Effects of in vivo benzo(a)pyrene treatment on liver microsomal mixed-function oxidase activities of gilthead seabream (Sparus aurata)

Benzo(a)pyrene [B(a)P] treatment of gilthead seabream, 25 mg/kg, i.p. for 5 consecutive days, did not cause any significant changes in ethylmorphine N-demethylase and aniline 4-hydroxylase activities of liver microsomes. The same treatment did not alter the liver microsomal cytochrome b5 content, NADH-cytochrome b5 reductase and NADPH-cytochrome P450 reductase activities. However, benzo(a)pyrene treatment caused a 2–3-fold increase in 7-ethoxyresorufin O-deethylase (7-EROD) activity of gilthead seabream liver microsomes. Although, upon treatment, total cytochrome P450 content of liver microsomes increased about 1.7-fold in 1990 fall, no such increase was observed in spring 1991. However, a new cytochrome P450 with an apparent M_r of 58,000 was observed on SDS-PAGE of liver microsomes obtained from benzo(a)pyrene treated gilthead seabream. Besides, in vitro addition of 0.2 × 10⁻⁶ M benzo(a)pyrene to the incubation mixture inhibited 7-ethoxyresorufin O-deethylase activity by 93%. Gilthead seabream liver microsomal 7-ethoxyresorufin O-deethylase activity was characterized with respect to substrate concentration, amount of enzyme, type of buffer used, incubation period and temperature.     

Characteristcs of microsomal enzyme controls in primary cultures of rat hepatocytes.

Cytochrome P-450, NADPH-cytochrome c reductase, biphenyl hydroxylase, and epoxide hydratase have been compared in intact rat liver and in primary hepatocyte cultures. After 10 days in culture, microsomal NADPH-cytochrome c reductase and epoxide hydratase activities declined to a third of the liver value, while cytochrome P-450 decreased to less than a tenth. Differences in the products of benzo[a]pyrene metabolism and gel electrophoresis of the microsomes indicated a change in the dominant form(s) of cytochrome P-450 in the cultured hepatocytes. Exposure of the cultured cells to phenobarbital for 5 days resulted in a threefold induction in NADPH-cytochrome c reductase and epoxide hydratase activities which was typical of liver induction of these enzymes. Exposure of the cells to 3-methylcholanthrene did not affect these activities. Cytochrome P-450 was induced over two times by phenobarbital and three to four times by 3-methylcholanthrene. The λ_max of the reduced carbon monoxide complex (450.7 nm) and analysis of microsomes by gel electrophoresis showed that the phenobarbital-induced cytochrome P-450 was different from the species induced by 3-methylcholanthrene (reduced carbon monoxide λ_max = 447.9 nm). However, metabolism of benzo[a]pyrene (specific activity and product distribution) was similar in microsomes of control and phenobarbital- and 3-methylcholan-threne-induced hepatocytes and the specific activity per nmole of cytochrome P-450 was higher than in liver microsomes. The activities for 2- and 4-hydroxylation of biphenyl were undetectable in all hepatocyte microsomes even though both activities were induced by 3-methylcholanthrene in the liver. Substrate-induced difference spectra and gel electrophoresis indicated an absence in phenobarbital-induced hepatocytes of most forms of cytochrome P-450 which were present in phenobarbital-induced rat liver microsomes. It is concluded that the control of cytochrome P-450 synthesis in these hepatocytes is considerably different from that found in whole liver, while other microsomal enzymes may be near to normal. Hormonal deficiencies in the culture medium and differential hormonal control of the various microsomal enzymes provide a likely explanation of these effects.     

Identification of cytochrome P450a (P450IIA1) as the principal testosterone 7 alpha-hydroxylase in rat liver microsomes and its regulation by thyroid hormones

In the preceding paper, evidence was presented that rat liver microsomes contain two structurally related isozymes of cytochrome P450, namely cytochromes P450a and P450m, that can both catalyze the 7 alpha-hydroxylation of testosterone. The aim of the present study was to determine the extent to which these two P450 isozymes are responsible for the 7 alpha-hydroxylation of testosterone catalyzed by rat liver microsomes. Four monoclonal antibodies against cytochrome P450a, designated A2, A4, A5, and A7, were prepared in BALB/c mice. Monoclonal antibodies A2 (an IgM), A4 (an IgG2b), and A5 (an IgG1) were determined to be distinct immunoglobulins, whereas A7 could not be distinguished from A5. All of the antibodies were highly specific for cytochrome P450a; none cross-reacted with cytochrome P450m or with 10 other P450 isozymes purified from rat liver microsomes. Competition experiments between unlabeled and horseradish peroxidase-conjugated antibodies revealed that each of the monoclonal antibodies recognized the same epitope on cytochrome P450a. None of the monoclonal antibodies bound to denatured cytochrome P450a, suggesting that they each bound to a spatial epitope. A monospecific, polyclonal antibody against cytochrome P450a was also prepared, as described in the preceding paper. The levels of cytochrome P450a in liver microsomes were determined by single radial immunodiffusion, Western immunoblot (with polyclonal antibody), and enzyme-linked immunosorbent assay with monoclonal antibody. The levels of cytochrome P450a declined with age in male but not female rats, and were inducible up to 10-fold by treatment of rats with various xenobiotics. The levels of cytochrome P450a (but not cytochrome P450m) were also elevated (approximately 3-fold) by thyroidectomy of mature male rats. Near normal levels of cytochrome P450a were restored by treatment of athyroid rats with triiodothyronine, whereas treatment with thyroxine was less effective in this regard. These changes in the levels of cytochrome P450a were highly correlated (r = 0.995) with changes in testosterone 7 alpha-hydroxylase activity. None of the monoclonal antibodies inhibited the catalytic activity of cytochrome P450a when reconstituted with NADPH-cytochrome P450 reductase and lipid. In contrast, the polyclonal antibody not only inhibited the catalytic activity of purified cytochrome P450a, but also completely inhibited (greater than 96%) the 7 alpha-hydroxylation of testosterone catalyzed by liver microsomes from immature and mature rats of both sexes and by liver microsomes from male rats treated with a variety of cytochrome P450 inducers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of n-hexane inhalation on the monooxygenase system in mice liver microsomes

When mice are continuously exposed to an atmosphere containing 2.5–3% n-hexane over a period of 4 days liver growth is observed after 24 h. Concomitantly the specific activities of cytochrome P450 and the cytochrome P450 reductase increase with a maximum after 38 h. The cytochrome b₅ levels in liver microsomes begin to rise at the second day and continue to increase even after the fourth day. No strict correlation is observed between the cytochrome P4SO content and the monooxygenation activity for cyclohexane. The pattern of hydroxylation products from n-hexane changes significantly upon pretreatment of the animals, suggesting that a qualitative alteration in the cytochrome P450 species had occurred.     

Spectral characteristics of a compound altering cytochrome P450 spectra from vertebrate microsomes suggest that it is a functional protein

A peak near 420 nm interfering with the spectral detection of cytochrome P450 has been reported for invertebrates and fish. It has been variously suggested to be a breakdown product of P450, or a hemoprotein with unknown functions. Similar spectra were observed in the present work with a neotropical fish, an amphibian, and rodents. Comparative analysis showed that difference spectra resulted from an unknown hemoprotein and neither from P420, nor from hemoglobin, that may contaminate animal microsomes. Seasonal appearance of this protein was observed and its spectrum described. This protein completely substituted P450 in spectra of liver microsomes of fish and rodents collected in the summer, while in the winter the same animals displayed either the classic P450 spectra (rodents) or those accompanied with the low-intensity 421-nm peak (fish). We suggest that the compound visualized in P450 spectra is a functional protein and not an artifact. The possibility that an unknown protein may substitute for cytochrome P450 in microsomes under certain environmental conditions and play a role in animal adaptation to unfavorable environmental fluctuations is discussed.     

Reconstitution of testosterone oxidation by purified rat cytochrome P450p (IIIA1)

Cytochrome P450p (IIIA1) has been purified from rat liver microsomes by several investigators, but in all cases the purified protein, in contrast to other P450 enzymes, has not been catalytically active when reconstituted with NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. We now report the successful reconstitution of testosterone oxidation by cytochrome P450p, which was purified from liver microsomes from troleandomycin-treated rats. The rate of testosterone oxidation was greatest when purified cytochrome P450p (50 pmol/ml) was reconstituted with a fivefold molar excess of NADPH-cytochrome P450 reductase, an equimolar amount of cytochrome b5, 200 micrograms/ml of a chloroform/methanol extract of microsomal lipid (which could not be substituted with dilauroylphosphatidylcholine), and the nonionic detergent, Emulgen 911 (50 micrograms/ml). Testosterone oxidation by cytochrome P450p was optimal at 200 mM potassium phosphate, pH 7.25. In addition to their final concentration, the order of addition of these components was found to influence the catalytic activity of cytochrome P450p. Under these experimental conditions, purified cytochrome P450p converted testosterone to four major and four minor metabolites at an overall rate of 18 nmol/nmol P450p/min (which is comparable to the rate of testosterone oxidation catalyzed by other purified forms of rat liver cytochrome P450). The four major metabolites were 6 beta-hydroxytestosterone (51%), 2 beta-hydroxytestosterone (18%), 15 beta-hydroxytestosterone (11%) and 6-dehydrotestosterone (10%). The four minor metabolites were 18-hydroxytestosterone (3%), 1 beta-hydroxytestosterone (3%), 16 beta-hydroxytestosterone (2%), and androstenedione (2%). With the exception of 16 beta-hydroxytestosterone and androstenedione, the conversion of testosterone to each of these metabolites was inhibited greater than 85% when liver microsomes from various sources were incubated with rabbit polyclonal antibody against cytochrome P450p. This antibody, which recognized two electrophoretically distinct proteins in liver microsomes from troleandomycin-treated rats, did not inhibit testosterone oxidation by cytochromes P450a, P450b, P450h, or P450m. The catalytic turnover of microsomal cytochrome P450p was estimated from the increase in testosterone oxidation and the apparent increase in cytochrome P450 concentration following treatment of liver microsomes from troleandomycin- or erythromycin-induced rats with potassium ferricyanide (which dissociates the cytochrome P450p-inducer complex). Based on this estimate, the catalytic turnover values for purified, reconstituted cytochrome P450p were 4.2 to 4.6 times greater than the rate catalyzed by microsomal cytochrome P450p.     

Catalytic activity of isolated cytochromes P-450d and P-450c

Cytochrome P-450d was isolated from isosafrol-induced rat liver microsomes by affinity chromatography on 1.8-diaminooctyl-Sepharose 4B and chromatography on hydroxylapatite using a linear potassium phosphate gradient (45-250 mM). The enzyme has a molecular mass of 54 kDa, CO-maximum 448 nm is characterized by a high spin state; the rate of 4-aminobiphenyl hydroxylation is 54 nmol/min/nmol of cytochrome P-450d (37 degrees C), those, of 7-ethoxyresorufin O-deethylation and benz (a) pyrene oxidation are 1 nmol/min/nmol of cytochrome P-450d (22 degrees C) and 2 nmol/min/nmol of cytochrome P-450d (37 degrees C), respectively. The properties of cytochrome P-450d were compared to those of cytochrome P-450c isolated from 3-methylcholanthrene-induced rats. The yield of these cytochromes under the conditions used (10% P-450d from isosafrol-induced microsomes and 15% P-450c from 3-methylcholanthrene-induced microsomes) was relatively high. Antibodies to cytochromes P-450d and P-450c were obtained. Using rocket immunoelectrophoresis the percentage of these hemoprotein forms in 3-methylcholanthrene-induced (P-450d-20%, P-450c-70%) and isosafrol-induced rat liver microsomes (P-450d-50%, P-450c-15%) was determined.     

Target organ-specific inactivation of drug metabolizing enzymes in kidney of hamsters treated with estradiol

Chronic treatment of hamsters with estradiol for several months has previously been shown to decrease the specific content of cytochrome P450 in the kidney, a target of hormonal carcinogenesis, but not in liver. The reason for this decrease in metabolic enzyme activity is unknown and has been examined in this investigation. We now report that the decrease in specific content of renal cytochrome P450 by 73% in response to estradiol was not affected by co-treatment with tamoxifen for 1 month. The subcutaneous infusion of 250 μg/day estradiol for 7 days lowered renal cytochrome P450 by 71% from control values and was therefore used for further mechanistic studies. This treatment decreased renal activities of estradiol 2- or 4-hydroxylase by 77 to 80%, of 7-ethoxycoumarin-O-deethylase by 66% of control values, respectively, and completely eliminated aryl hydrocarbon hydroxylase activities, whereas liver enzymes remained unaffected. After 7 days of infusion of estradiol, fluorescent products of lipid peroxidation were more than doubled in hamster kidney but remained unchanged in liver. The possibility of enzyme destruction by binding of estradiol 2,3-quinone to metabolizing enzymes was investigatedin vitro. In the presence of 2-hydroxyestradiol, cumene hydroperoxide, and microsomes, conditions known to favor the oxidation of the steroid to quinone, the binding of catechol estrogen metabolite to microsomal protein increased 60 fold over control values in the absence of cofactor. Purified rat liver cytochrome P450c also oxidized 2-hydroxyestradiol to 2,3-estradiol quinone. The rate of oxidation was linear for the first 2–3 min, but thereafter decreased with time. Under these incubation conditions, irreversible binding of catechol estrogen metabolite to cytochrome P450c increased for the first 2–3 min and then remained at this plateau level. It was concluded that enzyme destruction by a reactive estrogen metabolite or by lipid peroxides may be a major reason for the organ-specific decrease in cytochrome P450 enzymes in kidneys of estrogen-treated hamsters.     

Functional characteristics of cytochromes P-4501A1 and P-4501A2 in rodent liver

Antibodies to mouse liver cytochrome P3-450 (anti-P3-450) and antibodies to rat liver cytochrome P-450d (anti-P-450d-c) inhibit the 0-deethylation of 7-ethoxyresorufin (ER) in liver microsomes of benz(a)pyrene-induced (BP) mice but do not inhibit the 0-deethylase activity in liver microsomes of BP-induced rats. Anti-P3-450 and anti-P-450c inhibit BP-hydroxylation in BP-induced mouse liver microsomes by 20%, but they do not inhibit this reaction at all in BP-induced rat liver microsomes. In a reconstituted monooxygenase system isolated cytochrome P3-450 metabolized 7-ER and BP. In contrast, its homologue, cytochrome P-450d, did not metabolize these substrates. The fraction containing cytochrome P1-450 metabolized 7-ER at a low rate and BP at a rate of 3.6 nmol product/min/nmol cytochrome. Western blot analysis with anti-P-450c + d revealed two bands in SDS-PAGE gels containing BP-induced mouse liver microsomes. The interaction of mouse liver BP-microsomes with anti-P3-450 and anti-P-450d-c was accompanied by the appearance of a single band (cytochrome P3-450).     

The proximate carcinogen trans-3,4-dihydroxy-3,4-dihydro-dibenz[c,h]acridine is oxidized stereoselectively and regioselectively by cytochrome 1A1, epoxide hydrolase and hepatic microsomes from 3-methylcholanthrene-treated rats.

Metabolism of the proximate carcinogen trans-3,4-dihydroxy-3,4-dihydrodibenz[c,h]acridine has been examined with rat liver enzymes. The dihydrodiol is metabolized at a rate of 2.4 nmol/nmol of cytochrome P450 1A1/min with microsomes from 3-methylcholanthrene-treated rats, a rate more than 10-fold higher than that observed with microsomes from control or phenobarbital-treated rats. Major metabolises consisted of a diastereomeric pair of bis-dihydrodiols (68-83%), where the new dihydrodiol group has been introduced at the 8,9-position, tetraols derived from bay region 3,4-diol-1,2-epoxides (15-23%), and a small amount of a phenolic dihydrodiol(s) where the new hydroxy group is at the 8,9-position of the substrate. A highly purified monooxygenase system reconstituted with cytochrome P450 1A1 and epoxide hydrolase (17 nmol of metabolites/nmol of cytochrome P450 1A1/min) gave a metabolite profile very similar to that observed with liver microsomes from 3-methylcholanthrene-treated rats. Study of the stereoselectivity of these microsomes established that the (+)-(3S,4S)-dihydrodiol gave mainly the diol epoxide-1 diastereomer, in which the benzylic 4-hydroxyl group and epoxide oxygen are cis. The (-)-(3R,4R)-dihydrodiol gave mainly diol epoxide-2 where these same groups are trans. The major enantiomers of the diastereomeric bis-dihydrodiols are shown to have the same absolute configuration at the 8,9-position. Correlations of circular dichroism spectra suggest this configuration to be (8R,9R). The (8R,9S)-oxide may be their common precursor.     

Purification of two isozymes of rat liver microsomal cytochrome P450 with testosterone 7 alpha-hydroxylase activity

Cytochrome P450a was purified to electrophoretic homogeneity from liver microsomes from immature male Long-Evans rats treated with Aroclor 1254. Rabbit polyclonal antibody raised against cytochrome P450a cross-reacted with cytochromes P450b, P450e, and P450f (which are structurally related to cytochrome P450a). The cross-reacting antibodies were removed by passing anti-P450a over an N-octylamino-Sepharose column containing these heterologous antigens. The immunoabsorbed antibody recognized only a single protein (i.e., cytochrome P450a) in liver microsomes from immature male rats treated with Aroclor 1254 (i.e., the microsomes from which cytochrome P450a was purified). However, the immunoabsorbed antibody recognized three proteins in liver microsomes from mature male rats, as determined by Western immunoblot. As expected, one of these proteins (Mr 48,000) corresponded to cytochrome P450a. The other two proteins did not correspond to cytochromes P450b, P450e, or P450f (as might be expected if the antibody were incompletely immunoabsorbed), nor did they correspond to cytochromes P450c, P450d, P450g, P450h, P450i, P450j, P450k, or P450p. One of these proteins was designated cytochrome P450m (Mr approximately 49,000), the other cytochrome P450n (Mr approximately 50,000). Like cytochrome P450a, cytochrome P450n was present in liver microsomes from both male and female rats. However, whereas cytochrome P450a was detectable in liver microsomes from 1-week-old rats, cytochrome P450n was barely detectable until the rats were at least 3 weeks old. Furthermore, in contrast to cytochrome P450a, the levels of cytochrome P450n did not decline appreciably with age in postpubertal male rats. Cytochrome P450m was detectable only in liver microsomes from postpubertal (greater than 4 week-old) male rats. Cytochromes P450m and P450n were isolated from liver microsomes from mature male rats and purified to remove cytochrome P450a. When reconstituted with NADPH-cytochrome P450 reductase and lipid, cytochrome P450n exhibited little testosterone hydroxylase activity, whereas cytochrome P450m catalyzed the 15 alpha-, 18-, 6 beta-, and 7 alpha-hydroxylations of testosterone at 10.8, 4.6, 2.0, and 1.9 nmol/nmol P450/min, respectively. The ability of cytochrome P450m to catalyze the 7 alpha-hydroxylation of testosterone was not due to contamination with cytochrome P450a, which catalyzed this reaction at approximately 25 nmol/nmol P450a/min. Cytochrome P450m also converted testosterone to several minor metabolites, including androstenedione and 15 beta-, 14 alpha-, and 16 alpha-hydroxytestosterone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. II. Kinetic parameters of reductase and monooxygenase reactions.

The kinetic parameters of NADPH-dependent cytochrome P450 LM2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase. In this system substrate-free cytochrome P450 LM2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM2 reduction was found in the system. Vmax values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas Km values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio at a constant total protein concentration allowed the Kd of the NADPH-cytochrome P450 reductase/cytochrome P450 LM2 complex to be estimated as 6.4 +/- 0.5 microM. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.     

Catalysis of the oxidation of steroid and stilbene estrogens to estrogen quinone metabolites by the beta-naphthoflavone-inducible cytochrome P450 IA family.

Diethylstilbestrol (DES) or catecholestrogens are metabolized by microsomal enzymes to quinones, DES Q or catecholestrogen quinones, respectively, which have been shown to bind covalently to DNA and to undergo redox cycling. The isoforms of cytochrome P450 catalyzing this oxidation of estrogens to genotoxic intermediates were not known and have been identified in this study by (a) using microsomes of rats treated with various inducers of cytochrome P450; (b) using purified cytochrome P450 isoforms; and (c) examining the peroxide cofactor concentrations necessary for this oxidation by microsomes or pure isoenzymes. The highest rate of oxidation of DES to DES Q was obtained using beta-naphthoflavone-induced microsomes (14.0 nmol DES Q/mg protein/min) or cytochrome P450 IA1 (6.4 pmol DES Q/min/pmol P450). Isosafrole-induced microsomes or cytochrome P450 IA2 oxidized DES to quinone at one-third or one-fifth of that rate, respectively. Low or negligible rates of oxidation were measured when oxidations were catalyzed by microsomal rat liver enzymes induced by phenobarbital, ethanol, or pregnenolone-16 alpha-carbonitrile or by pure cytochromes P450 IIB1, IIB4, IIC3, IIC6, IIE1, IIE2, IIG1, or IIIA6. Cytochrome P450 IA1 also catalyzed the oxidation of 2- or 4-hydroxyestradiol to their corresponding quinones. The beta-naphthoflavone-induced microsomes and cytochrome P450 IA1 had the highest "affinity" for cumene hydroperoxide cofactor (Km = 77 microM). Cofactor concentrations above 250 microM resulted in decreased rates of oxidation. The other cytochrome P450 isoforms required much higher cofactor concentrations and were not inactivated at high cofactor concentrations. The data demonstrate that beta-naphthoflavone-inducible cytochrome P450 IA family enzymes catalyze most efficiently the oxidation of estrogenic hydroquinones to corresponding quinones. This oxidation may represent a detoxification pathway to keep organic hydroperoxides at minimal concentrations. The resulting quinone metabolites may be detoxified by other pathways. However, in cells with decreased detoxifying enzyme activities, quinones metabolites may accumulate and initiate carcinogenesis or cell death by covalent arylation of DNA or proteins.     

In vitro metabolism of FK-506 in rat, rabbit, and human liver microsomes: identification of a major metabolite and of cytochrome P450 3A as the major enzymes responsible for its metabolism.

The metabolism of the immunosuppressant FK-506 was shown to be catalyzed primarily by cytochrome P450 isozymes of the P450 3A subfamily. Antibodies against rat P450 3A inhibited FK-506 metabolism by 82% in rat liver microsomes and by 35-56% in liver microsomes from humans, dexamethasone-induced rats, and erythromycin-induced rabbits. Poor species cross-reactivity of the antibodies, metabolic switching, and/or some metabolism by P450 isozymes other than P450 3A may be responsible for the incomplete inhibition observed. Besides anti-rat P450 3A, antibodies against rat P450 1A also appeared to have some inhibitory effect implicating these particular cytochrome P450 isozymes as having a minor role in FK-506 metabolism. The formation of 13-desmethyl FK-506, identified here as a major metabolite of FK-506 in all types of microsomes examined, was inhibited completely by anti-P450 3A in liver microsomes from dexamethasone-induced rats and erythromycin-induced rabbits but only partially in human and control rat liver microsomes.     

Isosafrole-induced cytochrome P2-450 in DBA/2N mouse liver. Characterization and genetic control of induction

Mouse "cytochrome P2-450" is defined as that form of isosafrole-induced P-450 in DBA/2N liver most specifically correlated with isosafrole metabolism. Isosafrole pretreatment does not induce aryl hydrocarbon hydroxylase activity ("cytochrome P1-450") in C57BL/6N or DBA/2N mice, induces acetanilide 4-hydroxylase activity ("cytochrome P3-450") more than 3-fold in C57BL/6N but not in DBA/2N mice, and induces isosafrole metabolite formation more than 3-fold in both C57BL/6N and DBA/2N mice. P2-450 was, therefore, purified from isosafrole-treated DBA/2N liver microsomes having negligible amounts of contaminating P1-450 and P3-450. The apparent molecular weight of P2-450 is 55,000, and the protein appears homogeneous on sodium dodecyl sulfate-polyacrylamide gels. The Soret peak of the reduced purified cytochrome X CO complex is 448 nm. Purified P2-450, reconstituted in vitro, metabolizes acetanilide poorly and benzo[a]pyrene hardly at all. Anti-(P2-450) inhibits (90 to 100%) liver microsomal isosafrole metabolite formation, yet has no effect on aryl hydrocarbon hydroxylase, acetanilide 4-hydroxylase, biphenyl 2- or 4-hydroxylase, or 7-ethoxycoumarin O-de-ethylase activities. 3-Methylcholanthrene induces anti-(P2-450)-precipitable protein about 12-fold in C57BL/6N and 2-fold in DBA/2N liver; 2,3,7,8-tetrachlorodibenzo-p-dioxin (10 micrograms/kg), about 12-fold in both C57BL/6N and DBA/2N liver; isosafrole, more than 3-fold in both C57BL/6N and DBA/2N. Benzo[a]anthracene at maximal doses induces anti-(P2-450)-precipitable protein in C57BL/6N liver no more than 2-fold, yet is known to be a highly potent inducer of P1-450 mRNA in C57BL/6N liver. The sensitivity of the P2-450 induction process to isosafrole is inherited as an autosomal additive trait; studies of offspring from the C57BL/6N(DBA/N)F1 X DBA/2N backcross confirm involvement of the Ah locus or s closely segregating gene. In contrast, among crosses between C57BL/6N and DBA/2N, sensitivity of the P1-450 and P3-450 induction process to 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin is inherited as an autosomal dominant trait. These data suggest that, although P1-450, P2-450, and P3-450 proteins are controlled by the Ah locus, either a P-450 protein polymorphism exists between C57BL/6N and DBA/2N mice or subtle differences may exist in the interaction of various inducers with Ah receptor.     

Gender difference in enzymes related with alcohol consumption in hamster,an avid consumer of alcohol

Syrian golden hamster (Mesocricetus auratus) is extraordinary among laboratory rodents in its ability to drink alcohol. After being given a free choice between 15% ethanol and water for 5 days, both male and female hamsters derived at least 85% of the fluid intake from the ethanol solution. Analysis of the alcohol-metabolizing enzymes in alcohol-na??ve hamsters showed that the male had a higher activity of 57%, 58% and 34% in stomach alcohol dehydrogenase, liver cytochrome P450 1A2 and liver aldehyde dehydrogenase, respectively, compared with the female. The activity of lung angiotensin-converting enzyme, which influence fluid intake, was twofold higher in the male. After 4 weeks of ethanol consumption, the activities of the hepatic alcohol-metabolizing enzymes remained unchanged except cytochrome P450 2E1 which increased 42% and 88% in male and female hamsters, respectively. A reduction of ～80% in the activity of cytochrome P450 1A2 was observed in both genders. The activities of several other cytochrome P450 enzymes were also decreased. Although ethanol consumption did not increase plasma aminotransferase levels, it caused a significant increase in liver weight in female, but not male hamsters.     

Aflatoxin B1 metabolism by 3-methylcholanthrene-induced hamster hepatic cytochrome P-450s

We have studied the activation of aflatoxin B1 by hamster liver microsomes and purified hamster cytochrome P-450 isozymes using a umu mutagen test. The hamster liver microsomes or S-9 fractions were much more active than rat liver microsomes or S-9 fractions in the activation of umu gene expression by aflatoxin B1 metabolites. 3-Methyl-cholanthrene treatment increased aflatoxin B1 activation by hamster liver microsomes. Two major 3-methylcholanthrene-inducible cytochrome P-450 isozymes, P-450 MC1 (IIA) and P-450 MC4 (IA2), were purified from 3-methylcholanthrene-treated hamster liver microsomes, and the metabolism of aflatoxin B1 by these two cytochromes was studied. In the reconstituted enzyme system, both P-450 MC1 and P-450 MC4 were highly active in the activation of aflatoxin B1, and antibodies against these P-450s specifically inhibited these activities. Antibody against P-450 MC1 inhibited the activation of aflatoxin B1 by 20% in the presence of 3-methyl-cholanthrene-treated hamster liver microsomes. In contrast, antibody against P-450 MC4 stimulated the activity by 175%. These results indicated that hamster P-450 MC1 might convert aflatoxin B1 to more toxic metabolite(s), whereas P-450 MC4 might convert aflatoxin B1 to less toxic metabolite(s), than aflatoxin B1 in liver microsomes. The metabolite(s) produced by both hamster cytochrome P-450 MC1 and MC4 were genotoxic in the umu mutagen test.     

Characterization of covalent binding of [14C]-2-chloro-4-acetotoluidide to microsomes of starling liver and kidney

In this study, we have characterized the covalent binding of [14C]-2-chloro-4-acetotoluidide (CAT) radioactivity to microsomes of starling liver and kidney. The maximal velocity (Vmax) of covalent binding and apparent Michaelis constant (Km) for both tissues were similar. The Vmax for liver and kidney were 52.8 and 68.9 pmol/min/mg protein, and the apparent Kms were 0.54 and 0.87 mM, respectively. The covalent binding of radioactivity to heat-denatured microsomes of liver and kidney was reduced by 62% and 15%, respectively. Incubation at 0 degrees C reduced the binding by 80% to liver and 70% to kidney microsomes. Absence of nicotinamide adenine dinucleotide phosphate (NADP) and molecular O2 reduced the binding to liver microsomes by 36 and 53%, as opposed to 28% increase and 26% decrease in binding to kidney microsomes, respectively. Inducers of cytochrome P450 monooxygenase (P450), phenobarbital, and 3-methylcholanthrene (3-MC), had opposite effects on the covalent binding of [14C]-CAT radioactivity to hepatic and renal microsomes. Phenobarbital increased the binding to hepatic microsomes by 100% and had no effect on binding to renal microsomes. 3-MC, on the other hand, increased the binding to kidney microsomes by threefold and had no effect on the binding to hepatic microsomes. SKF 525A, an inhibitor of P450, inhibited the binding to hepatic microsomes by 60% at 0.5 mM but failed to have any effect on binding to renal microsomes. alpha-Naphthoflavone, another inhibitor of P450, had no effect on the covalent binding of [14C]-CAT radioactivity to microsomes of either tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction and regulation of cytochrome P450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation

The effects of starvation on rat renal cytochrome P-450s were studied. The content of spectrally measured cytochrome P-450 in the renal microsomes of male rats increased 2-fold with 72 h starvation, but cytochrome b5 and NADPH-cytochrome P-450 reductase were not induced. 7-Ethoxycoumarin O-dealkylation and aniline hydroxylation activities of the renal microsomes of control male rats were very low but were induced 2.5-3-fold by 72 h starvation. Aminopyrine N-demethylation and lauric acid hydroxylation activities were induced 1.5-2-fold by 72 h starvation. The changes in catalytic activities suggested that the contents of individual cytochrome P-450s in the renal microsomes were altered by starvation. The contents of some cytochrome P-450s were measured by Western blotting. P450 DM (P450IIE1), a typical form of cytochrome P-450 induced by starvation in rat liver, was barely detected in rat kidney and was induced 2-fold by 72 h starvation. P450 K-5, a typical renal cytochrome P-450 and lauric acid hydroxylase, accounted for 81% of the spectrally measured cytochrome P-450 in the renal microsomes of control male rats and was induced 2-fold by 72 h starvation. P450 K-5 was not induced in rat kidney by treatment with chemicals such as acetone or clofibrate. The renal microsomes of male rats contained 6-times as much P450 K-5 as those of female rats. These results suggest that P450 K-5 is regulated by an endocrine factor.     

Immunochemical characterization of some monooxygenase activities in liver microsomes from untreated and phenobarbital-treated rats

Two major forms of liver microsomal cytochrome P 450, one from untreated rats (P 450 A₂NI) and the other from phenobarbital-treated rats (P 450 B₂PB), were partially purified. Reconstitution of monooxygenase activities of purified enzymes and inhibition patterns of these activities by antibodies in microsomes gave the following results: 1) aniline hydroxylase activity is mainly supported by cytochrome P 450 A₂NI. This form is the major one in microsomes from control rats, but is also found at minute amounts in microsomes from phenobarbital-treated rats. It behaves as a constitutive form. 2) 4-nitroanisole-and benzphetamine-demethylase activities are mainly supported by cytochrome P 450 B₂PB which is predominant in phenobarbital-treated rats but is also present in control microsomes at low levels. 3) 4-nitroanisole-O-demethylase activity is less specific than benzphetamine-N-demethylase activity towards cytochrome P 450 B₂PB.