromes P-450, viz. metyrapone, SKF 525-A, and CO, inhibited the hepatic microsomal binding and metabolism of tetrachloroethylene. Free trichloroacetic acid was found to be the major metabolite of tetrachloroethylene from the hepatic microsomal cytochrome P-450 system. Neither 2.2,2-trichloroethanol nor chloral hydrate was produced in measurable amounts from tetrachloroethylene. A minor but significant metabolite of tetrachloroethylene by cytochrome P-450 was the trichloroacetyl moiety covalently bound to components of the hepatic microsomes. Incubation of tetrachloroethylene. an NADPH-generating system. EDTA and hepatic microsomes was without effect on the levels of microsomal cytochromes P-450, cytochrome b₅, beme, and NADPH-cytochrome c reductase. It is concluded that hepatic microsomal cytochromes P-450 bind and metabolize tetrachloroethylene. The major product of this interaction is trichloroacetic acid, which is also the major urinary metabolite of tetrachloroethylene in vivo. The forms of cytochrome P-450 that bind and metabolize tetrachloroethylene include those induced by pregnenolone-16α-carbonitrile and by phenobarbital. Cytochrome P-448. which was induced in rat liver by β-naphthoflavone, does not appear to spectrally bind or metabolize tetrachloroethylene. The metabolism and toxicity of tetrachloroethylene are considered in relation to other chlorinated ethylenes.     

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.     

Isozyme selectivity of the inhibition of rat liver cytochromes P-450 by chloramphenicol in vivo

The isozyme-selectivity of chloramphenicol as an inhibitor of rat liver cytochromes P-450 has been investigated. Untreated rats and rats treated with the inducers phenobarbital, beta-naphthoflavone, pregnenolone 16 alpha-carbonitrile, and clofibrate have been injected intraperitoneally with chloramphenicol, and inhibition of specific cytochrome P-450 isozymes has been assessed by monitoring the metabolism of warfarin, testosterone, isosafrole, or lauric acid in subsequently prepared hepatic microsomal preparations. Of eight major cytochrome P-450 isozymes which could be monitored in this fashion, three were inhibited by more than 50% by a dose of chloramphenicol of 300 mg/kg, whereas no evidence of inhibition of the remaining isozymes was obtained. P-450PB-C, an isozyme which is present in significant amounts in untreated rats and which is induced approximately 2-fold by phenobarbital, was the most susceptible cytochrome P-450 to inhibition by chloramphenicol both in vivo and in vitro. P-450PB-B, the major phenobarbital-inducible isozyme, and P-450UT-A, a male-specific testosterone 2 alpha- and 16 alpha-hydroxylase, were intermediate in their susceptibility to chloramphenicol. In contrast, the major isozymes induced by beta-naphthoflavone, pregnenolone 16 alpha-carbonitrile, and clofibrate, as well as a constitutive testosterone 7 alpha-hydroxylase, were not inhibited by chloramphenicol.     

Influence of chlordecone and mirex exposure on benzo[a]pyrene metabolism of rat-liver microsomes

The metabolism of benzo[a]pyrene (BP) in hepatic microsomes isolated from rats exposed to chlordecone or mirex was compared to that of untreated rats and rats treated with 3-methylcholanthrene (3-MC) or phenobarbital (PB). Treatment with chlordecone resulted in a two- to three-fold increase in cytochrome P-450 content but the BP-hydroxylase activity per mg microsomal protein was unaffected. Addition of alpha-naphthoflavone (alpha-NF) or chlordecone caused changes in BP-hydroxylase activity indicating that chlordecone-induced cytochromes P-450 were similar to control. H.p.l.c. analyses of BP metabolites confirmed this similarity. Treatment with mirex caused a two-fold induction of cytochrome P-450, and BP-hydroxylase activity expressed per mg microsomal protein was increased 1.3-fold. Addition of chlordecone or alpha-NF caused changes in BP-hydroxylase activity, indicating differences between control and mirex-induced cytochromes P-450. H.p.l.c. analyses of BP metabolites confirmed this difference. Treatment with chlordecone or mirex increased microsomal epoxide hydrolase activity three-fold. Chlordecone accumulated in hepatic nuclei.     

Inactivation of rat hepatic cytochrome P-450 isozymes by 3,5-dicarbethoxy- 2,6-dimethyl-4-ethyl-1,4-dihydropyridine

We have reported [Correia et al. (1987) Arch. Biochem. Biophys. 258, 436-443] that administration of 3,5-dicarbethoxy-4-ethyl-2,6-dimethyl-1,4-dihydropyridine (DDEP) to untreated, phenobarbital (PB) pretreated, or dexamethasone (DEX) pretreated rats results in relatively selective inactivation of cytochrome P-450 (P-450) isozymes h (CYP2C11), k (CYP2C6), and p (CYP3A). Such inactivation involves destruction of P-450 prosthetic heme predominantly by N-ethylation in untreated and PB-pretreated rats, whereas in DEX-pretreated rats, it also appears to be associated with prosthetic heme alkylation of the apocytochrome presumably at the active site. The cause for this differential course of DDEP-mediated P-450 heme destruction is unclear. Since this process is absolutely dependent on NADPH-mediated DDEP metabolism and can be reproduced in vitro, in search of mechanistic clues, we have examined DDEP metabolism by liver microsomes from the three rat sources as well as by isolated purified rat liver P-450h and P-450k. HPLC analyses of microsomal incubations of DDEP with NADPH, in the presence of an esterase inhibitor, revealed the presence of two major products: deethylated pyridine (DP) and 4-ethylpyridine (4-EDP) with product ratios (DP/4-EDP) of 1.4, 1.4, and 0.7 for reactions catalyzed by liver microsomes from untreated, PB-pretreated, and DEX-pretreated rats, respectively. The corresponding mean product ratios for P-450h- and P-450k-catalyzed reactions were 4.2 and 5.5, respectively. On the other hand, partition ratios (DP formed/P-450 destroyed) ranged from 12.0, 10.5, and 4.8, respectively, for incubations of liver microsomes from untreated, PB-pretreated, and DEX-pretreated rats to 9.5 and 28.9 for purified P-450h- and P-450k-catalyzed reactions, respectively. However, DP formation in all these microsomal systems was comparable, and although 4-EDP formation was greatly stimulated by DEX pretreatment, it does not appear to be a destructive pathway. In view of this, our findings reported herein suggest that the active site environment of P-450's h, k, and p apparently determines not only the pattern of DDEP metabolism but also the differential course of prosthetic heme destruction.     

Reversible inhibition of aromatic hydroxylation of methamphetamine in rat liver microsomal preparations pretreated with methamphetamine

The effects of a single and repeated administration of methamphetamine (MP) in vivo in rats on its own metabolism in vitro were investigated. In both cases, the p-hydroxylation of MP to p-hydroxymethamphetamine by a microsomal fraction from rat liver was inhibited for a period of 16 hr after the last injection of MP. This inhibition was diminished by dialysis of the microsomal preparations. In contrast, the reduced level of cytochrome P-450 in hepatic microsomes from rats pretreated with the SKF 525-A did not revert to the control value after dialysis. When microsomes were preincubated with N-hydroxymethamphetamine, which is the metabolite of MP and a potent substrate for the formation of a metabolic intermediate (MI) complex with cytochrome P-450, the content of the MI was increased and the MP-hydroxylation activity decreased in direct proportion to the length of the preincubation. These results suggest that the inhibition of MP-hydroxylation may be due to reduction of the level of cytochrome P-450 that accompanies the formation of the MI complex. Furthermore, it appears that the complex can be dissociated by dialysis.     

Effect of cytochrome P-450 and flavin-containing monooxygenase modifying factors on the in vitro metabolism of amiodarone by rat and rabbit

Experiments were conducted to affirm hepatic cytochrome P-450 involvement in the biotransformation of the class III antiarrhythmic agent, amiodarone (Am; Cordarone X) to its major metabolite, desethylamiodarone (DEA). Male Sprague-Dawley rats and male New Zealand white rabbits were treated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) (to induce cytochrome P-450 (PB-inducible cytochrome(s) P-450) or P-448 (MC-inducible cytochrome P-450). In vivo decreases in rat hepatic microsomal cytochrome P-450 were achieved either by a single ip dose of CCl4 or by a 2-day treatment with CoCl2. In vitro biotransformation of Am by hepatic microsomes from PB-induced and 3-MC-induced rats and PB-induced rabbits was significantly greater than that from noninduced animals. Conversely, in vitro DEA production was significantly decreased with hepatic microsomes from CCl4- and CoCl2-pretreated rats. The classic P-450 inhibitors, piperonyl butoxide, SKF 525A, n-octylamine, and CO provided a significant reduction in the in vitro formation of DEA by microsomes from induced animals. In vitro DEA formation by hepatic microsomes from PB- and 3-MC-induced rats was significantly decreased by 0.5 mM chloroquine (specific inhibitors of PB-inducible cytochrome(s) P-450) and 0.3 mM quinacrine (specific inhibitor of MC-inducible cytochrome(s) P-450), respectively. Further evidence for involvement of gut microsomal flavin-containing monooxygenase was provided by the inhibition of gut microsomal-mediated in vitro DEA formation in the presence of methimazole. Methimazole had no effect on hepatic microsomal DEA production in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the metabolism of N-nitrosodimethylamine and carbon tetrachloride by a common isozyme of cytochrome P-450

Carbon tetrachloride administration to rats produced a selective loss of hepatic cytochrome P-450-dependent catalytic activities. Of the cytochrome P-450-dependent catalytic activities tested, the metabolism of carbon tetrachloride to phosgene and the low Km N-nitrosodimethylamine demethylase were the most sensitive to destruction by carbon tetrachloride. A 50% or greater loss in these catalytic activities was observed 3 hr after giving 10 microliters carbon tetrachloride/kg. Related catalytic activities, such as the microsomal metabolism of carbon tetrachloride to chloroform and the high Km N-nitrosodimethylamine demethylase, were diminished less than 20% 3 hr after giving 10 microliters carbon tetrachloride/kg. To investigate further the relationship between the metabolism of N-nitrosodimethylamine and carbon tetrachloride, the effect of pyrazole, a known inducer of the low Km N-nitrosodimethylamine demethylase, on carbon tetrachloride metabolism was studied. Pyrazole treatment produced a 5.6-fold increase in the microsomal metabolism of carbon tetrachloride to phosgene and a 1.9-fold increase in the conversion of carbon tetrachloride to chloroform. The similarities between both the loss and the induction of the low Km N-nitrosodimethylamine demethylase and the metabolism of carbon tetrachloride to phosgene suggest that these catalytic activities represent a common isozyme of cytochrome P-450. Analysis of cytochromes P-450 by HPLC provided evidence for an isozyme of cytochrome P-450 inducible by pyrazole and destroyed by carbon tetrachloride.     

Identification of the cyanopregnenolone-inducible form of hepatic cytochrome P-450 as a catalyst of aldrin epoxidation

In light of recent suggestions that hepatic microsomal aldrin expoxidation activity selectively reflects the phenobarbital (PB)-inducible form(s) of cytochrome P-450 (P-450PB), we tested the effect of pregnenolone-16 alpha-carbonitrile (PCN), a synthetic steroid that induces P-450PCN, a form of the cytochrome biochemically and immunochemically distinguishable from P-450PB. In hepatic microsomes prepared from rats receiving PB, 3-methylcholanthrene (3-MC), or PCN, the latter compound produced a greater increase in aldrin epoxidation activity relative to control than did PB, whereas 3-MC decreased enzyme activity. Moreover, the aldrin epoxidation activity in microsomes prepared from PCN- or PB-pretreated rats was selectively inhibited by form-specific antibodies directed against P-450PCN or P-450PB, respectively, whereas anti-P-450MC antibodies gave no inhibition with microsomes prepared from induced or control animals. We conclude that P-450PCN, P-450PB, and probably other cytochromes P-450 catalyze aldrin epoxidation, precluding use of this enzyme as a specific marker of a single form of the cytochrome.     

Species differences in the hepatic formation of green pigments following the administration of norethindrone

Metabolic activation of the ethynyl substituent of the contraceptive steroid norethindrone to cause the loss of hepatic cytochrome P-450 and the formation of green pigments has been compared in vivo and in vitro in rat, hamster, guinea pig, rabbit, mouse and hen and with marmoset and human liver microsomal preparations in vitro. In vivo green pigment accumulation in the liver 4 hr after the administration of norethindrone (100 mg/kg, i.p.) varied 60-fold between species. Male rat was the most active in this respect, the hen was the least active. The accumulation of green pigments in female rats was 27% that of male animals. This sex-dependent difference was not seen in male and female mice. Cytochrome P-450 destruction in vivo was also greatest in the male rat given norethindrone, whereas no loss was detected in the hen. In other species, however, the correlation between green pigment accumulation and cytochrome P-450 destruction was not particularly good. When liver microsomes were incubated with norethindrone and an NADPH generating system in vitro, the ranking order between species with respect to the initial rates of green pigment formation was similar to that based on the hepatic accumulation of these compounds found in vivo. Human liver microsomes showed initial rates of green pigment formation which were only 2% of that seen in the male rat. No destruction of human microsomal cytochrome P-450 caused by norethindrone could be detected. The HPLC elution profile of the green pigments produced in the liver following the administration of norethindrone differed between species. Hepatic microsomal preparations in contrast, at least with short incubation times, formed only one green pigment. Results suggest that further metabolism of either norethindrone or the green pigment, involving a cytosolic factor(s), results in the varied HPLC patterns seen in vivo.     

Comparison of the effects of methyl-N-butyl ketone and phenobarbital on rat liver cytochromes P-450 and the metabolism of chloroform to phosgene

It was previously shown that treatment of rats with methyl-n-butyl ketone (MBK) produced an increase in the total level of liver microsomal cytochromes P-450 and an increase in the rate of metabolism of chloroform (CHCl3) to phosgene (COCl2). In the present study it was found that MBK also produced qualitative changes in the composition of microsomal cytochromes P-450 in rat liver as determined by anion-exchange chromatography. The degree of the chromatographic changes paralleled the effect of MBK on the rate of metabolism of CHCl3 to COCl2 and CHCl3-induced hepatotoxicity, suggesting that MBK potentiated the hepatotoxicity of CHCl3, at least in part, by inducing the formation of cytochromes P-450 that metabolized CHCl3 to the hepatotoxin COCl2. In this regard, reconstitution studies with a form of cytochrome P-450 isolated from rat liver microsomes from rats treated with MBK or phenobarbital (Pb) showed unequivocally that cytochrome P-450 can metabolize CHCl3 to COCl2. Although analysis of rat liver microsomes by SDS-polyacrylamide electrophoresis and anion-exchange chromatography suggested that MBK and Pb had similar effects on the composition of cytochromes P-450, metabolism studies indicated that differences did exist.     

Enhancement of hepatic microsomal drug metabolism in vitro following ethanol administration.

1. Administration of ethanol intraperitoneally at low dosages (10-25 mg/kg) to rats stimulates hepatic microsomal mixed-function oxidase activity in vitro. 2. Pretreatment with ethanol administered orally has no effect on in vivo drug metabolism as measured by pentobarbitone plasma half-life and has no effect on the excretion of ascorbic acid. Ethanol administration does not enhance its own binding to cytochrome P-450. 3. These observations suggest that the administration of ethanol, at moderate dosage, does not give rise to induction of hepatic cytochrome P-450. 4. Unwashed hepatic microsomes are contaminated with alcohol dehydrogenase, but pretreatment with ethanol does not increase microsomal generation of NADH. 5. Pretreatment with ethanol has no stimulatory effect on NADH-NADP+ transhydrogenation. 6. The stimulation of hepatic drug metabolism in vitro following administration of ethanol is not due to increased cytochrome P-450 nor to increased NADPH, per se, but appears to result from an increase in the activity of NADPH-cytochrome c reductase.     

Induction of rat hepatic microsomal cytochrome P-450 by 2,3',4,4',5,5'-hexachlorobiphenyl

The following evidence suggests that 2,3',4,4',5,5'-hexachlorobiphenyl resembles isosafrole as an inducer of hepatic microsomal cytochrome P-450d in the immature male Wistar rat. First, the major hepatic microsomal polypeptide (Mr = 52,000), intensified after treatment of rats with 2,3',4,4',5,5'-hexachlorobiphenyl, comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with cytochrome P-450d (i.e. the major isosafrole-inducible polypeptide) but had an electrophoretic mobility intermediate between cytochrome P-450b (Mr approximately equal to 51,500) and cytochrome P-450c (Mr = 56,000) (i.e. the major phenobarbital- and 3-methylcholanthrene-inducible polypeptides respectively). Second, when pairs of various xenobiotics were coadministered to rats at doses effecting maximal induction of hepatic microsomal cytochrome P-450, the inductive effects of 2,3',4,4',5,5'-hexachlorobiphenyl were additive with those of phenobarbital, 3-methylcholanthrene and pregnenolone-16 alpha-carbonitrile but not with those of isosafrole. The inductive effects of phenobarbital, 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile and isosafrole were all expressed additively with each other. Third, in contrast to phenobarbital and pregnenolone-16 alpha-carbonitrile treatment, treatment of rats with 2,3',4,4',5,5'-hexachlorobiphenyl, isosafrole or 3-methylcholanthrene failed to increase markedly the proportion of total cytochrome P-450 capable of forming a 446 nm-absorbing complex with metyrapone. Fourth, the in vitro metabolism of isosafrole, catalyzed by hepatic microsomes from rats treated with 2,3',4,4',5,5'-hexachlorobiphenyl, isosafrole or 3-methylcholanthrene, produced complexes between ferrous cytochrome P-450 and a methylenedioxyphenyl metabolite, the spectra of which were between 400 and 500 nm and were similar to each other but which were readily distinguishable from the spectra of the product adducts formed during the metabolism of isosafrole by hepatic microsomes from rats treated with corn oil (control), phenobarbital, or pregnenolone-16 alpha-carbonitrile.     

Growth hormone modulation of liver drug metabolic enzyme activity in the rat—I: Effect of the hormone on the content and rate of reduction of microsomal cytochrome P-450

The liver metabolism of hexobarbital and aniline was decreased 48 hr after the first injection of growth hormone (GH) in adult male rats. The content and rate of reduction of hepatic microsomal cytochrome P-450 were lowered in these rats as compared with control animals. Liver NADPH-cytochrome c reductase showed a similar decrease in activity after GH treatment. The decrease in hexobarbital metabolism paralleled the change in cytochrome P-450 reductase activity as measured with or without addition of this drug substrate to a suspension of liver microsomes from GH-treated rats. The change in aniline metabolism approximated the extent and rate of cytochrome P-450 reduction after GH treatment only when cytochrome P-450 reductase activity was measured without addition of aniline. Injection of GH produced a parallel decrease in the metabolism of both drugs as compared with cytochrome c reductase activity. Differences in optimal requirements for drug substrates (hexobarbital or aniline) or NADPH for cytochrome P-450 reductase were not detected. Preincubation studies showed no differences in microsomal drug metabolic enzyme system stability in rats injected with GH. Inhibitors of this system in vitro were not demonstrated in liver from GH-treated rats. GH is presumed to affect the level of liver drug metabolism through mechanisms in vivo operative at the first stage transfer of reducing equivalents to cytochrome P-450. An additional effect of this hormone on the level or catalytic properties of the hemoprotein cytochrome P-450 may contribute to the decrease in aniline metabolism.     

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.     

Inhibition of hepatic microsomal drug metabolism by the steroid hydroxylase inhibitor SU-10'603

SU-10'603 is a pyridine derivative that has been widely used as a steroid 17-hydroxylase inhibitor. Studies were done to compare the effects of SU-10'603 with those of the structurally related compound, metyrapone, on hepatic microsomal drug metabolism in vitro in rats and guinea pigs. In rat liver microsomes, SU-10'603 produced a concentration-dependent (0.01 to 1.0 mM) inhibition of ethylmorphine demethylation, aniline hydroxylation, and benzo[a]pyrene hydroxylation. A concentration of 0.1 to 0.2 mM decreased the metabolism of all three substrates by approximately 50%. SU-10'603 was a more potent inhibitor of ethylmorphine metabolism than metyrapone, and its relative potency was even greater with respect to aniline and benzo[a]pyrene metabolism. Similar results were obtained with guinea pig liver microsomes. SU-10'603 and metyrapone produced type II spectral changes in hepatic microsomes, but the apparent affinity of SU-10'603 for cytochrome(s) P-450 was greater than that of metyrapone. Both compounds inhibited the binding of type I substrates to microsomal cytochromes P-450; SU-10'603 was the more potent inhibitor. The results indicate that SU-10'603 is a potent inhibitor of hepatic microsomal monooxygenases whose mechanism of action is similar to that of metyrapone.     

Role of 3,5-dichlorophenyl methyl sulfone, a metabolite of m-dichlorobenzene, in the induction of hepatic microsomal drug-metabolizing enzymes by m-dichlorobenzene in rats

The increases in the hepatic microsomal aminopyrine N-demethylase activity and in the content of cytochrome P-450 produced by m-dichlorobenzene (m-DCB) occurred after increases in the hepatic concentration of 3,5-dichlorophenyl methyl sulfone, a minor metabolite. The extent of increases in aminopyrine N-demethylase activity and in the content of cytochrome P-450 at 48 hr after po administration of 200 mg/kg (1.36 mmol/kg) of m-DCB was almost equal to that 72 hr after the ip administration of 25 mumol/kg of the sulfone (Kimura et al., 1983). m-DCB in liver was not detectable at that time, and the concentration of sulfone was 63 to 70% of that 48 to 72 hr after the ip administration of 50 mumol/kg of sulfone. Administration of m-DCB (200 mg/kg) produced a significant reduction in hexobarbital sleeping time, but this reduction was less than that produced by administration of the sulfone (50 mumol/kg). The protein band patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the microsomes from rats treated with the sulfone and m-DCB were similar to those of phenobarbital-treated rats but were different from those of 3-methylcholanthrene-treated rats. The sulfone showed type I interaction with the cytochrome P-450 (Ks, 0.17 mM). The sulfone was formed from the sulfide but reduction of the sulfone was not observed when it was incubated in a hepatic microsomal preparation. The pattern of induction by the sulfone and m-DCB was similar to that by phenobarbital and differed from that by 3-methylcholanthrene. From these results, 3,5-dichlorophenyl methyl sulfone is considered to be a major contributing factor of the inducing activity of m-DCB and to be a potent phenobarbital-like inducer.     

Hypophysectomy differentially alters P-450 protein levels and enzyme activities in rat liver: pituitary control of hepatic NADPH cytochrome P-450 reductase

Pituitary-determined hormones regulate the expression of hepatic cytochromes P-450 through processes involving both negative and positive controls. Accordingly, protein levels of several P-450 forms are elevated in rat liver following hypophysectomy [P-450 forms designated 2a (gene IIIA2), RLM2 (gene IIA2), and PB-4 (gene IIB1)], whereas protein levels of others are suppressed [e.g., P-450 2c (gene IIC11)]. In the present study, microsomal steroid hydroxylase activities associated with these same P-450 forms were found to be decreased by hypophysectomy, despite elevations in protein levels for several of them. Studies were, therefore, undertaken to determine the biochemical basis for this decrease in microsomal P-450 enzyme specific activity. In vivo treatment of hypophysectomized rats with gonadotropin, under conditions that restore heme to testis P-450, and heme reconstitution experiments carried out with liver homogenates indicated that a deficiency in P-450-associated heme is unlikely to account for the observed decreases in liver P-450 enzyme specific activity. Analysis of the flavoprotein P-450 reductase, however, revealed that the reductase protein and its associated cytochrome c reductase activity are decreased by 50 to 75% in liver microsomes isolated from hypophysectomized rats. Moreover, supplementation of isolated liver microsomes with exogenous purified P-450 reductase stimulated microsomal steroid hydroxylase activity preferentially in the hypophysectomized rats, to levels consistent with the observed changes in P-450 protein levels. Thus, a deficiency in P-450 reductase, which is a rate-limiting component for many P-450-dependent hydroxylation reactions, appears to be responsible for the decrease in steroid hydroxylase specific activity in the hypophysectomized rats. Although growth hormone, adrenocorticotropic hormone, and chorionic gonadotropin were each ineffective at restoring hepatic P-450 reductase when administered to hypophysectomized rats, substantial restoration of P-450 reductase levels could be achieved by treatment of the hypophysectomized rats with thyroxine. Thyroxine treatment of these rats also elevated the microsomal steroid hydroxylase activities associated with the individual hepatic P-450 forms to levels commensurate with their respective P-450 protein levels. These results establish that hepatic P-450 reductase is subject to hormonal controls that are distinct from those governing cytochrome P-450 expression and further demonstrate the complexity of endocrine control of hepatic steroid hormone metabolism.