Biochemical changes in rat liver after 18.5 days of spaceflight

The effect of "weightlessness" on liver metabolism was examined using tissue from rats flown in earth orbit for 18.5 days aboard the Soviet Cosmos 936 biosatellite. Changes in the activities of certain carbohydrate and lipid enzymes were noted. Of the 28 hepatic enzyme activities assayed, two, palmitoyl-CoA desaturase and lactate dehydrogenase, increased, whereas five, glycogen phosphorylase, 6-phosphogluconate dehydrogenase, both acyltransferases which act on alpha-glycerolphosphate and diglycerides, and aconitate hydratase decreased. The remaining enzyme activities measured were unchanged. In addition, increased levels of liver glycogen and palmitoleate were noted which probably resulted from the lowered glycogen phosphorylase and increased palmitoyl-CoA desaturase activities, respectively, in those animals that experienced weightlessness. These changes caused by weightlessness were transient since all of the aforementioned alterations returned to normal values when measured in the livers of other rats which had flown in the biosatellite 25 days after recovery.     

Evidence for the involvement of multiple forms of cytochrome P-450 in the occurrence of sex-related differences of drug metabolism in the rat

Multiple forms of cytochrome P-450 in liver microsomes of untreated male and female rats could be divided into several fractions by the use of ω-amino-n-octyl Seph. 4B and DE-52 columns. Male cytochrome P-450 fractions (I-b - I-e) differed from female fractions (I-b - I-e) with respect to absorption peaks in carbon monoxide difference spectra and 7-prop-oxycoumarin O-depropylation activities. Although male and female I-a fractions showed quite similar protein bands on SDS-polyacrylamide gel electrophoresis, some protein bands in other male fractions (I-b - I-e) were absent in corresponding female fractions. Immunochemical examinations using immunoglobulin G raised to cytochrome P-450 purified from untreated male rats also showed that liver microsomes from male and female rats contain different forms of cytochrome P-450. Based on these results, we propose that sex-related differences of drug metabolizing activities in liver microsomes are caused by multiple forms of cytochrome P-450.     

Identification of molecular forms of cytochrome P-450 isolated from liver microsomes of rats induced with phenobarbital and 3-methylcholanthrene

Eight electrophoretically homogeneous forms of cytochrome P-450 were isolated from liver microsomes of phenobarbital (PB)- and 3-methylcholanthrene (MC)-induced male Wistar rats, using chromatography on 1.8-diaminooctyl-Sepharose, SEAE-Sephacel and hydroxylapatite. These cytochrome forms were compared to those described in literature in terms of their ability to metabolize androstenedione (AD), benzphetamine (BP) and 7-ethoxyresorufin (7-ER). Cytochrome P-450b capable of catalyzing with a high specificity the 16-hydroxylation of AD and N-demethylation of BP, and cytochrome P-450e immunologically related to P-450b but incapable of catalyzing these reactions were isolated from PB-microsomes. Besides, a male-specific cytochrome P-450h catalyzing the 16 alpha-hydroxylation of AD was isolated from PB-microsomes. Cytochrome P-450c possessing a high 7-ER-O-deethylase activity, and a high spin cytochrome P-450d as well as cytochrome P-450a specifically catalyzing the 7 alpha-oxidation of AD were isolated from MC-microsomes. Two forms of cytochrome P-450 isolated from PB-microsomes possessed no such activities. Data from immunochemical analysis suggest that one of these forms can be identified as cytochrome P-450k. It is concluded that the specificity of metabolism and the molecular activity of Wistar rat liver cytochrome P-450 forms are comparable with the corresponding parameters of hemoproteins isolated from other rat species. At the same time, data from metabolic analysis are suggestive of differences in the levels of certain cytochrome P-450 forms, in particular P-450a.     

Lack of inducibility of brain monooxygenase activities including parathion desulfuration

The ability of phenobarbital and beta-naphthoflavone to induce parathion desulfuration, aminopyrine N-demethylation, and NADPH-cytochrome-c reductase activity in the brain and liver of male and female rats was investigated. Activities of all three enzymes were found in similar levels in both the mitochondrial and microsomal fractions of brain. There were no sex differences in brain activities. Liver activities were from 10- to 30-fold higher than brain activities when computed on a tissue-wet-weight-equivalent basis. Although exposure to both inducers increased all three enzyme activities and cytochrome P-450 in liver, neither inducer increased the enzyme activities in mitochondrial or microsomal brain fractions of either sex. Thus, these brain monooxygenase activities appear to be refractory to induction by two classical types of cytochrome P-450 inducers. This lack of inducibility could serve to protect the animal against environmentally enhanced increases in the activation of xenobiotics to neurotoxic metabolites, such as parathion desulfuration to paraoxon.     

Food restriction prevents the loss of isosafrole inducible cytochrome P-450 mRNA and enzyme levels in aging rats

The influence of food restriction (FR) on the drug-inducible capacity of liver microsomal cytochrome P-450s IA1, IA2 and IIB1 and IIB2 was studied in 20-month-old male Fischer-344 rats. ELISA and Western Blotting revealed that the induction of the cytochrome P-450-IA1/IA2 and P-450-IIB1/IIB2 enzymes was considerably higher in the liver microsomes of FR rats than in their ad libitum (AL) fed counterparts. In order to determine whether the higher P-450 enzyme levels in FR rats were a reflection of an increased synthesis rate or a stabilization of these enzymes, hybridization studies were performed with a cDNA probe for P-450-IIB1/IIB2. These studies show markedly higher levels of P-450-IIB1/IIB2 mRNAs in the livers of FR rats as compared to AL animals. These results suggest that it is possible to prevent the age-dependent loss of drug-induced cytochrome P-450s by 40% dietary restriction which suggest FR may improve the drug-metabolizing capacity during aging.     

Digitoxin metabolism by liver microsomal cytochrome P-450 and UDP-glucuronosyltransferase and its role in the protection of rats from digitoxin toxicity by pregnenolone-16 alpha-carbonitrile

The aim of the present study was to investigate whether the mechanism by which pregnenolone-16 alpha-carbonitrile (PCN) protects rats from digitoxin toxicity was dependent on the induction of liver microsomal cytochrome P-450p and/or the UDP-glucuronosyltransferase active toward digitoxigenin monodigitoxoside (UDP-GT-dt1). Evidence is presented that suggests troleandomycin is a selective inhibitor of cytochrome P-450p in vivo, based on the pattern of inhibition observed when zoxazolamine paralysis time and hexobarbital sleeping time were measured in rats treated with different cytochrome P-450 inducers. A single dose of troleandomycin completely reversed the ability of PCN to protect rats from digitoxin toxicity, establishing the importance of cytochrome P-450p induction in the protective effect of PCN. The postpubertal decline in constitutive cytochrome P-450p levels in female but not male rats was paralleled by a female-specific, age-dependent decline in the rate of digitoxin sugar cleavage (i.e., digitoxosyl oxidation of digitoxin to 15'-dehydrodigitoxin and digitoxosyl cleavage to digitoxigenin bisdigitoxoside). This resulted in a marked sex difference in the rate of digitoxin sugar cleavage catalyzed by liver microsomes from mature rats (male/female approximately 6). However, no sex difference in digitoxin toxicity was observed in either immature or mature rats. In contrast to cytochrome P-450p, liver microsomal UDP-GT-dt1 activity increased dramatically with age in both male and female rats (mature/immature approximately 10). However, no age differences in digitoxin toxicity were observed in rats of either sex. The results indicate that cytochrome P-450p and UDP-GT-dt1 can be independently regulated in rat liver and that large changes in the constitutive levels of these microsomal enzymes have no effect on digitoxin toxicity. This suggests that the induction of cytochrome P-450p and UDP-GT-dt1 does not fully account for the mechanism by which PCN protects rats from digitoxin toxicity.     

Studies on the decrease of liver cytochrome P-450 content by triiodothyronine

In the rat liver, the microsomal content of cytochrome P-450 decreased by 50% after triiodothyronine (T3) administration. The molecular basis for the decreased cytochrome P-450 levels was investigated. The activities of the enzymes involved in heme synthesis or degradation were not altered by thyroid hormone administration. The incorporation of 3H-delta-aminolaevulinate into the liver microsomal heme was markedly reduced in T3-treated rats. The latter appeared not to reflect a lowered binding affinity of the apoprotein moiety of cytochrome P-450 for heme. The sodium dodecyl sulfate gel electrophoresis of the microsomal preparation showed a decrease in apocytochrome P-450. It is suggested that the amount of the apocytochrome may be the primary event affected in the formation of cytochrome P-450, by triiodothyronine treatment of thyroidectomized rats.     

Purification, composition, and some properties of rat liver carbamyl phosphate synthetase (ammonia).

Hepatic microsomes prepared from vitamin E deficient and supplemented rats were analyzed for cytochrome P-450 content and drug metabolizing activity. Reduced levels of benzo[α]pyrene hydroxylase and ethylmorphine N-demethylase activities were observed in microsomes derived from rats fed a diet deficient in vitamin E compared to those of control rats. NADPH-mediated destruction of P-450, and pentobarbital and zoxazolamine sleeping times were similar in the two groups. Induction with 3-methylcholanthrene raised the levels of benzo[α]pyrene hydroxylase activity of both supplemented and deficient rats to the same absolute levels. No differences were noted in cytochrome P-450 or P-448 content between control and tocopherol deficient rats, nor did the activity of liver catalase differ between the two dietary groups. Thus, these studies did not demonstrate any impairment of heme protein synthesis in vitamin E deficient rats.     

Time-course of effects of toluene on microsomal enzymes in rat liver, kidney and lung during and after inhalation exposure

Inhalation of toluene vapour of 2000 ppm increased the activities of aniline hydroxylase, aminopyrine N-demethylase, aryl hydrocarbon hydroxylase and NADPH-cytochrome c reductase and the concentrations of cytochromes P-450 and b5 in liver microsomes of adult male rats after an exposure period of 1 day or less. Repeated treatments, 8 h daily for 1-16 days, had only a slight further effect. In lung microsomes, the activities of monooxygenases and the concentration of cytochrome P-450 decreased after 6-24 h toluene exposure, but those of cytochrome b5 and NADPH-cytochrome c reductase did not change. In kidney microsomes the changes were mostly insignificant. After discontinuation of exposure the activities of enzymes and the concentrations of cytochromes returned to the control level in 1-4 days. The results obtained resemble the time-courses for the induction of monooxygenases by other inducers. The tissue differences suggest the unequal distribution of various cytochrome P-450 forms and their individual responsiveness to induction in liver, kidneys and lungs.     

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.     

Evidence that isoniazid and ethanol induce the same microsomal cytochrome P-450 in rat liver, an isozyme homologous to rabbit liver cytochrome P-450 isozyme 3a

Cytochrome P-450j has been purified to electrophoretic homogeneity from hepatic microsomes of adult male rats administered ethanol and compared to the corresponding enzyme from isoniazid-treated rats. The enzymes isolated from ethanol- and isoniazid-treated rats have identical chromatographic properties, minimum molecular weights, spectral properties, peptide maps, NH2-terminal sequences, immunochemical reactivities, and substrate selectivities. Both preparations of cytochrome P-450j have high catalytic activity in aniline hydroxylation, butanol oxidation, and N-nitrosodimethylamine demethylation with turnover numbers of 17-18, 37-46, and 15 nmol product/min/nmol of P-450, respectively. A single immunoprecipitin band exhibiting complete identity was observed when the two preparations were tested by double diffusion analysis with antibody to isoniazid-inducible cytochrome P-450j. Ethanol- and isoniazid-inducible rat liver cytochrome P-450j preparations have also been compared and contrasted with cytochrome P-450 isozyme 3a, the major ethanol-inducible isozyme from rabbit liver. The rat and rabbit liver enzymes have slightly different minimum molecular weights and somewhat different peptide maps but similar spectral, catalytic, and immunological properties, as well as significant homology in their NH2-terminal sequences. Antibody to either the rat or rabbit isozyme cross-reacts with the heterologous enzyme, showing a strong reaction of partial identity. Antibody against isozyme 3a specifically recognizes cytochrome P-450j in immunoblots of induced rat liver microsomes. Aniline hydroxylation catalyzed by the reconstituted system containing cytochrome P-450j is markedly inhibited (greater than 90%) by antibody to the rabbit protein. Furthermore, greater than 85% of butanol or aniline metabolism catalyzed by hepatic microsomes from ethanol- or isoniazid-treated rats is inhibited by antibody against isozyme 3a. Results of antibody inhibition studies suggest that cytochrome P-450j is induced four- to sixfold by ethanol or isoniazid treatment of rats. All of the evidence presented in this study indicates that the identical cytochrome P-450, P-450j, is induced in rat liver by either isoniazid or ethanol, and that this isozyme is closely related to rabbit cytochrome P-450 isozyme 3a.     

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.     

Effects of cage beddings on microsomal oxidative enzymes in rat liver

The purpose of the present studies was to evaluate the effects of some commercially available cage beddings on rat liver microsomal cytochrome P-450-dependent drug-metabolizing enzyme, ethylmorphine N-demethylase, and the carcinogen-metabolizing enzyme, benzo(a)pyrene hydroxylase. Sprague-Dawley rats were housed in cages containing cedar chip, corncob or heat-treated pinewood bedding for 3 weeks. Control rats were housed in cages on wire bottom floors containing no bedding material. Rats housed in cages containing cedar chip showed 18, 46 and 49% increases in liver cytochrome P-450 content, ethylmorphine N-demethylase and benzo(a)pyrene hydroxylase activities, respectively. The liver enzyme activities of rats housed in cages containing corncob bedding were similar to those obtained with control rats. In contrast, the pinewood-bedded rats showed a 21% decrease in ethylmorphine N-demethylase activity without affecting cytochrome P-450 content and benzo(a)pyrene hydroxylase activity. Hexobarbital-induced sleep times of the variously bedded rats were similar to those of control animals. These data suggest that the commercial bedding materials differ in their abilities to affect liver microsomal enzymes. Thus, interlaboratory variability in basal enzyme activities reported in the literature may be partly due to bedding materials used in the animal's cages.     

Regulation of cytochrome P-450j, a high-affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes

Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.     

Hormonal regulation of rat liver microsomal enzymes. Role of gonadal steroids in programming, maintenance, and suppression of delta 4-steroid 5 alpha-reductase, flavin-containing monooxygenase, and sex-specific cytochromes P-450

Neonatal gonadectomy studies and hormonal replacement regimens were employed to characterize the regulation of delta 4-steroid 5 alpha-reductase, microsomal flavin-containing monooxygenase, and several forms of rat hepatic microsomal cytochrome P-450, including three that are sexually differentiated. Rats of both sexes that had been gonadectomized at birth were either untreated or were administered testosterone propionate or estradiol benzoate neonatally (subcutaneous injection on days 1 and 3 of life), postpubertally (an implant of a hormone-packed capsule at 5 weeks of age), or both neonatally and postpubertally. At the age of 10 weeks, all rats were killed, and several liver microsomal enzymes were assayed using immunochemical and catalytic techniques. Expression in the 10-week-old male and female rats of two male-specific cytochrome P-450 forms, termed P-4502c/UT-A and P-4502a/PCN-E, and their associated respective 16 alpha- and 6 beta-steroid hydroxylase activities could either be imprinted (programmed) by androgen exposure during the early neonatal period or, alternatively, could be stimulated by continuous hormone treatment after the age of 5 weeks. By contrast, hepatic expression of two female-specific enzymes, P-4502d/UT-1 and delta 4-steroid 5 alpha-reductase, was only partially dependent on estradiol; birth-gonadectomized rats expressed as much as 30-50% of the enzyme levels present in untreated adult females. Expression of both female-specific enzymes was fully suppressed upon postpubertal exposure to testosterone. In another study, birth sham-operated female rats were administered testosterone using the same regimens described above for the birth-gonadectomized rats. Although neonatal testosterone treatment alone did not affect the expression in these females of the four sex-specific enzymes examined in this study, it did enhance significantly the masculinization effected by postpubertal androgen exposure. This resulted in expression of the male-specific enzymes P-4502c/UT-A and P-4502a/PCN-E in these females at levels comparable to those found in adult males, while simultaneously suppressing the two female-specific enzymes, P-4502d/UT-I and delta 4-steroid 5 alpha-reductase, by approximately 70-75% to levels characteristic of prepubertal rats of either sex. The levels of another microsomal enzyme, flavin-containing monooxygenase, were also measured and found to be regulated by testosterone, but the ontogenic profiles and the effects of gonadectomy and hormone replacement indicated clear differences in its regulation when compared to the other male-specific enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of cytochrome P-450-dependent activities in hagfish, dogfish, perch and spectacle caiman

1. Owing to interest in the original function and evolution of cytochrome P-450 this enzyme system was studied in four species each at different levels on the phylogenetic tree, representing Agnatha, Selachi, Teleostei, and Reptilia. 2. All species contained considerable amounts of hepatic microsomal cytochrome P-450. The content in dogfish, perch and spectacle caiman was 0.2-0.3 while in the dogfish it was 0.6 nmol per mg protein. 3. Hepatic microsomal cytochrome P-450 dependent ethylmorphine demethylase, benzo(a)pyrene hydroxylase, 7-ethoxycoumarin-O-deethylase and 7-ethoxyresorufin-O-deethylase activities have been measured and the assays were carefully characterized with respect to pH-, protein content-, incubation time- and temperature-activity relationships. 4. The study revealed species-dependent differences in the liver microsomal cytochrome P-450 enzyme system, which could be due to differences in cytochrome P-450 composition or in the microenvironment around the enzyme.     

Studies on in vitro antipyrine metabolism by 13C,15N double labeled method

The capacities of forms of cytochrome P-450 to oxidize antipyrine were compared. An isotope dilution gas chromatography/mass spectrometry/selected ion monitoring assay was developed to quantify the three main metabolites, norantipyrine, 3-hydroxymethylantipyrine and 4-hydroxyantipyrine. 13C,15N-Double labeled antipyrine was used as a substrate and the metabolites were analyzed as their trimethylsilyl derivatives. Among forms of cytochrome P-450 examined, a male-specific form of P-450, namely P-450-male, showed higher activity to form all the three metabolites. The other forms were responsible only for the formation of norantipyrine and 4-hydroxyantipyrine. The activities of liver microsomes from untreated male and female rats and rats treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyl were expressed dependent on the activities of forms of cytochrome P-450 examined.     

The effects of the continuous administration of N,N-dimethyl-4-phenylazoaniline (DAB) on the activities and the inducibilities of some drug-metabolizing enzymes in rat liver.

(1) The effect of feeding a relatively low-protein diet containing 0.06% DAB for 29 weeks on the activity of DAB-azoreductase, nitroreductase (p-nitrobenzoic acid), N-oxidase (N,N-dimethylaniline), N-demethylase (DAB), cytochrome P-450, NADPH-cytochrome c reductase, beta-glucuronidase and arylsulphatase A were studied. Rapid decreases occurred in the activities of the first six enzymes, reaching minimal values at between 4 and 8 weeks. Activities then increased in all cases to control or nearly control levels. This rate of increase was least for cytochrome P-450. At 4 weeks azoreductase activity with the chemotherapeutic agent CB10-252 (I) as substrate was significantly higher than in control rats. Early increases occurred in the activities of beta-glucuronidase and arylsulphatase A and the activity of the latter never dropped below the control level. (2) An investigation was made of the differential effects of dye feeding on some of the enzyme activities in the two major liver lobes and differences were found. (3) The effect of phenobarbital (PB) pretreatment on the DAB-fed rats was studied at 4-week intervals. The activities of DAB-azoreductase and of nitroreductase increased throughout the whole period, while the activities of the lysosomal enzymes were decreased. (4) After feeding DAB for 4 weeks the effect of PB and 3-methylcholanthrene (MC) on the activities of DAB-azoreductase, CB10-252-azoreductase and components of the azoreductases-cytochrome P-450, NADPH-cytochrome c reductase, the CO-CB10-252-azoreductase was not induced by PB or MC, and CO did not inhibit its reduction. Its reduction depended only slightly on NADH. CO caused a greater relative decrease in the activity of DAB-azoreductase in dye-fed animals and also in animals following PB and MC pretreatment, implying a greater role of cytochrome P-450 in dye-fed animals.     

NH2-terminal sequence analyses of four rat hepatic microsomal cytochromes P-450

Cytochromes P-450f, P-450g, P-450h, and P-450i are four hepatic microsomal hemoproteins that have been purified from adult rats. Whereas cytochromes P-450g and P-450h appear to be male-specific hemoproteins, cytochrome P-450i is apparently a female-specific enzyme purified from untreated adult female rats. Cytochrome P-450f has been purified from adult male and female rats with equivalent recoveries. Amino-terminal sequence analyses of the first 15-20 amino acid residues of each of these cytochromes P-450 has been accomplished in the current investigation. Each protein possesses a hydrophobic leader sequence consisting of 65-87% hydrophobic amino acids, and only one charged amino acid (Asp) in the amino-terminal region. Although differences in the amino-terminal sequences of cytochromes P-450f, P-450g, P-450h, and P-450i are identified, these hemoproteins all begin with Met-Asp, and marked structural homology is observed among certain of these enzymes. Cytochromes P-450g and P-450h, two male-specific proteins, have 11-12/15 identical residues with cytochrome P-450i, a female-specific isozyme. Cytochromes P-450f and P-450h have 16/20 identical amino-terminal residues. Only limited sequence homology is observed between the amino-terminal sequences of cytochromes P-450f-i compared to rat liver cytochromes P-450a-e. The results demonstrate that cytochromes P-450f, P-450g, P-450h, and P-450i are isozymic to each other and five additional rat hepatic microsomal cytochrome P-450 isozymes (P-450a-e).     

Purification and characterization of cytochromes P-450 from liver microsomes of 3-methylcholanthrene-treated crab-eating monkeys

Two forms of cytochrome P-450 (P-450MC1 and P-450MC2) were purified from liver microsomes of crab-eating monkeys (Macaca irus) treated with 3-methylcholanthrene (MC). Monkey P-450MC1 preparation had a specific content of 14.0 nmol/mg protein and showed a main protein band with a minimum molecular weight of 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monkey P-450MC2 preparation had a specific content of 12.1 nmol/mg protein and a minimum molecular weight of 54,000. The carbon monoxide-reduced difference spectral peaks of monkey P-450MC1 and P-450MC2 were at 448 and 447 nm, respectively. In the reconstituted system, monkey P-450MC2 had high activities for benzo[a]pyrene 3-hydroxylation and 7-ethoxycoumarin O-deethylation. Monkey P-450MC1 had low activities toward these two substrates and a high activity for benzphetamine N-demethylation. Monkey P-450MC1 and P-450MC2 were detected by immunoblotting using an antibody prepared against rat cytochrome P-450c, which is a major form of cytochrome P-450 in liver microsomes of MC-treated rats. These results suggested that the molecular properties of cytochrome P-450 in liver microsomes of crab-eating monkeys treated with MC are similar to those in rats.