Induction of cytochrome P-450 forms in liver microsomes of rats in the early neonatal period after administration of phenobarbital and 3-methylcholanthrene

The activity of cytochrome P-450 dependent monooxygenase system from rat liver microsomes after induction by phenobarbital and 3-methylcholantrene in early neonatal period (3-16 days after birth) was studied. It was found that the total amount of cytochrome P-450 increases after injection of these inducers in neonatal rats of all age groups. In parallel, in the case of 3-methylcholantrene induction the benz(a)pyrene hydroxylase and 7-ethoxyresorufin deethylase activities increase; phenobarbital induction causes a rise in the benzphetamine-N-demethylase and benz(a)pyrene hydroxylase activities. Immunochemical analysis involving the use of antibodies specifically directed against cytochrome P-450 of adult rats revealed that the level of cytochrome P-450 in the case of 3-methylcholantrene induction increases from 5 to 50%, whereas that of cytochrome P-450 upon phenobarbital induction increases from 5 to 40% in liver microsomes of 3- and 16-day-old rats. The mode of inhibition of various substrates metabolism by antibodies in neonatal rat microsomes suggests that the 3-methylcholantrene-induced cytochrome P-448, like in adult rats, participates in the hydroxylation of benz(a)pyrene and O-deethylation of 7-etoxyresorufin. The participation of phenobarbital-induced cytochrome P-450 in the metabolism of benzphetamine and aldrin in neonatal rats is much lower than in the adult ones. The metabolism of benz(a)pyrene in phenobarbital-induced neonatal rat microsomes in all age groups is not inhibited by antibodies. The age-dependent differences in inhibition of metabolism and the increase in the benz(a)pyrene hydroxylase activity in phenobarbital-induced rats suggest that the spectrum of inducible forms of cytochrome P-450 in neonatal rats differ from that in adult animals.     

Phenobarbital-type induction of cytochrome P-450 in liver microsomes by perfluorodecalin

Cytochrome P-450 induction in hepatic microsomes after injections of rats with a fluorocarbon emulsion containing perfluorodecalin was studied in comparison with phenobarbital and methylcholanthrene type inductions. It was shown that perfluorodecalin injection as well as the phenobarbital one cause an increase in the cytochrome P-450 content, NADPH-cytochrome c reductase activity, the rates of benzphetamine N-demethylation and aldrin epoxidation in the microsomes. Using the Ouchterlony double immunodiffusion test with antibodies against cytochrome P-450b, an immunological identity of cytochrome P-450 isoforms during perfluorodecalin and phenobarbital inductions was shown. Upon "rocket" immunoelectrophoresis the recovery of cytochrome P-450 which is immunologically indistinguishable from cytochrome P-450b was approximately 72% in perfluorodecalin-induced microsomes. The activity of benzphetamine demethylase and aldrin epoxidase was inhibited by antibodies against cytochrome P-450b. These results suggest that in rat hepatic microsomes perfluorodecalin induces the cytochrome P-450 isoform whose immunological properties and substrate specificity correspond to those of phenobarbital-type cytochrome P-450.     

Induction of P-450 isoenzyme activities in Syrian golden hamster liver compared to rat liver as probed by the rate of 7-alkoxyresorufin-O-dealkylation

The activities of 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-deethylase (PROD), 7-ethoxycoumarin-O-deethylase (ECOD) and aromatic hydrocarbon hydroxylase (AHH) were measured in hepatic microsomes from male and female Wistar rats and Syrian golden hamsters in order to probe the basal activity and the inducibility by phenobarbital (PB) and 3-methylcholanthrene (MC) of different P-450 isoenzymes. The basal activities of EROD and ECOD, but not PROD and AHH, were higher in male hamsters than in male rats. No sex-related difference in enzyme activities was observed with hamsters, whereas male rats had a higher ECOD and AHH activity than female rats. Induction by PB led to a 450-fold and 250-fold increase in PROD activity in male and female rat liver microsomes, respectively, while MC had a more pronounced inductive effect on EROD activity in this species. In hamsters, EROD activity was induced by MC but not by PB. Unexpectedly PROD activity in male and female hamster liver microsomes was only moderately induced by PB, the extent being lower than on induction by MC. Therefore, the activity of PROD, which is useful as a specific enzymatic assay for P-450 IIB in the rat liver, cannot be used to probe PB-like inducers in the hamster liver.     

Dose-related effects of phenobarbital on hepatic microsomal enzymes

To study the relationship between the dose of phenobarbital (PB) and the magnitude of its effects on microsomal enzymes, cytochrome P-450, UDP-glucuronyl transferase (UDPGT), and glucose-6-phosphatase (G6P) activities were determined in liver homogenate and microsome preparations from control rats and rats treated for 6 days with PB at doses ranging from 1 to 125 mg/kg/day. Both P-450 and UDPGT activities were enhanced by PB in a dose-related fashion. However, while the lowest dose of the drug to produce significant induction of both enzymes was the same (3 mg/kg), maximal induction of P-450 (214%) and UDPGT (285%) was obtained with different doses of PB, namely 75 and 125 mg/kg, respectively. UDPGT induction could equally be demonstrated regardless of whether "native" enzyme or enzyme activated by UDP-N-acetyl glucosamine, digitonin or deoxycholate was employed. In contrast to these inducing effects of the drug on P-450 and UDPGT, PB treatment resulted in a dose-related inhibition of G6P activity. The inhibitory effect was observed with both "native" and deoxycholate-activated enzymes, and could be demonstrated whether the data were expressed as enzyme specific activity (nanomoles per minute per milligram microsomal protein) or as total G6P activity (micromoles per minute per 100 g body weight). These results indicate that: (I) enzyme induction by PB is dose-related; (ii) induction of both P-450 and UDPGT is obtained in the rat with doses of the drug similar to those given to man; and (iii) observed inhibition of G6P activity by PB does not solely reflect an enzymatic dilution secondary to the proliferated endoplasmic reticulum.     

Liver microsomal cytochromes P-450 and azoreductase activity.

Hepatic microsomal azoreductase activity with amaranth (3-hydroxy-4[(4-sulfo-1-naphthalenyl)azo]-2,7-naphthalenedisulfonic acid trisodium salt) as a substrate is proportional to the levels of microsomal cytochrome P-450 from control or phenobarbital-pretreated rats and mice or cytochrome P-448 from 3-methylchol-anthrene-pretreated animals. In the "inducible" C57B/6J strain of mice, 3-methylcholanthrene and phenobarbital pretreatment cause an increase in cytochrome P-448 and P-450 levels, respectively, which is directly proportional to the increase of azoreductase activity. However, in the "noninducible" DBA/2J strain of mice, only phenobarbital treatment causes the increase both in cytochrome P-450 levels and azoreductase activity, while 3-methylcholanthrene has no effect. These experiments suggest that the P-450 type cytochromes are responsible for azoreductase activity in liver microsomes.     

Effect of excessive intake of ascorbic acid on hepatic and extra-hepatic phase I and phase II drug metabolism in rat

Guinea pig is the animal model of choice for studies on effects of ascorbic acid (AA). However, rat is one of the largely used animals for investigations related to chemical carcinogenesis. Therefore, the present study was designed to evaluate the changes induced by high intake of the vitamin in xenobiotic and carcinogen metabolizing status of the organs. Male Wistar rats, dosed daily with 50 mg AA/100 g body weight for 10 weeks, demonstrated a small non-significant increase in hepatic, pulmonary and colon cytochrome P-450 (Cyt. P-450) contents, which was accompanied with a significant increase in hepatic and pulmonary arylhydrocarbon hydroxylase (AHH) activities. Phase II enzymes of drug metabolism responded in different ways to increased intake of AA. UDP-glucuronyltransferase (UDPGT) activity was unaffected in liver and colon, but it was increased (p less than 0.005) in lung. Activities of glutathione S-transferase (GST) were decreased in the three organs. Inducibility of AHH by 3-methylcholanthrene (MCA) or phenobarbital (PB) was largely reduced due to AA feeding. Besides this, MCA and PB had differential effects on enzymatic levels in AA fed rats. When compared with our earlier observations in guinea pig, it was found that rat responded similarly to guinea pig to increased intake of AA with regard to hepatic AHH, Cyt. P-450, UDPGT and GST, pulmonary AHH, Cyt. P-450 and Cyt. b5, and all studied colon enzymes, except GST.     

Inhibition of CCl4 metabolism by oxygen varies between isoenzymes of cytochrome P-450

Oxygen inhibition of CCl4 metabolism by different isoenzymes of cytochrome P-450 was assessed by studying liver microsomes isolated from control rats and rats treated with phenobarbital or isoniazid. Rates of CCl4 metabolism were similar for all microsomes under a nitrogen atmosphere. An air atmosphere inhibited metabolism by microsomes from control rats to 12% of the value under nitrogen and metabolism by microsomes from rats treated with phenobarbital to 5%. It inhibited metabolism by microsomes from rats treated with isoniazid only to 32%. Rats treated with phenobarbital, which increases hepatic cytochrome P-450 content, or isoniazid, which does not increase hepatic cytochrome P-450 content, both metabolized more CCl4 than control rats as indicated by exhalation of greater quantities of CCl4 metabolites and by an increase in CCl4 toxicity. These results indicate that some isoenzymes of cytochrome P-450 are more effective than others in metabolizing CCl4 when oxygen is present.     

Role of cytochrome P-450IIE1 in N-nitroso-N-methylaniline induced hepatocyte cytotoxicity.

1. The cytotoxicity of N-nitrosomethylaniline (NMA) towards hepatocytes isolated from rats was prevented by acetone or ethanol (inhibitors for cytochrome P-450IIE1) but not by metyrapone or SKF525A (inhibitors for cytochrome P-450IIB1/2). Various alcohols, secondary ketones and isothiocyanates that induced cytochrome P-450IIE1 were also found to be protective. Various aromatic and chlorinated hydrocarbon solvents that are substrates or inducers of cytochrome P-450IIE1 also prevented NMA cytotoxicity. Nitrogen-containing heterocycles that induced cytochrome P-450IIE1 were less effective. Further evidence that cytochrome P-450IIE1 was responsible for the activation of NMA was the marked increase in hepatocyte susceptibility if hepatocytes from pyrazole-induced rats were used. 2. NMA was more cytotoxic to hepatocytes isolated from phenobarbital-pretreated rats than uninduced rats. However, metyrapone now prevented and SKF525A delayed the cytotoxicity whereas ethanol, acetone, allyl isocyanate, isoniazid or trichloroethylene had no effect on the susceptibility of phenobarbital-induced hepatocytes. Furthermore, microsomes isolated from phenobarbital-pretreated rats had higher NMA-N-demethylase activity which was more inhibited by metyrapone and SKF525A than that of uninduced microsomal activity. By contrast the N-demethylase activity of phenobarbital induced microsomes was more resistant to acetone, ethanol, hexanal, trichloroethylene and toluene than uninduced microsome. 3. The above results suggest that cytochrome P-450IIE1 catalyses the cytotoxic activation of NMA in normal or pyrazole-induced hepatocytes whereas cytochrome P-450IIB1/2 is responsible for cytotoxicity in phenobarbital-induced hepatocytes.     

Further consideration of phenobarbital effects on cytochrome P-450 activity in the killifish, Fundulus heteroclitus

1. Ethoxyresorufin O-deethylase (EROD) activity, aldrin epoxidase (AE) activity, cytochrome P-450 content, and levels of cytochrome P-450E (the major BNF-inducible P-450 form and primary EROD catalyst in scup) or its homologues were measured in hepatic microsomes isolated from Fundulus heteroclitus, scup (Stenotomus chrysops) and brook trout (Salvelinus fontinalis) treated with beta-naphthoflavone (BNF) or phenobarbital (PB). 2. In all three teleost species, BNF treatment caused expected increases in P-450 content, EROD activity and P-450E level; but either no change or a slight decrease in AE turnover rate (nmol/min/nmol P-450). 3. Polyclonal antibodies to P-450E did not inhibit AE activity in microsomes from BNF-treated scup, confirming that this major BNF-inducible P-450 form does not catalyze AE activity in fish. 4. In contrast, PB treatment did not affect hepatic AE activity, P-450 content or levels of "P-450E" in F. heteroclitus, but did variably affect EROD activity which was suppressed in one experiment and elevated in another. 5. The results indicate that (i) contrary to previous reports, neither PB nor MC-type inducers increase AE activity in F. heteroclitus, (ii) MC-type inducers do not affect AE activity in the other teleost species examined, and (iii) AE activity is not a reliable indicator of P-450 induction by environmental chemicals. 6. We emphasize the need to establish the mechanism of PB action, and the nature of any fish P-450 forms analogous to PB-inducible forms in mammals in order to conclusively evaluate PB-responses in fish.     

Effects of phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls on sex-specific forms of cytochrome P-450 in liver microsomes of rats

The effects of treatment with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls (PCB) on the amounts of sex-specific forms of cytochrome P-450, namely P-450-male and P-450-female, in male and female rats were studied. Although treatment with phenobarbital, 3-methylcholanthrene or PCB markedly increased the total amount of hepatic cytochrome P-450, P-450-male and P-450-female were rather decreased or not significantly changed. Thus, the percentages of P-450-male and P-450-female in the total cytochrome P-450 were decreased in liver microsomes from the treated rats. The increases in specific cytochrome P-450, such as P-448-H, P-448-L, and P-450I-c accounted for the increase in the total amount of cytochrome P-450 in the treated rats. The treatment with phenobarbital or PCB increased the activities of testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase and aminopyrine N-demethylase more markedly in female rats than in male rats. Similarly, the treatment with 3-methylcholanthrene increased benzo(a)pyrene hydroxylase more markedly in female rats. Therefore, the sex-differences in testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase, and aminopyrine N-demethylase activities became smaller after the drug treatment. These results indicate that sex-specific P-450-male and P-450-female were unaffected, or even depressed by the agents in some cases.     

Isolation and characterization of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats

Chromatography on 1.8-diaminooctyl-Sepharose and DEAE-Sephacel resulted in 4 fractions of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats. All the four fractions differed in terms of their absorption maxima in the CO-reduced state, Mr and catalytic activity. Only one cytochrome fraction (cytochrome P-450 C) possessed a high activity upon benz(a)pyrene hydroxylation. All cytochrome P-450 forms were characterized by a low rate of aminopyrine N-demethylation. Antibodies against cytochrome P-450 C (P-448) (anti-P-448) were raised. Cytochromes of fractions A, B1 and B2 in the Ouchterlony reaction of double immunodiffusion did not give precipitation bands with anti-P-448. Neither of the four cytochrome P-450 forms interacted with the antibodies raised against cytochrome P-450 isolated from liver microsomes of rats induced with phenobarbital. The procedure developed is applicable to the isolation of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced rats. Using rocket immunoelectrophoresis, cytochrome P-450 C possessing a high (as compared to benz(a)pyrene metabolism) activity (18 nmol/min/nmol cytochrome) and a high (60-70%) content in 3-methylcholanthrene-induced rat liver microsomes was shown to give a relatively high yield.     

Constitutive testosterone 6 beta-hydroxylase in rat liver

The cytochrome P-450 that was purified from hepatic microsomes of male rats treated with phenobarbital and designated P450 PB-1 (Funae and Imaoka (1985) Biochim. Biophys. Acta 842, 119-132) had high testosterone 6 beta-hydroxylation activity (turnover rate, 13.5 nmol of product/min/nmol of P-450) in a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5, and a 1:1 mixture of lecithin and phosphatidylserine in the presence of sodium cholate. In ordinary conditions in the reconstituted system with cytochrome P-450, reductase, and dilauroylphosphatidylcholine, P450 PB-1 had little 6 beta-hydroxylase activity. The catalytic activities toward testosterone of two major constitutive forms, P450 UT-2 and P450 UT-5, were not affected by cytochrome b5, phospholipid, or sodium cholate. P450 PB-1 in rat liver microsomes was assayed by immunoblotting with specific antibody to P450 PB-1. P450 PB-1 accounted for 24.4 +/- 5.6% (mean +/- SD) of the total spectrally-measured cytochrome P-450 in hepatic microsomes of untreated adult male rats, and was not found in untreated adult female rats. P450 PB-1 was induced twofold with phenobarbital in male rats. P450 PB-1 was purified from untreated male rats and identified as P450 PB-1 from phenobarbital-treated rats by its NH2-terminal sequence, peptide mapping, and immunochemistry. These results showed that P450 PB-1 is a constitutive male-specific form in rat liver. There was a good correlation (r = 0.925) between the P450 PB-1 level and testosterone 6 beta-hydroxylase activity in rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coordinate induction of multiple mRNAs specific for rat liver phenobarbital-inducible cytochromes P-450

Utilizing two-dimensional gel electrophoresis, the polypeptide composition of a purified microsomal cytochrome P-450 preparation isolated from phenobarbital-treated Long-Evans rats obtained from Charles River Laboratories has been examined. The purified protein consists of three polypeptides with nearly identical subunit molecular weights (approximately 52,000) but differing in net charge. These three polypeptides can be detected in liver microsomes isolated from phenobarbital-treated rats by immunoblot analysis but are virtually absent in microsomes isolated from untreated rats. All three polypeptides appear to be products of distinct mRNAs since they can be immunoprecipitated from rabbit reticulocyte lysates programmed with poly(A+)-RNA isolated from phenobarbital-treated rats. The amount of functional mRNA specific for the P-450 polypeptides increases dramatically in response to an acute administration of phenobarbital; however, in untreated rats the amount of functional mRNA was below the level of detection by the translational assay. These data are consistent with the very low level of the phenobarbital-inducible cytochromes P-450 in liver microsomes isolated from untreated rats. Finally, the data indicate that all three cytochrome P-450 mRNAs increase rapidly in response to phenobarbital administration and are regulated coordinately.     

Comparison of the inducing effect of triphenyldioxan, bis-(dichloropyridyloxy)benzene and phenobarbital on the liver monooxygenase

The induction by triphenyldioxane (TPD) of cytochrome P-450 in rat liver microsomes was studied. It was demonstrated that TPD injection in a single dose (10 mg/kg of body mass) is associated with a marked induction of cytochromes P-450 b/e (cytochrome PB-forms) in rat liver microsomes and a significant increase in the benzphetamine-N-demethylase activity typical of cytochrome P-450b. In other words, TPD is a potent inducer of PB-type, the inducing effect being attained by an injection of a single dose of TPD which is by one order of magnitude less than that of phenobarbital. It can be assumed that this compound shows a high affinity for the hypothetical receptor responsible for cytochrome P-450b synthesis. It was shown also that TPD does not induce the monooxygenase system of mouse liver, whereas 1,4-bis[2-(dichloropyridyloxy)]benzene (DPB) is a potent inducer of PB-type in mice, being fairly ineffective in rats. Hence, the species-specific effect of TPD and DPB appears to be opposite.     

Studies on the interaction of furan with hepatic cytochrome P-450

In vitro incubation of rat liver micro-somes with [¹⁴C]-furan in the presence of NADPH resulted in the covalent incorporation of furan-derived radioactivity in microsomal protein. Compared to microsomes from untreated rats a two- to threefold increase in binding was observed with microsomes from phenobarbital-treated rats and a four- to five-fold increase was observed with microsomes from rats pretreated with imidazole or pyrazole. Covalent binding was reduced with microsomes from rats pretreated with β-naphthoflavone. Chemicals containing an amine group (semicarbazide), those in which the amine group is blocked but have a free thiol group (N-acetylcysteine), and those which have both an amine and a thiol group (glutathione) effectively blocked binding of [¹⁴C]-furan to microsomal protein. A decrease in cytochrome P-450 (P-450) content and decreases in the activities of P-450-dependent aniline hydroxylase, 7-ethoxycoumarin-O-deethylase (BCD), and 7-ethoxyresorufin-O-deethylase (ERD) was observed 24 hours after a single oral administration of 8 or 25 mg/kg of furan, suggesting that the reactive intermediate formed during P-450 catalyzed metabolism could be binding with nucleophilic groups within the P-450. In vitro studies indicated a significant decrease in the activity of aniline hydroxylase in pyrazole microsomes and BCD in phenobarbital microsomes without any significant change in the CO-binding spectrum of P-450 or in the total microsomal heme content, suggesting that furan inhibits the P-450s induced by PB and pyrazole. An almost equal distribution of furan-derived radioactivity in the heme and protein fractions of the CO-binding particles after In vitro treatment of microsomes with furan suggests binding of furan metabolites with heme and apoprotein of P-450, and, probably, due to this interaction, furan is acting as a suicide inhibitor of P-450.     

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.     

Effect of different dietary triglycerides on 7alpha-hydroxylation of cholesterol and other mixed-function oxidations

The effect of a diet containing triglycerides of different fatty acid composition on hepatic 7alpha-hydroxylation of cholesterol was studied. 7alpha-Hydroxylation of exogenous as well as endogenous cholesterol was significantly lower in the liver of rats fed trilinolein and triolein than in those fed tripalmitin and trierucin. The concentration of cytochrome P-450 in liver microsomes was significantly lower in the rats fed tripalmitin and trierucin than in those fed triolein and trilinolein. The inhibitory effect of triolein and trilinolein on 7alpha-hydroxylation of cholesterol and the stimulatory effect of these triglycerides on the concentration of cytochrome P-450 was not due to the small amounts of peroxides present in the unsaturated triglycerides. Thus addition of the antioxidant butylated hydroxyanisol did not change the general pattern with respect to 7alpha-hydroxylation and concentration of cytochrome P-450. However, a diet consisting of peroxidized linoleic acid further decreased 7alpha-hydroxylation of cholesterol. The difference between the effect obtained with triolein and trilinolein on the one hand and trierucin and tripalmitin on the other was observed also in experiments with lower concentrations of fat in the diet and in experiments with different lighting conditions and feeding patterns. The inverse relation between cytochrome P-450 and 7alpha-hydroxylation of cholesterol, as well as results obtained with substrates for mixed-function oxidation other than cholesterol suggest that most of the changes observed due to the different diets are specific for 7alpha-hydroxylation of cholesterol. The level of cholesterol 7alpha-hydroxylase activity was found to be better related to the degree of absorption of fat than to total amount of absorbed fat or degree of unsaturation of the fat. The results are discussed in relation to previous knowledge concerning mechanisms regulating biosynthesis of bile acid.     

Comparative characteristics of isolated forms of cytochrome P-450 induced by phenobarbital and perfluorodecalin

Two forms of cytochrome P-450 were isolated from liver microsomes of perfluorodecalin-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from phenobarbital-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromatographic behaviour on 1.8-diaminooctyl-Sepharose 4B and DEAE-Sephacel columns, molecular mass determined by SDS polyacrylamide gel electrophoresis, spectral properties, immunoreactivity, peptide mapping, catalytic activity. These findings suggest that in rat liver microsomes perfluorodecalin and phenobarbital which differ in their chemical structure induce identical forms of cytochrome P-450.     

Oxidation of uroporphyrinogen by methylcholanthrene-induced cytochrome P-450. Essential role of cytochrome P-450d.

We have previously shown that uroporphyrinogen is oxidized to uroporphyrin by microsomes (microsomal fractions) from 3-methylcholanthrene-pretreated chick embryo liver [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We report here that a specific antibody to chick liver methylcholanthrene-induced cytochrome P-450 (P-450) inhibited both uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in chick-embryo liver microsomes. 3-Methylcholanthrene-pretreatment of rats and mice markedly increased uroporphyrinogen oxidation in hepatic microsomes as well as P-450-mediated ethoxyresorufin de-ethylation. In rodent microsomes, uroporphyrinogen oxidation required the addition of NADPH, whereas chick liver microsomes required both NADPH and 3,3',4,4'-tetrachlorobiphenyl. Treatment of rats with methylcholanthrene, hexachlorobenzene and o-aminoazotoluene increased uroporphyrinogen oxidation and P-450d, whereas phenobarbital did not increase either. The contribution of hepatic P-450c and P-450d to uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in methylcholanthrene-induced microsomes was assessed by using specific antibodies to P-450c and P-450d. Uroporphyrinogen oxidation by methylcholanthrene-induced rat liver microsomes was inhibited up to 75% by specific antibodies to P-450d, but not by specific antibodies to P-450c. In contrast, ethoxyresorufin de-ethylation was inhibited only 20% by anti-P450d but 70% by anti-P450c. Methylcholanthrene-induced kidney microsomes which contain P-450c but non P-450d did not oxidize uroporphyrinogen. These data indicate that hepatic P-450d catalyses uroporphyrinogen oxidation. We suggest that the P-450d-catalysed oxidation of uroporphyrinogen has a role in the uroporphyria caused by hexachlorobenzene and other compounds.     

The effect of alpha-ecdysone and phenobarbital on the alpha-ecdysone 20-monooxygenase of house fly larva

The NADPH-dependent cytochrome P-450 20-monooxygenation of alpha-ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica, L. larvae, but about 50% of the activity is associated with mitochondria and 37% with microsomes. The mitochondrial activity is increased by pretreatment with alpha-ecdysone with a concomitant decrease in Km values. This effect is not observed in microsomes. Induction with phenobarbital represses the mitochondrial 20-monooxygenase but does not change the microsomal activity, although a large increase in cytochrome P-450 is observed in the latter fraction. It is concluded that only the mitochondrial 20-monooxygenase appears to be regulated by alpha-ecdysone which suggests that mitochondrial cytochrome P-450 forms are involved in the moulting phenomenon; whereas, microsomal cytochrome P-450 activity may be of a nonspecific nature and not relevant to development.