Specificity of rat liver cytochrome P-450 isozymes in the mutagenic activation of benzo[a]pyrene, aromatic amines and aflatoxin B1

The ability of three purified forms of rat liver cytochromeP-450 to metabolically activate benzo[a]pyrene, trans-benzo-[a]pyrene-7,8-dihydrodiol,2-aminofluorene, afiatoxin B₁, dimethylnitrosamine, and a pyrolysisproduct of tryptophan(3-amino-l-methyl-5H-pyrido(4,3-b)indole)(Trp-P-2) to muta-genic products was examined using Salmonellatyphimurium strains TA98 and G46 in a reconstituted monooxygenasesystem. The isozymes examined were cytochrome P-450-PB (themajor phenobarbital inducible form), and the two major 3-MCinducible forms (cytochromes P-448₅₂ and P-448₅₅). CytochromesP-448₅₂ and P-448₅₅ preferentially metabolize 2-aminofluoreneand Trp-P-2 to mutagenic products. However, only cytochromeP-448₅₅ metabolizes benzo[a]pyrene and its 7,8-dihydrodiol derivativeto mutagenic products. Both cytochrome P-448₅₂ and P-448₅₅ metabolizeafiatoxin B, to mutagenic products at a much faster rate thancytochrome P-450-PB. Dimethylnitrosamine was not activated byany of the isozymes tested.     

Species difference among experimental rodents in the activity and induction of cytochrome P-450 isozymes for mutagenic activation of carcinogenic aromatic amines

The expressions of hepatic microsomal cytochrome P-450 isozymes in male rats, mice, hamsters and guinea pigs were studied comparatively with or without an ip injection of a cytochrome P-450 inducer. The activity and quantity of microsomal cytochrome P-450 isozymes were determined respectively by a bacterial mutation assay with Salmonella typhimurium TA98 and immunochemical assays using monoclonal antibodies against rat cytochrome P-450 isozymes. 3-Methoxy-4-aminoazobenzene (3-MeO-AAB), 2-amino-3-methyl-9H-pyrido[2,3-b]indole acetate (MeA alpha C) and 3-methylcholanthrene were used as cytochrome P-450 inducers, and 7 carcinogenic aromatic amines including 3-MeO-AAB and MeA alpha C were used as substrates for the mutation assay. By means of these assays, we examined the species differences among rodents in the activity and induction rate of hepatic cytochrome P-450 isozymes responsible for the mutagenic activation of carcinogenic aromatic amines.     

Immunochemical characterization of cytochrome P-450 isozymes responsible for benzene oxidation in the rat liver

The contribution of cytochrome P-450 isozymes to benzene metabolismin liver microsomes from fed, fasted, pyrazole-, pbenobarbital(PB)- and ethanol-treated rats and in respective isocaloriccontrols was investigated using monoclonal antibodies (mAbs).Clone 1-7-1 mAb did not inhibit benzene metabolism, whereasclone 2-66-3 inhibited only in PB-induced microsomes at a highconcentration of benzene (6.26 mM), and clone 1-91-3 mAb inhibitedbenzene metabolism in all cases. The degree of inhibition wasas follows: fed isocaloric control PB < fasted < pyrazole ethanol. The pattern of inhibition was similar with clone 1-91-3for low (0.23 mM) and high concentrations of benzene, exceptin PB-induced mkrosomes. Western blot analysis showed that clone1-7-1 mAb did not bind any liver mkrosomal protein in the regionof cytochrome P-450s, whereas with clone 2-66-3 a clear-cutband was seen only in liver microsomes from PB-treated rats,with clone 1-98-1, a band was detected in mkrosomes from alltreated groups, in the following order: PB = isocaloric control< fed < fasted < pyrazole < ethanol. These resultsindicate that (i) cytochromes P-450b,e and P-450J contributeto benzene metabolism in rat liver; (ii) the former has a lowaffinity to benzene and is induced by PB; and (iii) P-450J hasa high affinity to benzene and is induced by 1-day fasting,pyrazole and ethanol, but decreased by PB treatment.     

A new form of cytochrome P-450 responsible for mutagenic activation of 2-amino-3-methylimidazo[4,5-f]quinoline in human livers.

Antibodies to P-450IA2 strongly inhibited the mutagenic activation of 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate but not aflatoxin B1 in human liver microsomes. The anti-rat P-450IA2 antibodies were capable of recognizing two proteins which show different mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of human liver microsomes. A new form of cytochrome P-450 (designated P-450-HM4) cross-reactive with anti-rat P-450IA2 antibodies showing that the smaller molecular weight was purified from human liver microsomes by means of the fast-performance liquid chromatography system. The molecular weight of P-450-HM4 was estimated to be 49,000, which was apparently different from that of P-450PA (human P-450IA2). The antibodies to P-450-HM4 did not cross-react with P-450PA (human P-450IA2) but inhibited to various extents the mutagenic activation of IQ in microsomes from human livers. In addition, P-450-HM4 showed significant mutagen-producing activity from IQ in a reconstituted system. Together with these and other results reported previously, it is concluded that at least two forms of cytochrome P-450 [P-450-HM4 and P-450PA (human P-450IA2)] are involved in the mutagenic activation of IQ in human liver.     

Androgen-dependent renal microsomal cytochrome P-450 responsible for N-hydroxylation and mutagenic activation of 3-methoxy-4-aminoazobenzene in the BALB/c mouse

A murine renal microsomal enzyme responsible for the mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) was characterized by its catalytic activity for the mutagenic and metabolic conversion of 3-MeO-AAB. Incubation of 3-MeO-AAB with a renal or hepatic microsome fraction from male BALB/c mice in the presence of NADPH and NADH yielded N-hydroxy and 4'-hydroxy metabolites of 3-MeO-AAB as determined by two-dimensional thin layer chromatography, and the enzyme responsible for the N-hydroxylation was named 3-MeO-AAB N-hydroxylase. A mutagenicity test using Salmonella typhimurium TA98 bacteria as a tester strain has revealed that N-hydroxy-3-MeO-AAB is a potent direct mutagen but that 4'-hydroxy-3-MeO-AAB is not mutagenic. Although 3-MeO-AAB N-hydroxylase activity in liver microsomes showed no sex difference, the enzyme activity in the kidney was detected from male mice but not from females. However, administration of testosterone to female mice induced the enzyme in the kidney. Castration of male mice depressed the activity of 3-MeO-AAB N-hydroxylase in renal microsomes but it little affected the hepatic activity, and on administration of testosterone to the castrated mice the depressed renal microsomal activity recovered to a normal level. The activity of 3-MeO-AAB hydroxylase and the amount of cytochrome P-450 in renal microsomes showed a close correlation. Both renal and hepatic microsomes required NADPH as a main cofactor to mutagenize 3-MeO-AAB and to yield N-hydroxy-3-MeO-AAB from 3-MeO-AAB, and the enzyme activity was strongly inhibited by 7,8-benzoflavone. When the activities of renal and hepatic 3-MeO-AAB N-hydroxylase were compared on the basis of the amount of cytochrome P-450, the renal type enzyme showed about 8 times greater activity than hepatic type enzyme. These results indicate that the kidney contains an androgen-dependent microsomal 3-MeO-AAB hydroxylase which is different from an isozyme present in the liver and which is a new type of cytochrome P-450 isozyme.     

Mutagenic activation of carcinogenic N-nitrosopropylamines by rat liver: evidence for a cytochrome P-450 dependent reaction

Mutagenic potential of carcinogenic N-nitrosopropylamines wasexamined by the Ames's liquid incubation assay, using rat liver9000 g supernatant (S9) fraction for metabolic activation. N-Nitrosobis(2-hydroxypropyl)amine,N-nitroso(2-hydroxypropyl)-(2-oxopropyl)amine (HPOP), N-nitrosobis(2-oxopropyl)amine(BOP), N-nitrosobis(2-acetoxypropyl)amine, N-nitroso-2,6-dimethylmorpholine,N-nitrosomethyl-(2-hydroxypropyl)amine and N-nitrosomethyl(2-oxopropyl)amineall showed positive mutagenicity in strain TA100 in the presenceof liver S9 while being negative in strain TA98. With the exceptionof HPOP and BOP, which were also mutagenic in TA100 withoutS9 metabolic activation, these N-nitrosopropylamines requiredthe presence of microsomes as a source of enzymes as well asNADP⁺ as a cofactor for mutagenic activation. Treatment of ratswith polychlorinated biphenyls or phenobarbital (PB) resultedin a marked increase in the ability of S9 to activate the sevenN-nitrosamines tested whereas 3-methylcholanthrene (3-MC) inductionwas not effective. All the mutagenic activities were considerablydecreased by preincubation in an atmosphere of either carbonmonoxide or nitrogen gas or by adding cytochrome c to the S9mixture. Metyrapone, a specific inhibitor of PB-inducible majorcytochrome P-450, considerably inhibited mutagenicity, whereas7,8-benzoflavone, a specific inhibitor of 3-MC-inducible majorcytochrome P-448, was totally lacking this effect. These resultsdemonstrate a correlation between rat liver S9 dependent mutagenicityof six N-nitrosopropylamines and their known carcinogenicityin rat in vivo experiments, and that the PB-inducible majorcytochrome P-450 is involved in the mutagenic activation. BOPwas also shown to be activated by extrahepatic (lung, kidney,pancreas) tissue S9, blood S9 and bovine serum albumin (BSA)to the extent of 50% of that activity obtained with liver S9.A possible mechanism of BSA-mediated activation of BOP is discussed.     

Metabolic activation of heterocyclic aromatic amines catalyzed by human arylamine N-acetyltransferase isozymes (NAT1 and NAT2) expressed in Salmonella typhimurium

Heterocyclic aromatic amines formed during the cooking of meatand meat-derived products can be activated to reactive metaboliteswhich bind to DNA, induce mutations and cause tumors in animals.A principal route of metabolic activation is N-oxidation tohydroxylamines, and their subsequent activation by acetyltransferase-catalyzedO-acetylation. We have used mutagenicity assays to study O-acetylationof heterocyclic arylhydroxylamines by the two isozymes of humanN-acetyltransferase, NAT1 and NAT2, expressed in Salmonellatyphimurium. N-Acetylation was also examined, using an HPLCmethod. In addition, Salmonella strains with endogenous acetyltransferaseand lacking this activating activity were used. Hydroxylaminesof nine heterocyclic aromatic amines, IQ, isoIQ, MeIQ, MeIQx,NI, PhIP, Glu-P-1, Glu-P-2, and Trp-P-2 were generated in situby rat liver S9 mix. The strains expressing human NAT1 and lackingacetyltransferase activity showing little or no ability to activatethese substrates. The strains expressing human NAT2 and Salmonellaacetyltransferase supported to different extents the activationof all the compounds except PhIP and Trp-P-2. N-Acetylationof IQ, MeIQx and PhIP was slow or not detectable. In conclusion,human NAT2 but not NAT1 can O-acetylate heterocyclic hydroxylamines.NAT2 probably plays a key role in the genotoxic effects of theabove heterocyclic amines except for PhIP and Trp-P-2, whichhave NAT2-independent mutagenic activity.     

Metabolic activation of mutagenic tryptophan pyrolysis products (Trp-P-1 and Trp-P-2) by a purified cytochrome P-450-dependent monooxygenase system

Tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, were activated to metabolites mutagenic to Salmonella typhimurium by cotychorome P-450 purified from rat liver microsomes. Of the four purified cytochrome P-450 preparations tested, PCB P-448 and MC P-448 showed high activity, while PCB P-450 and PB P-450 were less active. The number of revertants was proportional to the amount of cytochrome PCB P-448 added under the conditions used.     

Hepatic cytochrome P-450 isozyme(s) induced by dietary carcinogenic aromatic amines preferentially in female mice of DBA/2 and other strains

DBA/2, BALB/c or (BALB/cxDBA/2)F₁ (CDF₁) mice of both sexeswere treated for 1 week with a dietary hepatocarcinogenic tryptophanpyrolysate component (Trp P-1 or Trp P-2), and the activityof hepatic microsomal enzyme(s) for mutagenk activations ofTrp P-1 and Trp P-2 were assessed by means of a mutation testwith Salmonella typhimurium TA98. In both Ah-responsive (BALB/cand CDF₁) and Ah-nonresponsive (DBA/2) mice, the dietary treatmentwith Trp P-l or Trp P-2 resulted in a significant increase ofthe enzyme activity for mutagenic activations of Trp P-1 andTrp P-2 in females but not in males, except the case of maleBALB/c mice treated with dietary Trp P-1 Also induction of enzyme(s)in female mice was suppressed by an administration of testosterone.The induced hepatic microsomal enzyme(s) was demonstrated tobe cytochrome P-450 isozyme(s) (mol. wt of 55 000 daltons) byimmunoblots with use of an anti-rat cytochrome P-448 monoclonalantibody and by selective inhibition of the activity by additionof 7,8-benzoflavone into the mutation assay system. These findingsindicate that carcinogic aromatic amines such as Trp P-1 andTrp P-2 are able to induce hepatic cytochrome P-450 isozyme(s)not only in Ah-responsive mice (BALB/c and CDF₁) but also inAh-nonresponsive DBA/2 mice and that the cytochrome P-450 inductionis controlled by androgen(s).     

Metabolic activation of a protein pyrolysate promutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by rat liver microsomes and purified cytochrome P-450

The enzymatic activation of a promutagenic pyrolysate, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx), was studied using the Ames mutagenesis test system.The enzyme catalyzing the mutagenic activation of MeIQx is mainlylocalized in the microsomal fraction. A large number of revertantswas observed in the presence of hepatic microsomes obtainedfrom 3-methylcholanthrene (3-MC)- or polychlorinated biphenyl(PCB)-treated rats but only a minimal number with the hepaticmicrosomes from untreated or phenobarbital (PB)-treated rats.In addition, the microsomal activation was reduced efficientlyby known inhibitors of cytochrome P-450- mediated reactionssuch as 7,8-benzoflavone, ellipticine and flavone. Among fiveforms of purified rat cytochrome P-450, the highest sp. act.(no. of revertants induced/nmol cytochrome P-450) for the activationof MeIQx was observed with a high-spin form of cytochrome P-450,P-448-H, followed by the low-spin form, P-448-L, and to a lesserextent by PB-inducible forms, P-450b and P-450e. P-450-male,which is a main constitutive form of cytochrome P-450 In malerat livers, showed considerable catalysis for the mutagenicactivation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) andMeIQx. These results Indicate that the metabolic activationof MeIQx is catalyzed mainly by two forms of cytochrome P-450,P-448-H and P-488-L, in the livers of PCB- or 3-MC-treated rats,but also that P-450-male may play an important role in the activationin livers of Intact male rats.     

Benzene metabolism by ethanol-, acetone-, and benzene-inducible cytochrome P-450 (IIE1) in rat and rabbit liver microsomes

Ethanol is known to exert a synergistic effect on the toxicity of benzene. In the present investigation it was found that benzene was metabolized at a rate 20-65-fold higher in liver microsomes from ethanol- or acetone-treated rats than in microsomes from control animals. One high affinity site [Km = 19 +/- 5 (SD) microM] and one low affinity site [Km = 0.3 +/- 0.1 mM] for benzene metabolism were present in microsomes of acetone-treated rats, and similar sites were seen in microsomes from control or ethanol-treated rats. Treatment of the animals with either ethanol or acetone mainly influenced the Vmax values for benzene metabolism. Also benzene treatment of rats caused an increased rate of microsomal benzene metabolism. The hepatic microsomal NADPH-dependent metabolism of benzene was inhibited by compounds known to interact with the ethanol-inducible form of P-450 such as imidazole, ethanol, aniline, and acetone but was unaffected by addition of metyrapone. Anti-IgG against ethanol-inducible cytochrome P-450 from rat (P-450j) or rabbit liver (P-450 LMeb) inhibited the microsomal benzene metabolism effectively in rat or rabbit liver microsomes, respectively, whereas preimmune IgG was without effect. The level of rat ethanol-inducible P-450 (P-450j) was induced to an extent similar to that for the microsomal benzene metabolism, by either benzene, acetone, or ethanol. The data indicate that benzene is metabolized mainly by the ethanol-inducible P-450 form in liver microsomes and that the induction of this isozyme by ethanol can provide an explanation for the synergistic action of ethanol on benzene toxicity.     

Metabolic oxidation of the carcinogens 4-aminobiphenyl and 4,4'-methylene-bis(2-chloroaniline) by human hepatic microsomes and by purified rat hepatic cytochrome P-450 monooxygenases

Metabolic N-oxidation and ring-oxidation of carcinogenic arylamines by hepatic cytochromes P-450 are generally regarded as critical activation and detoxification pathways, respectively. Two arylamines with known human exposure, 4-aminobiphenyl (ABP) and 4,4'-methylene-bis(2-chloroaniline) (MOCA), have been examined as substrates for 10 different purified rat hepatic cytochromes P-450 and for human liver microsomal preparations from 22 individuals. Metabolites were analyzed by high-performance liquid chromatography and flow scintillation techniques. As reported for certain other carcinogenic arylamines, the isosafrole-inducible isozyme, P-450ISF-G, had the highest catalytic activity for ABP N-oxidation (13.6 nmol/min/nmol P-450), but P-450BNF-B, P-450UT-A, P-450UT-F, and P-450PB-B also showed appreciable activity. Ring-oxidation of ABP occurred only to a minor extent. In contrast, N-oxidation of MOCA was preferentially catalyzed by the phenobarbital-inducible enzymes, P-450PB-B and P-450PB-D (9.0 and 6.6 nmol/min/nmol P-450, respectively). MOCA ring-oxidation and methylene carbon oxidation showed varied cytochrome P-450 selectivity and accounted for 14 to 79% of total oxidation products. There was a 44-fold variation in rates of ABP N-oxidation in the 22 human liver microsomal preparations, while rates of N-oxidation of MOCA varied only 8-fold. Ring/methylene carbon-oxidation of MOCA accounted for 6-19% of total oxidation products in the case of the human microsomal preparations, whereas ring-oxidation of ABP accounted for less than 7% of total oxidation. In addition, there was a strong correlation (R = 0.90) between rates of ABP N-oxidation and phenacetin O-deethylation, which is considered a human genetic polymorphism. Moreover, both the ABP N-oxidation and phenacetin O-deethylation activities of human liver microsomes showed a good correlation (R = 0.72) with the levels of cytochrome P-450 immunochemically related to rat P-450ISF-G. These data indicate that specific inducible and constitutive cytochromes P-450 are involved in the metabolic activation and detoxification of the carcinogens ABP and MOCA. Therefore, individual profiles of cytochromes P-450, affected by both environmental and genetic factors, may be significant determinants of individual susceptibility to arylamine carcinogenesis.     

Mutagenic activation of carcinogenic N-nitrosopropylamines by liver S9 fractions from mice, rats and hamsters: evidence for a cytochrome P-450-dependent reaction

The mutagenic potential of nine carcinogenic N-nitrosopropylamineswas examined by Ames preincubation assay using liver 9000 gsupernatant (S9) fractions from female rats and male hamstersand mice for metabolic activation. N-Nitrosobis(2-hydroxypropyl)amine,N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine, N-nitrosobis(2-oxopropyl)amine,N-nitrosobis(2-acetoxypropyl)amine, N-nitroso-2,6-dimethylmorpholine,N-nitrosomethyl(2-hydroxypropyl)amine, N-nitrosomethyl(2-oxopropyl)amine,N-nitroso(2,3-dihydroxypropyl)(2-hydroxypropyl)amine and N-nitrosomethyl(2,3-dihydroxypropyl)amineall showed positive mutagenicity in strain TA100 in the presenceof liver S9 from three animal species pretreated with polychlorinatedbiphenyls or phenobarbital (PB). The S9-mediated mutagenicityof these N-nitrosamines was almost completely diminished bythe removal of NADP⁺ from the assay system. All the activitieswere considerably decreased by preincubation in an atmosphereof carbon monoxide or adding cytochrome c to the S9 mixture.Metyrapone considerably inhibited mutagenicity, whereas 7,8-benzoflavonewas totally lacking this effect. These results demonstrate acorrelation between the mutagenicity of nine N-nitrosopropylaminesmediated by liver S9 from three animal species and their knowncarcinogenicity in rodent in vivo experiments, and that thePB-inducible major cytochrome P-450 is selectively involvedin the mutagenic activation. A relationship between mutagenicpotencies of the N-nitrosamines and their known carcinogenicpotencies in rats and hamsters is discussed     

Species, sex and organ differences in induction of a cytochrome P-450 isozyme responsible for carcinogen activation: effects of dietary hepatocarcinogenic tryptophan pyrolysate components in mice and rats

Both sexes of BALB/cxDBA/2 F₁ mice and F344 rats were treatedfor 1 week with a diet containing 0.02% of hepatocarcinogenictryptophan pyrolysate component (Trp P-1 or Trp P-2), and changesin the carcinogen activation enzyme activity in various organswere examined comparatively using a mutation test with Salmonellatyphimurium TA98 as a tester bacterium. Hepatic enzymes fromuntreated mice and rats showed a definite catalytic activityfor mutagenic activations of Trp P-1 and Trp P-2, whereas theactivities of other organs —such as lung, kidney, smallintestine and colon—were undetectable or very low. Inboth mice and rats either the Trp P-1 or Trp P-2 feeding resultedin induction of cytochrome P-450 isozyme(s), which could mediatein the liver but not in other organs the mutagenic activationof the carcinogen itself. As to the sex difference, the inductionof the activation enzyme(s) was greater in the female animalsthan in the males. Species difference in the activity of hepaticenzymes catalyzing the Trp P-1 and Trp P-2 mutageneses was alsoobserved in animals treated with the basal diet; the activitywas higher in mice than in the sex-matched rats (Trp P-1, {smalltilde}1.5-fold; Trp P-2, {small tilde}7-fold). When diet containingTrp P-1 or Trp P-2 was fed for 1 week, the activity of the ratliver for Trp P-1 mutagenesis was of a level similar to thatof the sex-matched mice, but for Trp P-2 mutagenesis it wasless than half that in the mice. The induced hepatic enzymesin mice and rats were suggested to be 3-methylcholanthrene-induciblecytochrome P-448 isozymes as determined by mutation tests withTrp P-1, Trp P-2 and two other substrates and by immunochemicalanalyses of rat hepatic cytochrome P-450 using monoclonal antibodiesagainst rat cytochrome P-448 isozymes. These results indicatethat a form of cytochrome P-450 responsible for activation ofTrp P-1 and Trp P-2 is inducible by dietary treatment of miceor rats with these carcinogens and that the amount of the cytochromeP-450, including resident and induced forms, is related to thespecies, sex and organ differences in their carcinogenic susceptibilityto these chemicals.     

N,N-diethyl-2-[4-(phenylmethyl)phenoxy] ethanamine (DPPE), a chemopotentiating and cytoprotective agent in clinical trials: interaction with histamine at cytochrome P450 3A4 and other isozymes that metabolize antineoplastic drugs

Purpose: N,N-diethyl-2-[4-(phenylmethyl)phe- noxy]ethanamine · HCl (DPPE), an intracellular histamine (HA) antagonist with chemopotentiating and cytoprotective properties, is currently in phase 2 and 3 clinical trials in breast and prostate cancer. DPPE modulates growth at in vitro concentrations that antagonize HA binding to cytochromes P450 in rat liver microsomes. HA inhibits P450 metabolism of some drugs. Recent in vitro studies in human colon cancer cells have linked DPPE enhancement of paclitaxel, doxorubicin and vinblastine cytotoxicity to inhibition of the P-glycoprotein (P-gp) pump. Many substrates of P-gp are also substrates of CYP3A4, a P450 isozyme that metabolizes a variety of antineoplastic agents and is highly expressed in some malignant tissues. Therefore, we assessed whether (a) DPPE and HA interact at CYP3A4 and other P450 human isozymes, and (b) DPPE inhibits the catalytic activity of CYP3A4. Methods: Using spectral analysis, we measured DPPE and HA binding to insect microsomes that express human P450 isozymes 1A1, 2B6, 2D6 or 3A4. Employing thin-layer chromatography, we assessed the metabolism of DPPE by each isozyme and DPPE inhibition of testosterone metabolism by CYP3A4 and by rat liver microsomes. Results: (1) DPPE evoked “type I” (substrate site binding) absorbance-difference spectra with CYP2D6 (K_s=4.1 ± 0.4 μM), CYP3A4 (K_s= 31 ±15 μM) and CYP1A1 (K_s=40 ± 9 μM), but not with CYP2B6. (2) In correspondence with the binding studies, DPPE was metabolized by CYP2D6, CYP3A4 and CYP1A1; no metabolism occurred with CYP2B6. (3) HA evoked “type II” (heme iron binding) absorbance-difference spectra with all four isozymes, with K_s values in the range 80–600 μM. DPPE inhibited HA (600 μM) binding to CYP2D6 (IC₅₀=4 μM, 95% CI=1.8–8.9 μM) and CYP1A1 (IC₅₀=135 μM: 95% CI=100–177 μM), but stimulated HA (500 and 1000 μM) binding to CYP3A4 (EC₅₀=155 μM, 95% CI=104–231 μM). DPPE did not affect HA binding to CYP2B6. (4) DPPE inhibited the metabolism of testosterone by CYP3A4. The concentration/effect curve was biphasic: DPPE inhibited metabolism by 30% at the first site (IC₅₀=3 μM, 95% CI=0.5–25.5 μM), and an additional 70% inhibition occurred at the second site (IC₅₀=350 μM, 95% CI=215–570 μM). A similar result was observed with rat liver microsomes. Conclusion: DPPE is a substrate for CYP3A4, CYP2D6 and CYP1A1, but not CYP2B6. DPPE inhibits testosterone metabolism by interacting at two sites on CYP3A4, the first correlating with its K_s value to bind the substrate site and the second, with its EC₅₀ value to enhance HA binding to the heme iron. We postulate that (1) the inhibitory effect of DPPE on CYP3A4 activity is mediated directly at the substrate site and indirectly by its enhancement of the binding of HA to the heme moiety; (2) in tumor cells that express high constitutive levels of CYP3A4, potentiation of chemotherapy cytotoxicity by DPPE results, in part, from inhibition of CYP3A4-mediated metabolism and P-gp-mediated efflux of antineoplastic drugs; (3) in normal cells that express low constitutive levels of the isozyme, cytoprotection by DPPE results, in part, from induction of CYP3A4 and P-gp, resulting in an increase both in metabolism and efflux of antineoplastic drugs. Received: 26 April 1999 / Accepted: 3 September 1999     

Participation of rat liver cytochrome P450 2E1 in the activation of N-nitrosodimethylamine and N-nitrosodiethylamine to products genotoxic in an acetyltransferase-overexpressing Salmonella typhimurium strain (NM2009).

The possible roles of cytochrome P450 (P450) enzymes in the metabolic activation of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) by rat liver microsomes have been examined in a system containing the bacterial tester strain Salmonella typhimurium NM2009, a newly developed strain showing high O-acetyltransfer activities. The DNA-damaging activity could be determined by measuring expression of the umu gene in a plasmid containing the fused umuC-lacZ gene construct in the bacteria. The following lines of evidence support the view that both NDMA and NDEA are principally oxidized to reactive products by P450 2E1 in rat liver microsomes. First, NDMA and NDEA were activated by rat liver microsomes in a protein- and substrate-dependent manner and the former chemical was more active than the latter; both activities were induced in rats treated with P450 2E1 inducers such as ethanol, acetone and isoniazid and by starvation. Second, activation of NDMA and NDEA were both inhibited significantly by antibodies raised against rat P450 2E1 and by P450 2E1 inhibitors such as diethyldithiocarbamate and 4-methylpyrazole in rat liver microsomes. Finally, in reconstituted monooxygenase systems containing purified rat P450 enzymes, P450 2E1 gave the highest rates of the activation of both NDMA and NDEA; the addition of rabbit cytochrome b5 to the system caused about a 1.5-fold increase in both reactions. In separate experiments we also found that N-nitrosomethylacethoxymethylamine, a compound that reacts with DNA after ester cleavage, is more genotoxic in S.typhimurium NM2009 than in S.typhimurium NM2000, a strain that is defective in O-acetyltransferase activity. Part of the pathway involved in the activation of nitrosamines is suggested to be acetylation of alkyldiazohydroxides formed by P450 or acetylesterase, because the genotoxic activity of N-nitrosomethylacethoxymethylamine in S.typhimurium NM2009 could be inhibited by the O-acetyltransferase inhibitor pentachlorophenol. These results indicate that NDMA and NDEA are oxidized to gentoxoic products by rat liver microsomes and that a P450 2E1 enzyme plays a major role in the activation of these two potent carcinogens. The activation pathway of N-nitrosodialkylamines through acetylation by O-acetyltransferase has been proposed. This simple bacterial system for measuring genotoxicity should facilitate studies on the activation of N-nitroso alkylamines.     

Activation of amino-alpha-carboline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and a copper phthalocyanine cellulose extract of cigarette smoke condensate by cytochrome P-450 enzymes in rat and human liver microsomes

The ability of cigarette smoke condensate to induce a genotoxic response has been measured in liver microsomal and reconstituted monooxygenase systems containing rat and human cytochrome P-450 (P-450) enzymes, as determined by umu gene expression in Salmonella typhimurium TA1535/pSK1002. The reactivities of amino-alpha-carboline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), two compounds known to be present at considerable levels in cigarette smoke condensate, were also determined and compared with regard to genotoxicity. Amino-alpha-carboline and PhIP are activated principally by P-450 1A2 enzymes in human and rat liver microsomes: (a) activation of both compounds was catalyzed efficiently by liver microsomes prepared from rats treated with 5,6-benzoflavone, isosafrole, or the commercial polychlorinated biphenyl mixture Aroclor 1254, and the activities could be considerably inhibited by antibodies raised against P-450 1A1 or 1A2; (b) the rates of activation of these compounds were correlated with the amount of human P-450 1A2 and of phenacetin O-deethylation activity in different human liver microsomal preparations, and these activities were inhibited by anti-P-450 1A2; (c) reconstituted enzyme systems containing P-450 1A enzymes isolated from rats and humans showed the highest rates of activation of amino-alpha-carboline and PhIP. In rat liver microsomes PhIP may also be activated by P-450 3A enzymes; activity was induced in rats treated with pregnenolone 16 alpha-carbonitrile and was inhibited by anti-human P-450 3A4. However, in humans the contribution of P-450 3A enzymes could be excluded as judged by the very low effects of anti-P-450 3A4 on the microsomal activities and poor correlation with P-450 3A4-catalyzed activities in various liver samples. Cigarette smoke condensate strongly inhibited the activation of several potent procarcinogens by human liver microsomes, particularly the reactions catalyzed by P-450 1A2, but was not so inhibitory of the activation reactions catalyzed by P-450 3A4 and of P-450 2D6-catalyzed bufuralol 1'-hydroxylation. Genotoxic components of the cigarette smoke condensate were extracted by using copper phthalocyanine cellulose (blue cotton). Genotoxicity of this extract was observed only after activation by P-450, and the inhibition of P-450 1A2 activities by these extracts was slight.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of benzo(a)pyrene by subcellular fractions of rat liver: evidence for similar patterns of cytochrome P-450 in rough and smooth endoplasmic reticulum but not in nuclei and plasma membrane

Since our earlier work (P. Stasiecki, F. Oesch, G. Bruder, E.D. Jarasch, and W.W. Franke, Eur. J. Cell Biol., 21: 79-92, 1980) had shown that carcinogen-metabolizing monooxygenase activity was present in almost all investigated cellular membranes, the possibility of differential control of the various metabolic pathways in the individual cellular membranes arose. Using high pressure liquid chromatography we have now studied the benzo(a)pyrene metabolites formed by rough and smooth endoplasmic reticulum, nuclei, and plasma membrane as well as mitochondrial fractions and investigated the metabolic cooperation between the monooxygenases and epoxide hydrolase in these fractions. Since various cytochrome P-450 isozymes catalyze the oxidative attack on the benzo(a)pyrene molecule at defined preferential sites, this analysis also provides an indirect trace of potential differences in the pattern of cytochrome P-450 isozymes present in the individual membranes. The metabolic profiles produced by the two most active fractions, smooth and rough endoplasmic reticulum, were very similar to each other but different from those produced by the other three preparations. The metabolite pattern produced by incubations containing nuclear fractions differed slightly from that produced by the fractions of endoplasmic reticulum, but plasma membrane and mitochondria produced markedly different patterns. Since the similarity of the benzo(a)pyrene metabolite pattern produced by the smooth and rough endoplasmic reticulum suggested similar cytochrome P-450 isozyme patterns in these two subfractions, they were further investigated by the use of selective inducers as well as a broad spectrum substrate, 7-ethoxy-coumarin, in the absence and presence of selective inhibitors. Treatment of animals with trans-stilbene oxide or phenobarbital (a) increased the total amount of metabolites per protein mass and time, (b) changed the pattern of metabolites, but (c) induced a pattern of metabolites which was again very similar in rough and smooth endoplasmic reticulum. Even more distinct changes were found following treatment with 3-methylcholanthrene or beta-naphthoflavone. Both of these compounds (a) preferentially induced the activity of rough endoplasmic reticulum, (b) changed the profile of metabolites, but (c) again did not disturb the similarities of the benzo(a)pyrene metabolite pattern between both fractions.(ABSTRACT TRUNCATED AT 400 WORDS)