Hydroxylation of benzo[a]pyrene and binding of (−)trans 5-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to deoxyribonucleic acid catalyzed by purified forms of rabbit liver microsomal cytochrome P-450: Effect of 7,8-benzoflavone,butylated hydroxytoluene and ascorbic acid

The catalytic activities of hepatic microsornes from untreated, phenobarbital-treated and 3-methylcholanthrene-treated adult rabbits with respect to benzo[a]pyrene hydroxylation and the activation of (?)(rflw-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene[(?)trans-7,8-diol] to DNA-binding metabolites were determined in the absence and presence of mixed-function oxidase inhibitors and compared to the corresponding activities of the individual enzyme systems. Treatment of rabbits with phnobarbital led to induction of P-450LM₂ and a concomitant 3-fold enhancement in microsomal benzo[a]pyrene hydroxylase activity, whereas the conversion of (?)trans-7,8-diol to DNA-binding products was unaffected. Homogeneous phenobarbital-inducible P-450LM₂ exhibited the highest activity and specificity toward benzo[a]pyrene and the lowest activity toward (?)trans-7,8-diol. Conversely, P-450LM₄ was the major form of cytochrome P-450 induced in rabbit liver by 3-methylcholanthrene or β-naphthoflavone, and this was associated in microsomes with an increase in the metabolism of (?)trans-7, 8-diol but not of benzo[a]pyrene. Homogeneous P-450LM₄ preferentially Catalyzed the oxygénation of (?)trans-7,8-diol, but was largely ineffective with benzo[a]pyrene. Partially purified P-450LM₇ lacked substrate specificity, for it metabolized both benzo[a]pyrene and (?)trans-7, S-diol at comparable rates. Additionally, 7,8-benzoflavone strongly inhibited benzo[a]pyrene hydroxylation by P-450LM₄ and phenobarbital-induced microsomes, as well as (?)trans-7,8-diol metabolism by P-450LM₄ and 3-methyl-cholanthrene-induced microsomes; in contrast, the activity of control microsomes with either substrate, and the activities of P-450LM₄ and LM₂ with benzo[a]pyrene and (?)trans-7 ,8-diol, respectively, were only partially or slightly decreased by 7,8-benzoflavone. Unlike 7,8-benzoflavone, butylated hydroxytoluene inhibited benzo[a]pyrene hydroxylation only. Thus, different forms of rabbit liver microsomal cytochrome P-450 were involved in the metabolism of benzo[a]pyrene and its 7,8-dihydrodiol. The results also demonstrate that the changes in substrate specificity and inhibitor sensitivity seen in phenobarbital- and 3-methylcholanthrene-induced microsomes relative to control rabbit liver microsomes can be accounted for by the catalytic properties of a specific form of cytochrome P-450 that prevails in these preparations, P-450LM₂ and LM₄, respectively.     

Immunological and enzymatic comparison of hepatic cytochrome P-450 fractions from phenobarbital-, 3-methylcholanthrene-, β-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats

A comparison of the cytochrome P-450 forms induced in rat liver microsomes by phenobarbital on the one hand, and 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin on the other hand, was performed using specific antibodies: anti-P-450 B₂ PB IG (against the phenobarbital-induced cytochrome P-450) and anti-P-450 B₂ BNF IG (against the β-naphtoflavone-induced cytochrome P-450). On DEAE-cellulose chromatography, four cytochrome P-450 fractions were separated, called P-450 A (non-adsorbed), P-450 Ba, P-450 Bb and P-450 Bc, from control, phenobarbital-, 3-methylcholanthrene, /gb-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats. Cytochrome P-450 A fractions appeared to be unmodified by the inducers, whereas the specifically induced cytochrome P-450 forms were always recovered in Bb fractions. The P-450 Bb fractions induced by 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibited common antigenic determinants, comparable catalytic activities (benzphetamine, N-demethylase, benzo[a]pyrene hydroxylase) and similar mol. wts. Moreover, the inhibition patterns by the two antibodies of benzphetamine N-demethylase and benzo[a]pyrene hydroxylase activities catalysed by 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin microsomes or by the corresponding P-450 Bb fractions in a reconstituted system were quite identical. By these different criteria, β-naphtoflavone, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin seem to induce a common cytochrome P-450 species in rat liver.     

Further studies on the increase in drug-metabolizing capacity adjacent to intrahepatic Morris hepatomas

Microsomal cytochrome P-450 content was higher in histologically non-tumorous liver adjacent to intrahepatically implanted Morris hepatomas 5123D or 7795 than in histologically normal liver far removed from each tumor. V_max values for microsomal benzo[a]pyrene monooxygenase activity and cyclophosphamide activation were also significantly higher in tumor-adjacent liver than in normal liver far removed from tumor. K_m values of these reactions were unchanged. After intrahepatic implantation, inert spheres of several different materials produced no regional differences in hepatic microsomal cytochrome P-450 content. Both intrahepatic Morris hepatomas exhibited markedly reduced cytochrome P-450 content and benzo[a]pyrene monooxygenase activity. Cyclophosphamide biotransformation could not be detected in microsomes from either Morris hepatoma. Similar recoveries from microsomes of far-removed and tumor-adjacent liver indicated that differences between these regions in drug-metabolizing activity could not be attributed to different stabilities or sedimenting properties of their microsomes. Although microsomal recovery was significantly less from hepatomas than from far-removed or tumor-adjacent liver, this loss of tumor microsomes accounted for only a small part of the reductions in cytochrome P-450-mediated monooxygenases observed within tumors. Compared to control rats. tumor-bearing rats exhibited no change in hepatic drug-metabolizing capacity measured in vivo by hexobarbital sleeping times and antipyrine elimination rates. Phenobarbital (PB) pretreatment of tumor-bearing rats induced cytochrome P-450 to different extents within far-removed liver, tumoradjacent liver, and both hepatomas. The same differential inducibility occurred with PB pretreatment for cyclophosphamide activation. After PB induction, differences in drug-metabolizing activity between far-removed and tumor-adjacent liver disappeared; though induced, these activities remained lower in the hepatomas than in other regions. These changes in drug-metabolizing activity in both basal and PB-induced states of various hepatic regions were related to changes in cellularity of tumor-adjacent tissue. Hepatocellular nuclei prepared from tumor-containing liver were separated into diploid and tetraploid classes by sucrose density gradient centrifugation. Compared to far-removed liver, tumoradjacent liver contained significantly more diploid nuclei and less tetraploid nuclei.     

Induction of rat hepatic epoxide hydratase by dietary antioxidants.

Supplementation of rat diet with butylated hydroxytoluene (BHT), butylated hydroxyanisole, or ethoxyquin resulted in increased liver epoxide hydratase activity. The increase was obvious at 0.1% and amounted to 200–400% at 0.5%. Increased activity was accompanied by increased proportion of the epoxide hydratase band in SDS polyacrylamide gels, indicating induction of the enzyme. Ethoxycoumarin deethylase activity and cytochrome b₅ concentrations were moderately elevated while cytochrome P-450 concentrations and aryl hydrocarbon hydroxylase activity remained at control levels. Preferential inhibition of monooxygenase activity by metyrapone and not 7,8-benzoflavone, as well as increased affinity of the reduced cytochrome P-450 for metyrapone as a ligand, indicated that the cytochrome P-450 population after BHT treatment was similar to that found after phenobarbital treatment. The antioxidants used in this study had no in vitro effect on epoxide hydratase activity and inhibited monooxygenase activity only in phenobarbital-stimulated microsomes but not in 3-methylcholanthrene-stimulated microsomes. Combined treatment with dietary antioxidants and intraperitoneally administered 3-methylcholanthrene resulted in marked induction of epoxide hydratase activity while the 3-methylcholanthrene-mediated increase in aryl hydrocarbon hydroxylase activity was partially depressed. Covalent binding of tritiated benzo[a]pyrene to calf thymus DNA was less effectively catalyzed by liver microsomes from animals fed antioxidants. The depression of covalent binding was marked after combined treatment with antioxidants and 3-methylcholanthrene. The shift in the microsomal enzyme pattern caused by antioxidants may be related to their inhibitory effects on chemical carcinogenesis.     

Enhanced aryl hydrocarbon hydroxylase activity after interaction between solubilized cytochrome P-448 and microsomes low in endogenous cytochrome P-450

The effects of cumene hydroperoxide on microsomal mixed-function oxidase components and enzyme activities were determined. In vitro cumene hydroperoxide treatment decreased cytochrome P-450 content, benzphetamine N-demethylase activity and aryl hydrocarbon hydroxylase activity of hepatic and renal microsomes from adult male and female rats, and of hepatic microsomes from fetal rats. Cumene hydroperoxide-treated microsomes, as well as fetal liver and adult renal microsomes, which are naturally low in cytochrome P-450 and mixed-function oxidase activity, were used to incorporate partially purified hepatic cytochrome P-448 isolated from 2,3,7,8-tetrachlorodibenzo-p-dioxin-pretreated immature male rats. This resulted in an enhanced rate of benzo[a]pyrene hydroxylation. Aryl hydrocarbon hydroxylase activity was increased 12-, 26-. 31- and 53-fold when 1.0 nmole of partially purified cytochrome P-448 was incubated with fetal liver microsomes, microsomes from kidney cortex of female rats, and cumene hydroperoxide-pretreated hepatic microsomes from female and male rats, respectively. The increased rate of benzo[a]pyrene hydroxylation was linear with cytochrome P-448 over the range 0.25 to 1.0 nmole. Because cumene hydroperoxide-pretreated microsomes from male rat liver and the hepatic and renal microsomes from female rats have a combination of high NADPH-cytochrome c reductase activity and low mixed-function oxidase activity, they are an attractive choice for catalytic studies of the interaction between cytochrome P-448 and microsomes.     

Biochemical and ultrastructural changes in livers of cadmium-treated rats.

The effect of ethoxyquin in vivo and in vitro on drug metabolism in rat liver microsomes was studied. In feeding experiments, a threshold dose of induction was found at 0.05% ethoxyquin for 14 days. At 0.5% ethoxyquin, relative liver weight, cytochrome P-450 content, cytochrome b₅ content, ethylmorphine demethylation, and ethoxycoumarin deethylation were increased by a factor of 1.5 to 2. Aryl hydrocarbon hydroxylase activity was, however, not induced but even decreased by 0.5% ethoxyquin in food. Induction of epoxide hydratase was marked, amounting to 400% of control after 0.5% ethoxyquin. The induced enzyme was similar to the phenobarbital-inducible cytochrome P-450 in its CO spectrum, in its affinity for the ligand metyrapone, in the preferential inhibition of monooxygenase activity by metyrapone and not α-naphthoflavone, and in the increase in the phenobarbital-inducible band after gel electrophoresis of microsomal proteins. Mixed pretreatment with ethoxyquin and 3-methylcholanthrene led to formation of cytochrome P-448. However, monooxygenase activities were slightly lower and epoxide hydratase activity was considerably higher than after treatment with 3-methylcholanthrene alone. Ethoxyquin inhibited benzo(a)pyrene hydroxylation and ethoxycoumarin deethylation in vitro. These reactions were less sensitive to ethoxyquin in 3-methylcholanthrene-stimulated microsomes than in phenobarbital-stimulated microsomes, indicating that ethoxyquin does not only preferentially induce but also preferentially inhibits phenobarbital-type cytochrome P-450.     

Tissue and sex differences in the activation of aromatic hydrocarbon hydroxylases in rats

Betamethasone and α-naphthoflavone produced similar activation of biphenyl 2-hydroxylase and benzo[a]pyrene 3-hydroxylase in control male rat liver microsomes. In small intestinal epithelial microsomes, betamethasone had no effect whereas α-naphthoflavone caused a pronounced activation of benzo[a]pyrene hydroxylation and a lesser activation of biphenyl 2-hydroxylation. In lung microsomes, betamethasone had no effect on either enzyme activity whereas α-naphthoflavone had no effect on biphenyl 2-hydroxylase but inhibited benzo[a]pyrene hydroxylase. In kidney cortex microsomes from male rats both compounds caused inhibition or had no effect whereas in kidney cortex microsomes from female rats betamethasone activated whereas α-naphthoflavone had no effect.Activation also occurred in isolated viable hepatocytes from male rats. The response of biphenyl 2-hydroxylase was very similar to that found in male rat liver microsomes but benzo[a]pyrene hydroxylase was more sensitive to activation and less sensitive to inhibition than in microsomes. The findings are interpreted as demonstrating the presence of more than one ‘latent’ aromatic hydrocarbon hydroxylase in rodents.     

Functional heterogeneity of UDP-glucuronyltransferase in rat tissues

Tissue distribution of UDP-glucuronyltransferase was investigated using two substrate groups which were shown to be conjugated by two different forms of this enzyme in previous studies with rat liver. These enzyme forms were found to be differentially inducible by 3-methylcholanthrene (form 1) and phenobarbital (form 2). Group 1 substrates (conjugated by form 1) include 1-naphthol, N-hydroxy-2-naphthylamine and 3-hydroxybenzo[a]pyrene; group 2 substrates (conjugated by form 2) comprise 4-hydroxybiphenyl, morphine and chloramphenicol. Group 1 substrates are conjugated in a number of tissues, for example, liver, kidney, small intestinal mucosa, lung, skin, testes and spleen. However, conjugation of group 2 substrates is detectable only in liver and intestine to an appreciable extent. It is concluded that enzyme(s) efficient in the conjugation of group 1 substrates is ubiquitous in the investigated organs, whilst only liver and intestine possess enzyme(s) efficient in the conjugation of group 2 substrates.In contrast to 3-hydroxybenzo[a]pyrene, benzo[a]pyrene 7,8-dihydrodiol cannot be clearly associated with only one of the 2 substrate groups. Glucuronidation of benzo[a]pyrene 7.8-dihydrodiol is enhanced by both phenobarbital and 3-methylcholanthrene in liver. Conjugation of the dihydrodiol is detectable in all tissues examined. However, enzyme activity towards the dihydrodiol is much lower than that towards 3-hydroxybenzo[a]pyrene. It is disproportionately low with skin microsomes.     

The effect of long term treatment with disulfiram on content of cytochrome P-450 and on benzo(a)pyrene monooxygenase activity in microsomes isolated from the rat small intestinal mucosa

Disulfiram (DS) was administered perorally once a day to rats for 30 days to investigate the effects on cytochrome P-450 content and benzo(a)pyrene (BP) monooxygenase activity in microsomes isolated from the small intestinal mucosa. 50 mg or 100 mg DS/kg body weight caused a dose-related increase in BP monooxygenase activity, whereas the content of cytochrome P-450 was increased at the higher dose only. Similar absorption characteristics of cytochrome P-450 and turnover rates for BP on the basis o f cytochrome P-450 was observed among the different microsomal preparations. The addition of DS or diethyldithiocarbamate (DDTC) to incubates of intestinal microsomes inhibited BP monooxygenase activity. Microsomes isolated from DS-treated rats were however less sensitive to in vitro inhibition by DS.     

Benzo[a]pyrene metabolism in the Mongolian gerbil: influence of chlordecone and mirex induction

1. The metabolism of benzo[a]pyrene (BP) by gerbil hepatic microsomes is increased following induction by phenobarbital (PB), chlordecone, mirex and 3-methylcholanthrene (3-MC).

2. By several criteria including the influence of α-naphthoflavone (α-NF) on BP-hydroxylase activity and BP-metabolite profiles, the cytochromes P-450 responsible for benzo[a]pyrene metabolism appear to be similar in microsomes isolated from PB-, chlordecone-, or mirex-treated gerbils. The cytochromes P-450 present in microsomes isolated from control animals and those treated with 3-MC are different from each other and from those present in PB, chlordecone, or mirex microsomes by the same criteria.

3. Of the inducers used, only PB induced microsomal epoxide hydrolase activity.     

Hepatic and extrahepatic microsomal electron transport components and mixed-function oxygenases in the marine fish Stenotomus versicolor.

NADPH-cytochrome c reductase, benzo[a]pyrene hydroxylase and aminopyrine demethylase activities in hepatic microsomes from the marine fish scup (Stenotomus versicolor) were characterized according to dependence of Ph, temperature, ionic strength and Mg²⁺. The kinetic properties of benzo[a] pyrene hydroxylase were variable, depending on protein and substrate concentration, with measured K_m values for benzo[a]pyrene between 4 × 10^?7 M and 4 × 10^?5 M. The K_m for aminopyrine was 7 × 10^?4 M, and NADPH-cytochrome c reductase had K_m values of 2.1 × 10^?5 M and 1.3 × 10^?5 M for cytochrome c and NADPH. respectively. NADH supported benzo[a]pyrene hydroxylation at 10 per cent of the rate seen with NADPH, and no synergism was observed. Aminopyrine demethylation proceeded at least as well with NADH as with NADPH, and there was synergism when combined. NADPH- and NADH-cytochrome c reductases were detected in “microsomes” from fourteen extrahepatic tissues, including kidney, testis, foregut, gill, heart, red muscle, hindgut, buccal epidermis, pyloric caecum, spleen, brain, lens, ovary and white muscle. Benzo[a]pyrene hydroxylase was detected in all but white muscle, while cytochrome P-450 and aminopyrine demethylase were detectable in fewer tissues. Reduced, CO-ligated absorption maxima in the Soret region were 450 nm for all those but liver (occasionally 449 nm) and heart (about 447 nm). The estimated turnover numbers for benzo[a]pyrene hydroxylase and aminopyrine demethylase, and the influence of 7,8-benzoflavone in vitro on benzo[a]pyrene hydroxylase indicate that the cytochromes P-450 in different fish tissues are not catalytically equivalent.     

Morphology of oocyte and follicle destruction by polycyclic aromatic hydrocarbons in mice

The carcinogenic polycyclic aromatic hydrocarbons, benzo(a)pyrene, 3-methylcholanthrene, and 7,12-dimethylbenz(a)anthracene, destroy primordial oocytes in the mouse ovary. The rate of oocyte destruction was proportional to the activity of the ovarian microsomal cytochrome P-450-dependent monooxygenase, aryl hydrocarbon (benzo(a)pyrene) hydroxylase (EC 1.14.14.2) as well as to the carcinogenicity of the polycyclic hydrocarbon. After treatment with benzo(a)pyrene or 3-methylcholanthrene only primordial oocyte destruction occurred, and no evidence of toxicity was observed in surrounding granulosa or ovarian stromal cells. 7,12-Dimethylbenz(a)anthracene was much more toxic and destroyed large follicles and oocytes in addition to primordial oocytes and primary follicles. Seven weeks after treatment with 3-methylcholanthrene the ovary had the bland afollicular appearance characteristic of ovarian failure. These three polycyclic aromatic hydrocarbons are capable of producing premature ovarian failure in rodents.     

Ethoxyquin as an inducer and inhibitor of phenobarbital-type cytochrome P-450 in rat liver microsomes

The effect of ethoxyquin in vivo and in vitro on drug metabolism in rat liver microsomes was studied. In feeding experiments, a threshold dose of induction was found at 0.05% ethoxyquin for 14 days. At 0.5% ethoxyquin, relative liver weight, cytochrome P-450 content, cytochrome b₅ content, ethylmorphine demethylation, and ethoxycoumarin deethylation were increased by a factor of 1.5 to 2. Aryl hydrocarbon hydroxylase activity was, however, not induced but even decreased by 0.5% ethoxyquin in food. Induction of epoxide hydratase was marked, amounting to 400% of control after 0.5% ethoxyquin. The induced enzyme was similar to the phenobarbital-inducible cytochrome P-450 in its CO spectrum, in its affinity for the ligand metyrapone, in the preferential inhibition of monooxygenase activity by metyrapone and not α-naphthoflavone, and in the increase in the phenobarbital-inducible band after gel electrophoresis of microsomal proteins. Mixed pretreatment with ethoxyquin and 3-methylcholanthrene led to formation of cytochrome P-448. However, monooxygenase activities were slightly lower and epoxide hydratase activity was considerably higher than after treatment with 3-methylcholanthrene alone. Ethoxyquin inhibited benzo(a)pyrene hydroxylation and ethoxycoumarin deethylation in vitro. These reactions were less sensitive to ethoxyquin in 3-methylcholanthrene-stimulated microsomes than in phenobarbital-stimulated microsomes, indicating that ethoxyquin does not only preferentially induce but also preferentially inhibits phenobarbital-type cytochrome P-450.     

The mechanistic plurality of cytochrome P-450 and its biological ramifications

1. The mechanistic plurality of the microsomal cytochrome P-450 enzyme system is illustrated by studies of the oxidative metabolism of benzo[a]pyrene, 3-hydroxybenzo[a]pyrene and arachidonic acid.

2. Rat liver microsomal metabolism of benzo[a]pyrene or 3-hydroxy-benzo[a]pyrene, supported by cumene hydroperoxide, generates benzo[a]pyrene quinones via molecular oxygen-dependent and -independent pathways.

3. Arachidonic acid is metabolized by rat liver microsomal fractions to a variety of oxygenated products, including cis-trans diene conjugated monohydroxy-acids, epoxy-acids as well as ω- and ω — 1 -oxidation products. The chemistry of the different reaction products is discussed in terms of the possible mechanisms responsible for their formation and the role of the haemoprotein during catalysis.

4. An integrated view for the reaction cycle of cytochrome P-450 is presented.     

Effect of cadmium on phenobarbital and polycyclic hydrocarbon-induced alterations in hepatic microsomal monooxygenase activity in the rat

The heavy metal, cadmium, is a potent inhibitor of the hepatic microsomal monooxygenase enzyme system in male, but not female, rats. The selectivity of this inhibitory effect of Cd was further examined by utilizing rats treated with inducers of this drug metabolizing enzyme system. Animals received phenobarbital (PB) sodium (100 mg/kg, ip, 72, 48, and 24 hr before sacrifice), or 3-methylcholanthrene or benzo[a]pyrene (20 mg/kg, ip, 72 and 48 hr prior to sacrifice) as inducers. Designated groups of these animals also received cadmium (1.0 mg Cd²⁺/kg), ip) either 120 or 72 hr before sacrifice. Noninduced male rats exhibited significant decreases in cytochrome P-450 content and drug-metabolizing enzyme activity following Cd treatment. The magnitude of the reductions in drug-metabolizing enzyme activity produced by Cd paralleled the magnitude of the sex difference in biotransformation of the substrate examined. Phenobarbital-treated male rats receiving a simultaneous Cd injection (72 hr prior to sacrifice) were also sensitive to Cd-induced inhibitions in cytochrome P-450 content and monooxygenase activity, although the extent of the reductions produced by Cd in PB-treated animals was less than that observed in noninduced male rats. In PB-treated male rats receiving a prior dose of Cd (120 hr before sacrifice), only the metabolism of the highly sex-dependent substrate, ethylmorphine, was significantly reduced. Cytochrome P-448 levels, and cytochrome P-448-mediated biotransformations which are elevated following hydrocarbon treatment, were not decreased by either simultaneous or prior Cd administration to male rats. Control, PB-treated, and hydrocarbon-treated female rats were not susceptible to Cd-induced reduction in hemoprotein content or inhibition of drug-metabolizing enzyme activity with the exception of those animals receiving both benzo[a]pyrene and Cd, which displayed slight but significant reductions in the oxidation of sex-dependent substrates. These results demonstrate the selective nature of the inhibitory effects of Cd upon drug metabolism in the rat.     

Identification and partial purification of hamster microsomal cytochrome P-450 isoenzymes

We have identified and partially purified three forms of cytochrome P-450 from hamster liver microsomes. Phenobarbital (PB) treatment induced three major polypeptides with relative mobilities (M_r) of 47,000, 50,000 and 51,500. The 47,000 polypeptide was assigned as epoxide hydrolase, since it was also enhanced by trans-stilbene oxide (TSO) treatment. Two polypeptides (M_r = 48,500 and 53,500) were induced by both 3-methylcholanthrene (3-MC) and β-naphthoflavone (BNF) treatments. Treatment with Aroclor 1254 induced three polypeptides (M_r = 48,500, 50,000 and 53,500), indicating the induction of both drug- and carcinogen-inducible cytochrome P-450s. Liver microsomal benzo[a]pyrene hydroxylase activity was not affected significantly by any of these inducers. In contrast, it was induced 2- to 3-fold in lung microsomes by 3-MC, BNF or Aroclor 1254 treatment. Benzphetamine N-demethylase and 7-ethoxycoumarin O-deethylase activities, expressed as nmoles of product formed per min per mg of liver microsomal protein, were increased 3- to 4-fold by either PB or Aroclor treatment. The activity of 7-ethoxycoumarin O-deethylase was the only one enhanced significantly by 3-methylcholanthrene or β-naphthoflavone treatment in liver microsomes. Pregnenolone-16-α-carbonitrile (PCN) and TSO did not alter any of these activities. The major polypeptides induced by PB (M_r = 50,000) and 3-MC (M_r = 48,500 and 53,500 respectively) were partially purified, to a specific content of 6–10 nmoles P-450/mg of protein and were active in catalyzing N-demethylation of benzphetamine, hydroxylation of benzo[a]pyrene, and O-deethylation of 7-ethoxycoumarin with different substrate specificity. None of these isoenzymes immuno-cross-reacted with antibodies prepared against rabbit cytochrome P-450_LM2 or P-450_LM4.     

Inhibition of cytochrome P-450c-mediated benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by dihydrosafrole

1. Inhibitory activity of dihydrosafrole towards benzo[a]pyrene (BP) hydroxylase activity in hepatic microsomes from β-naphthoflavone (BNF)-induced rats, and in reconstituted systems containing cytochrome P-450c, increased dramatically on preincubation of the inhibitor with NADPH; no inhibition occurred without preincubation. The level of BP hydroxylase inhibition was associated with the progressive formation of the 456 nm dihydrosafrole metabolite-cytochrome P-450c spectral complex during preincubation.

2. Inhibition of BP hydroxylase by dihydrosafrole in control microsomes, and inhibition of ethoxyresorufin O-deethylase (EROD) in microsomes (control or BNF-induced) and in reconstituted systems with cytochrome P-450c, did not require preincubation and apparently was not dependent on prior formation of the dihydrosafrole metabolite-cytochrome P-450 complex.

3. Kinetic studies established that, following preincubation with NADPH, dihydrosafrole was a noncompetitive inhibitor of both BP hydroxylase and EROD activities. In the absence of preincubation, dihydrosafrole was an effective competitive inhibitor of EROD in BNF-induced microsomes and in reconstituted systems with cytochrome P-450c.

4. Both ethoxyresorufin and benzo[α]pyrene inhibited the development of the type 1 optical difference spectrum of dihydrosafrole in reconstituted systems containing cytochrome P-450c. Inhibition by ethoxyresorufin was competitive while that caused by benzo[α]pyrene was noncompetitive in nature.

5. The type II ligand phenylimidazole was an effective noncompetitive inhibitor of EROD activity but failed to exert any inhibitory effect on cytochrome P-450c-mediated BP hydroxylase activity. Phenylimidazole inhibited formation of the dihydrosafrole type 1 optical difference spectrum non-competitively.

6. The results indicate that ethoxyresorufin and benzo[α]pyrene may occupy different binding sites on cytochrome P-450c and that dihydrosafrole binds primarily to the site utilized by ethoxyresorufin.     

Hydrogen peroxide-supported oxidation of benzo [a]pyrene by rat liver microsomal fractions

In the presence of liver microsomes from phenobarbital-pretreated rats, hydrogen peroxide oxidized benzo [a]pyrene to a number of biologically significant products at a rate that was approximately 20 per cent as fast as that seen by us and others with NADPH and oxygen. As with NADPH-dependent reactions [J. Capdevila, R. W. Estabrook, and R. A. Prough, Archs. Biochem. Biophys.200, 186 (1980)], the hydrogen peroxide-dependent reactions resulted in the production of relatively large quantities of dihydrodiols as metabolites. This was in marked contrast to the product distribution observed when cumene hydroperoxide was utilized as a cosubstrate (foregoing reference). The formation of the various organic-soluble metabolites was dependent on the presence of functional liver microsomal cytochrome P-450 in the reaction mixture. Approximately 48 per cent of the benzo[a]pyrene metabolites, however, was observed to be bound to microsomal protein, and inhibition of cytochrome P-450 function, by metyrapone or N-octylamine did not affect the extent of covalent binding of the hydrocarbon to the microsomal protein. The differences noted during benzo[a]pyrene metabolism using hydrogen peroxide strongly suggest that at least two distinct mechanisms exist to account for the oxidation of the hydrocarbon, i.e. epoxidation and one-electron oxidation reactions.     

Induction of monooxygenase activity in the intestine of spot (Leiostomus xanthurus), a marine teleost, by dietary polycyclic aromatic hydrocarbons

The response of intestinal monooxygenases to dietary polycyclic aromatic hydrocarbon (PAH) exposure was evaluated in spot (Leiostomus xanthurus), a marine teleost fish. Ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) activities were highest in the pyloric caeca and in the proximal half of the intestine. Intestinal microsomes from fish given control diets had very low levels of EROD and AHH activities relative to those in liver. After exposure to a diet containing 10 mg of 3-methylcholanthrene/kg of food, the levels of intestinal EROD and AHH activities increased 36-fold and 17-fold, respectively, such that intestinal monooxygenase activity exceeded that of the liver, which was not induced by this treatment. A significant increase in intestinal monooxygenase activity occurred in fish receiving dietary benzo[a]pyrene (BP) at concentrations as low as 10 micrograms of BP/kg food. A 5-fold increase in intestinal AHH and EROD activities was observed within 3 hr after administration of dietary BP. A plateau in gut monooxygenase activity occurred after approximately 3 days of PAH exposure; these activities decreased to control levels within 3 days after replacing the PAH diet with the control diet. Starvation resulted in disappearance of detectable monooxygenase activity. Monoclonal antibody (MAB 1-12-3) against the major PAH-inducible cytochrome P-450 (P-450E) in the liver of the marine teleost (Stenotomus chrysops) [Park et al. Arch. Biochem. Biophys. 249, 399 (1986)] recognized a single protein band in intestinal microsomes, with Mr near 54,000, which we conclude is the spot counterpart to cytochrome P-450E.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome P-448 and the activation of toxic chemicals and carcinogens

1. The metabolic activation of carcinogens and some toxic chemicals appears to involve oxygenation in conformationally hindered positions in the chemical molecules.

2. Oxygenation of xenobiotics in hindered positions is effected by cytochrome P-448 (LM₄) but not by cytochrome P-450 (LM₂).

3. Substrate-interaction spectra show that cytochrome P-448 has an active site with a conformation different from that of cytochrome P-450.

4. Induction of cytochrome P-448, as specifically measured by ethoxyresorufin O-de-ethylase activity, occurs in rat liver, kidney and lung after administration of the carcinogens, 3-methylcholanthrene, Aroclor 1254, 2-anthramine, safrole, 7,12-dimethylbenz[a]an-thracene, MNNG and 2-acetamidofluorene. The doubtful carcinogens, saccharin, DDT and aldrin, resulted in no significant induction. The drugs paracetamol, antipyrine, imipramine and rifampicin resulted in diminished enzyme activity, indicating the absence of any induction of cytochrome P-448.

5. In studies with the matched pairs of carcinogens and non-carcinogens, benzo[a]pyrene and benzo[e]pyrene, and 1,2,5,6-dibenzanthracene and anthracene, only the carcinogenic analogue resulted in induction of cytochrome P-448. With α- and β-naphthylamine, both resulted in marked induction of cytochrome P-448 in liver, kidney and lung, indicating that both isomers might be carcinogenic.