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 alpha-naphthoflavone (alpha-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.     

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

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

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.     

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.     

Qualitative and quantitative differences in the induction and inhibition of hepatic benzo[a]pyrene metabolism in the rat and hamster

The present study compared the induction and inhibition of the metabolism of the prototype polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in rat and hamster liver microsomes. The production of total polar metabolites was quantitated by separating 3H-metabolites from [3H]-BaP using reverse-phase thin-layer chromatography. The rate of hepatic microsomal BaP metabolism was similar in the rat and hamster (0.81 vs 0.72 nmol/min/nmol cytochrome P-450 respectively). In the rat, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 5 micrograms/kg, i.p.) and 3-methylcholanthrene (3-MC; 50 mg/kg, i.p., X 3 days) pretreatments doubled the rate of BaP metabolism, whereas phenobarbital pretreatment (PB; 80 mg/kg, i.p., X 3 days) had no effect. In contrast, hamster hepatic microsomal BaP metabolism was elevated 2.3-fold by PB pretreatment, whereas TCDD and 3-MC pretreatments had no effect. Isosafrole pretreatment (ISO; 150 mg/kg, i.p., X 3 days) elevated the rate by almost 2-fold in each species. Another cytochrome P-448-mediated activity, 7-ethoxyresorufin O-deethylase (EROD), was induced by the same compounds that induced BaP metabolism in the rat. In hamster liver microsomes, in contrast to BaP metabolism, EROD was induced by TCDD and 3-MC but not PB or ISO pretreatments. The results suggest differences in the substrate specificity of the cytochromes P-448-450 induced by TCDD, 3-MC and PB in these species. This was supported by the different selectivity of the in vitro inhibitors, metyrapone and 7,8-benzoflavone, towards BaP metabolism and EROD in hepatic microsomes from TCDD- or PB-pretreated rats and hamsters. Reverse-phase HPLC analysis indicated that, while 3-hydroxy-BaP was the major metabolite formed by the untreated rat, untreated hamster liver microsomes formed predominantly BaP-4,5-diol. Microsomes from TCDD-treated rats generated elevated levels of all BaP-diols, diones and 3-hydroxy-BaP, with the major metabolites being BaP-9,10- and BaP-7,8-diols. In contrast, the metabolite profile from TCDD-pretreated hamsters was unchanged from the control. PB-treated hamster microsomes produced elevated levels of BaP-diones and 3-hydroxy-BaP. However, the major hepatic metabolite formed by PB-pretreated hamsters was BaP-4,5-diol, while BaP-9,10- and BaP-7,8-diols were not detected. The results of the study indicate differences in the induced cytochrome P-450s and the generation of toxic BaP metabolites in the liver of the rat and hamster.     

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.     

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.     

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.     

Differential effects of disulfiram and diethyldithiocarbamate on small intestinal and liver microsomal benzo[a]pyrene metabolism

Microsomes isolated from rat small intestinal mucosa and liver were used to study the effects of disulfiram and diethyldithiocarbamate on benzo[a]pyrene monooxygenase activity. This activity was decreased in the intestinal microsomes to 25 per cent of control 24 hr after a single oral dose of disulfiram. In contrast, daily administration of disulfiram for 5 days produced a dose related increase of benzo[a]pyrene monooxygenase activity, above control level. The elevated activities were accompanied by a concomitant increase in the concentration of cytochrome P-450. This benzo[a]pyrene monooxygenase activity was further stimulated by addition of α-naphthoflavone to the incubation medium. Furthermore, the absorption maximum of this cytochrome was at 450 nm in the CO bound reduced difference spectrum. These observations indicate that the disulfiram induced cytochrome P-450 was of the control type. Daily pretreatment with diethyldithiocarbamate impaired both intestinal and liver microsomes at benzo[a]pyrene monooxygenase activities. Pretreatment with a single dose of 3-methylcholanthrene resulted in a more than 10-fold increase of intestinal benzo[a]pyrene monooxygenase activity after 24 hr. Administration of disulfiram 24 hr before treatment appeared to potentiate the 3-methylcholanthrene induced increase of intestinal benzo[a]pyrene monooxygenase activity. In vitro addition of disulfiram and diethyldithiocarbamate to incubates of intestinal or liver microsomes inhibited benzo[a]pyrene metabolism to various extents; the liver being more sensitive. Disulfiram was approximately 50-fold more potent as an inhibitor than diethyldithiocarbamate. The in vitro inhibition of intestinal benzo[a]pyrene monooxygenase activity obtained with disulfiram appeared to be caused both by direct interaction with the monooxygenase system and through NADPH dependent metabolic activation of disulfiram, while the inhibition of diethyldithiocarbamate may be a result of the latter process only.     

Variations among untreated rabbits in benzo(a)pyrene metabolism and its modulation by 7,8-benzoflavone

The metabolism of benzo(a)pyrene by rabbit liver microsomes can be stimulated or inhibited by 7,8-benzo(a)flavone (ANF) depending on the distribution of specific P-450 enzymes present within the microsomes. Treatment of rabbits with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or rifampicin leads to an increase of hepatic microsomal metabolism of benzo(a)pyrene. ANF stimulates the rate of benzo(a)pyrene metabolism catalyzed by microsomes isolated from rabbits treated with rifampicin by 3-fold. In contrast, ANF moderately inhibits the activity of microsomes from TCDD-treated rabbits. Variations in the benzo(a)pyrene hydroxylase activity of microsomes from untreated rabbits apparently reflect differences in the expression of P-450 1, a constitutive form of P-450. Thus, the benzo(a)pyrene hydroxylase activity of microsomes from untreated rabbits, which varies from 0.40 to 1.5 nmol/min/mg of protein, is directly correlated with the microsomal concentration of P-450 1. The metabolism of benzo(a)pyrene by microsomes containing high concentrations of P-450 1 is inhibited by a monoclonal antibody specific for this cytochrome to approximately the rate exhibited by microsomes with a low concentration of P-450 1. The benzo(a)pyrene activity stimulated by ANF in microsomes with a low concentration of P-450 1 is not inhibited by the monoclonal antibody. The activity of P-450 1 is inhibited by ANF at concentrations that stimulate other constitutive forms of P-450. Thus, ANF produces offsetting effects on benzo(a)pyrene metabolism in microsomes from untreated animals by stimulating the activity of at least one cytochrome and inhibiting P-450 1-mediated activity.     

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

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

Studies on the properties of highly purified cytochrome P-448 and its dependent activity benzo[a]pyrene hydroxylase,from Saccharomyces cerevisiae

1. The yeast Saccharomyces cerevisiae, produces a cytochrome P-450 enzyme with a Soret peak in the reduced-CO difference spectrum at 448 nm. The enzyme purified to homogeneity (88–97% pure on a specific content basis) has a molecular wt. of 55 500 as determined by SDS-PAGE.

2. Amino acid analysis of yeast cytochrome P-448 revealed 407 amino acid residues per molecule with a 43% complement of hydrophobic residues. Although the number of residues is smaller than cytochrome P-448 enzymes from mammalian sources, the percentage of hydrophobic residues is almost identical. Estimation of the haem content of yeast cytochrome P-448 showed that one haem group was present per molecule. Phospholipid was present at very low levels. The molecular wt. of the polypeptide chain plus an estimated 5–6 units of hexose and of hexosamine is in good agreement with the molecular wt. value obtained from SDS-PAGE.

3. A reconstituted system of purified cytochrome P-448, purified NADPH-cytochrome P-450 (c) reductase and phospholipid showed aryl hydrocarbon hydroxylase activity towards benzo[a]pyrene. Both protein components, NADPH and dilauroyl phosphatidylcholine (or emulgen 911) were necessary for full activity. The NADPH requirement could be replaced by cumene hydroperoxide or H₂O₂ generated in situfrom a glucose oxidase system; in each case V_max is increased, but the apparent affinity for benzo[a]pyrene, as measured by an increased K_m, is lowered.

4. The spin state of purified yeast cytochrome P-448 was 94% low spin (22°C) as determined from the temperature-dependent spin-state equilibrium. The addition of benzo[a]pyrene to this enzyme resulted in a change to higher spin state (18% high spinat 22°C).

5. Equilibrium gel filtration analysis of the number of benzo[a]pyrene binding sites per mole of enzyme monomer showed a value of 1 for purified yeast cytochrome P-448 and 6 for this enzyme in microsomal form. The corresponding values for purified and microsomal cytochrome P-450 from phenobarbital-pretreated rats are 1 and 6, respectively. However, purified cytochrome P-448 from β-naphthoflavone-induced rats gave a value of 6 benzo[a]pyrene binding sites.

6. Type I binding spectra with purified yeast cytochrome P-448 were observed with benzo[a]pyrene, lanosterol, ethylmorphine, dimethylnitrosamine, sodium phenobarbitone and perhydrofluorene. Type II spectral changes were observed with imidazole, aniline and benzphetamine.

7. Cytochrome P-448 from Saccharomyces cerevisiae is identified as a distinct enzyme of the P-450 family. This enzyme however has many properties in common with cytochrome P-448 from mammalian sources.

8. A more specific and efficient form of benzo[a]pyrene hydroxylase is induced by the addition of benzo[a]pyrene to the yeast growth medium at zero time. The efficiency of the enzyme, as indicated by the V_max / K_m ratio, increases progressively with concentration of benzo[a]pyrene. This indicates that multiple forms of yeast cytochrome P-448 occur. Induction of more efficient forms occurs at the expense of less efficient forms as little increase in total enzyme concn. is observed.     

Induction of cytochrome P-450 1A1 in human hepatoma HepG2 and lung carcinoma NCI-H322 cells by motorcycle exhaust particulate

The effects of motorcycle exhaust particulate (MEP) on human cytochrome P-450 (P-450)-dependent monooxygenases were determined using human hepatoma cell line HepG2 and lung carcinoma cell line NCI-H322 treated with organic extracts of MEP from a two-stroke engine. Gas chromatography and mass spectrometry analysis of MEP extract revealed the presence of carcinogens benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[g,h,i]perylene, chrysene, and indeno[1,2,3-c,d]pyrene in the chemical mixture. Treatment with MEP extract produced concentration- and time-dependent increases of monooxygenase activity in HepG2 cells. Treatment of the cells with 100 microg/ ml MEP extract for 24 h markedly increased benzo[a]pyrene hydroxylation, 7-ethoxycoumarin, and 7-ethoxyresorufin O-deethylation activities in microsomes. Immunoblot analysis of microsomal proteins using mouse monoclonal antibody 1-12-3 against P-450 1A1 revealed that MEP extract induced a P-450-immunorelated protein in the hepatoma cells. RNA blot analysis of cellular total RNA using a human P-450 1A1 3'-end cDNA probe showed that MEP extract increased the level of a hybridizable P-450 mRNA. These P-450 1A1 inductive effects of MEP extract were similar to those from treatment with 10 microM benzo[a]pyrene or 3-methylcholanthrene (3-MC) in HepG2 cells. Treatment of lung carcinoma NCI-H322 cells with 100 microg/ml MEP extract, 10 microM benzo[a]pyrene, or 3-MC resulted in induction of monooxygenase activity, protein, and mRNA of P-450 1A1, similar to the induction observed with the hepatoma cells. The present study demonstrates that MEP extract has the ability to induce human hepatic and pulmonary P-450 1A1 in the liver- and lung-derived cell lines, and the induction involves a pretranslational mechanism. Induction of the human hepatic and pulmonary P-450 1A1 in vitro may provide important information in the assessment of MEP metabolism and toxicity in humans.     

Spectral and inhibitory interactions of methylenedioxyphenyl and related compounds with purified isozymes of cytochrome P-450

1. Spectral and inhibitory interactions of two methylenedioxyphenyl (MDP) compounds (dihydrosafrole (DHS) and 4,5-dichloro-1,2-methylenedioxybenzene (DCMB)) and 4-n-butyl dioxolane (BD) were studied in vitro in reconstituted systems incorporating cytochromes P-450b and P-450c, purified respectively from hepatic microsomes of phenobarbital (PB)- and β-naphthoflavone (βNF)-treated rats.

2. In NADPH-fortified reconstituted systems containing P-450b, DHS yielded a stable type III spectral complex with peaks at 428 and 458 nm; a complex with a single 456?nm peak was formed in systems containing cytochrome P-450c. DCMB formed unstable 456–458?nm spectral complexes with both isozymes, and BD generated an unstable complex with a single Soret peak near 428?nm with cytochrome P-450b; no spectral interaction occurred between BD and cytochrome P-450c. Carbon monoxide was formed in incubations of DCMB with both isozymes but was not observed with either DHS or BD.

3. Marked selectivity was observed in the ability of the test compounds to inhibit selected mono-oxygenase reactions in the reconstituted systems. Thus, while DHS was an effective inhibitor of cytochrome P-450b-mediated ethoxycoumarin O-deethylase (ECD), it failed to inhibit aldrin epoxidase (AE) in the same system; DCMB and BD inhibited both of these reactions. In reconstituted systems incorporating cytochrome P-450c, DHS and DCMB, but not BD, were effective inhibitors of ethoxyresorufin O-deethylase (ERD) activity but none of the compounds showed any inhibitory activity towards aryl hydrocarbon (benzo[a]pyrene)hydrolase (AHH) activity.

4. The results indicate that metabolite complex formation with cytochrome P-450 is not the sole criterion for inhibition of mono-oxygenase activity by MDF and related compounds, and that in some cases type I competitive interactions at the substrate binding sites may be the primary contributing factor.     

Effects of acetone administration on cytochrome P-450-dependent monooxygenases in hamster liver,kidney, and lung

The effects of acetone on liver, kidney, and lung monooxygenases were studied using hamsters administered 8% acetone in drinking water. Binding of aniline to liver microsomes induced a type II difference spectrum, and the spectral binding was enhanced in hamsters pretreated with acetone. Administration of acetone caused significant increases of cytochrome P-450 and cytochromeb ₅ contents in liver microsomes. The increases of the hemeproteins were associated with induction of monooxygenase activities toward test substrates, aniline, N-nitrosodimethylamine, benzphetamine, benzo(a)pyrene, and 7-ethoxycoumarin. In the kidneys, acetone administration increased microsomal contents of the hemeprotein and monooxygenase activities toward aniline, N-nitrosodimethylamine, and 7-ethoxycoumarin, but not benzphetamine or benzo(a)pyrene. In the lungs, acetone pretreatment increased aniline hydroxylase activity without affecting the levels of N-nitrosodimethylamine demethylase, cytochromes P-450 andb ₅. In marked contrast to the inductive effects in the liver, acetone administration markedly decreased lung microsomal benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase activities. Gel electrophoresis of liver and kidney microsomes from control and acetone-treated hamsters revealed that acetone treatment enhanced the intensity of a protein band(s) in the cytochrome P-450 molecular weight region. Immunoblotting of the microsomal proteins showed that the protein band induced by acetone in hamster liver, kidney and lung was cross-reactive with antibody raised against ethanol-inducible human liver cytochrome P-450. These results demonstrate that acetone has the ability to uniformly induce a specific form of cytochrome P-450, designated as IIE1, and to cause differential changes of monooxygenase activities in the hamster tissues. The complex effects of acetone on hepatic and extrahepatic monooxygenase systems may be important determinants of tissue-specific chemical toxicity.The nomenclature of P-450 used in this report follows the system recommended by Nebert et al. (1987, 1989). P-450 IIE1 has also been referred to as P-450ac by Patten et al. (1986), P-450j by Ryan et al. (1986), and as isozyme 3a and P-450_ALC by Coon and Koop (1987) in various species.     

Relationship Between Activities of Cytochrome P-450 Monooxygenases in Human Placental Microsomes and Binding of Benzo(A)Pyrene Metabolites to Calf Thymus DNA

ABSTRACT

Benzo(a)pyrene metabolism in human placental microsomes from smokers was studied. Benzo(a)pyrene metabolites were separated using high pressure liquid chromatographic technique. Reaction of benzo(a)pyrene with a microsomal fraction of placenta from individuals who smoke cigarettes during pregnency yields 7,8 dihydroxy benzo(a)pyrene as a major metabolite, while 3′-hydroxy benzo(a)pyrene, 4,5 dihydroxy benzo(a)pyrene and quinones constitute minor metabolites. The activities of arylhydrocarbon hydroxylase and 7-ethoxycoumarine deethylase exhibited much higher activities in smokers than in nonsmokers. Examination of specific binding of monoclonal antibodies to cytochrome P-450 isozymes in placental microsomes revealed that cigaratte smoking specifically enhanced the level of cytochrome P-450 c and d isozymes in human placental microsomes. Coincubation of ³H-benzo(a)pyrene and calf thymus DNA with placental microsomes yielded acid insoluble ³H-B(a)P from smokers, suggesting that cigarette smoking may induce placental enzymes which convert benzo(a)pyrene into ultimate metabolites to form carcinogen-DNA adducts.     

Studies of the mechanisms of metabolism of diethyl p-nitrophenyl phosphorothionate (parathion) by rabbit liver microsomes

Based on the protein content of microsomes, the administration of 3-methylcholanthrene (3-MC) and phenobarbital (PB) to adult rabbits leads to an increased rate of metabolism of parathion (diethyl 4-nitrophenyl phosphorothionate) by rough-surfaced and whole microsomes but not by smooth-surfaced microsomes. Although prior administration of both PB and 3-MC increased the cytochrome P-450 content of the microsomes, when the rate of metabolism of parathion was calculated on the basis of the concentration of cytochrome P-450 in these microsomes, there is no difference in the rate of metabolism of parathion by rough-surfaced and smooth-surfaced microsomes from the untreated, 3-MC-treated and PB-treated animals. However, based on the cytochrome P-450 concentration, the rate of metabolism of parathion by whole microsomes from 3-MC and PB-treated animals is less than the rate with whole microsomes from untreated animals. Further studies have shown there is no correlation between the concentration of high spin or low spin cytochrome P-450 in any of the microsomal fractions or subfractions and the rate of metabolism of parathion to paraoxon or diethyl phosphorothionate.     

Cytochrome P-450 and oxidative metabolism in molluscs

1. Cytochrome P-450 and the associated components and oxidative activities of a mixed-function oxidase (MFO) system are localized primarily in the microsomes of the digestive gland of molluscs.

2. Cytochrome P-450 and putative cytochrome P-450-catalysed oxidative activities, measured in vitro and/or in vivo, have variously been detected in 23 species of mollusc.

3. Cytochrome P-450 and other MFO components and activities may be increased by exposure to xenobiotics, but the results are variable and no correlation is obvious between changes in cytochrome P-450 content and measured MFO activities (benzo[a]pyrene hydroxylase (BPH) and 7-ethoxycoumarin O-deethylase (ECOD)).

4. Type II binding compounds (clotrimazole, miconazole, ketoconazole, metyrapone and pyridine) give type II difference spectra with mussel digestive gland microsomal P-450, whereas type I binding compounds (testosterone, 7-ethoxycoumarin, α-naphthoflavone, SKF525-A) give apparent reverse type I difference spectra.

5. The existence of multiple or particular forms (P450 IVA or LAw) of cytochrome P-450 is indicated from enzyme kinetics and inhibition studies, seasonality, purification studies and cDNA probes.

6. Microsomal MFO activities are observed even in the absence of added or generated NADPH, and the NADPH-independent BPH, ECOD and N,N-dimethylaniline N-demethylase activities are inhibited by reducing agents, including NADPH.

7. The major metabolites of microsomal benzo[a]pyrene metabolism are quinones.

8. One-electron oxidation is considered to be one possible mechanism of molluscan cytochrome P-450 catalytic action.     

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.     

Role of hepatic microsomal cytochrome P-450 in the toxicity of fluorinated ether anesthetics

To evaluate the role of cytochrome P-450 in anesthetic toxicity, we investigated the effects of hepatic microsomal cytochrome P-450 inducers [phenobarbital (PB), 3-methylcholanthrene (3-MC) and pregnenolone-16 α-carbonitrile (PCN)] and inhibitors [SKF 525-A, metyrapone, and 2allyl2isopropylacetamide (ALA)] on the potentiation of lethal effects to rats of i.p. administered 2,2,2-trifluoroethyl vinyl ether (TFVE), ethyl 2,2,2-trifluoroethyl ether (TFEE), allyl 2,2,2-trifluoroethyl ether (TFAE) and 2,3-epoxypropyl 2,2,2-trifluoroethyl ether (EPTFE). The time courses of tail-vein blood anesthetic concentrations and quantities of exhaled anesthetics together with the in vitro metabolism of the anesthetics and their binding to microsomal cytochromes P-450 were also determined. The results indicate that (1) the majority of the administered anesthetics make a single pass through the liver prior to exhalation and apparently are metabolized to toxic products, (2) the epoxide (EPTFE) exerts its lethal effects independently of cytochrome P-450 catalyzed metabolism and does not lie on the major path of TFAE metabolism, (3) all the anesthetics yield 2,2,2-trinuoroethanol (TFE) on metabolism in vitro but lethality does not always correlate with the rates of TFE formation, (4) PB induced cytochromes P-450 potentiate lethal effects of TFVE and TFEE but not of TFAE, and inhibitors differentiate mechanisms of TFVE and TFEE lethality, (5) PCN induced cytochromes P-450 potentiate the toxicity of TFVE, TFAE, and TFEE in a similar manner, and (6) 3-MC induction potentiates TFEE and TFAE lethality apparently independently of cytochrome P-450 catalyzed metabolism.