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 inactivation of N-nitrosamines by cytochrome P-450 in vitro and in vivo

Nitrite was formed on incubation of N-nitrosamines with both microsomal systems and a reconstituted system consisting of cytochrome P-450 and NADPH P-450 reductase from pig liver. Nitrite was not obtained when the nitrosamines were incubated with NADPH P-450 reductase alone or when molecular oxygen or NADPH was omitted. Various inhibitors of the microsomal monooxygenase decreased nitrite generation. Furthermore, nitrite and a substantially higher amount of nitrate could be found in the urine of rats given N-nitrosodiphenylamine. Diphenylamine was also detected. From in-vitro studies, it is concluded that denitrosation of N-nitrosamines is a cytochrome P-450-dependent process, which also occurs in vivo.     

A comparative study of the mutagenic activation of N-nitrosopropylamines by various animal species and man: evidence for a cytochrome P-450 dependent reaction

The mutagenic potential of seven carcinogenic N-nitrosopropylamines was examined by means of Ames' preincubation assay using liver 9000 g superanatant (S9) fractions from rats, hamsters, mice, rabbits, monkeys and humans for metabolic activation. N-Nitroso(2-hydroxypropyl) (2-oxopropyl)amine (HPOP), N-nitrosobis(2-oxopropyl)amine (BOP), N-nitrosobis(2-acetoxypropyl)amine (BAP), N-nitroso-2,6-dimethylmorpholine, N-nitrosomethyl(2-hydroxypropyl)amine (MHP) and N-nitrosomethyl(2-oxopropyl)amine all showed positive mutagenicity in strain TA100 in the presence of liver S9 from each of the uninduced animals, but N-nitrosobis(2-hydroxypropyl)amine was negative. The mutagenic activity of MHP was highest with liver S9 from the hamster, but that of BAP was lowest with hamster liver S9. With regard to the activities of the other N-nitrosopropylamines, there were no significant differences among five animal species. In the presence of liver S9 from humans, HPOP, BOP and MHP showed positive mutagenicity. With the exception of HPOP and BOP, the animal or human liver S9-mediated mutagenicity of these N-nitrosopropylamines was almost completely lost upon removal of NADP+ from the assay system, preincubation in an atmosphere of carbon monoxide, or addition of cytochrome c to the S9 mixture. metyrapone decreased the activities of five compounds (except for BOP) by between 29 and 71%, whereas 7,8-benzoflavone was totally lacking in this effect. These results demonstrated that the phenobarbital-inducible major cytochrome P-450 in animal and human livers is involved in the mutagenic activation of the N-nitrosopropylamines.     

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     

Localization of a cytochrome P-450 isozyme (cytochrome P-450 PB-B) and NADPH-cytochrome P-450 reductase in rat nasal mucosa

Antibodies raised against cytochrome P-450 PB-B, the major phenobarbital-inducible isozyme of rat hepatic microsomal cytochrome P-450, and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) were employed to determine the cellular localizations of these enzymes within the nasal mucosa of untreated rats. Immunohistochemical staining for each enzyme was detected at the light microscopic level within the respiratory and olfactory epithelia, duct and acinar cells of seromucous glands in the respiratory region, and duct and acinar cells of Bowman's glands in the olfactory region. These findings demonstrate that a number of different cell types in rat nasal mucosa contain enzymes which participate in the monooxygenations of chemical carcinogens and other xenobiotics.     

Metabolic activation of pyrolysate arylamines by human liver microsomes; possible involvement of a P-488-H type cytochrome P-450

Metabolic activating capacity of human livers for carcinogenic heterocyclic arylamines has been studied using a Salmonella mutagenesis test. A large individual variation was observed among 15 liver samples in the capacities of activation of Glu-P-1 (2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole), IQ (2-amino-3-methylimidazo[4,5-f]quinoline) and MeIQx (2-amino-3,8-dimethyl-3 H-imidazo[4,5-f]quinoxaline). The average numbers of revertants induced by the three heterocyclic arylamines were nearly the same or rather higher in the presence of hepatic microsomes from human than those from rat. In high-performance liquid chromatography, formation of N-hydroxy-Glu-P-1 was detected and accounted for more than 80% of the total mutagenicity observed in the human microsomal system with Glu-P-1, indicating that, similarly to experimental animals, N-hydroxylation is a major activating step for heterocyclic arylamines in human. Addition of flavone or 7,8-benzoflavone to human liver microsomes showed effective inhibition of the mutagenic activation of Glu-P-1, although the treatment rather enhanced microsomal benzo[a]pyrene hydroxylation in human livers. Mutagenic activation of Glu-P-1 by human liver microsomes was also decreased by the inclusion of anti-rat P-448-H IgG, and was well correlated with the content of immunoreactive P-448-H in livers, suggesting the involvement of a human cytochrome P-450, which shares immunochemical and catalytic properties with rat P-448-H, in the metabolic activation of heterocyclic arylamines in human livers.     

Metabolic processing of 2-acetylaminofluorene by microsomes and six highly purified cytochrome P-450 forms from rabbit liver

Kinetic analysis of oxidative metabolism of 2-acetylaminofluorene(AAF) was studied in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)-induced microsomes and with six highly purified cytochromeP-450 isoenzymes from rabbit liver. Kinetic parameters weredefined for 7-, 5-, 3-, 1- and N-hydroxylations of AAF. 7-Hydroxylationwas best defined by a two enzyme system, displaying a high affinityand relatively low capacity and a low affinity high capacitycomponents in both control and TCDD induced microsomes. Allthe purified cytochrome P-450 isoenzymes were capable of catalyzingthe 7-hydroxylation of AAF and, with the exception of form 4,this was the only oxidation on the AAF molecule catalyzed bythese forms. It is probable that forms 1, 4 and 6 accountedfor a substantial part (25%) of total metabolic capacity correspondingto the high affinity component of 7-hydroxylation, whereas forms3b and 3c accounted for <5% of the metabolic capacity displayedby the low affinity component in control microsomes. However,forms 4 and 6 could account for >90% of the metabolic capacityof the high affinity component of 7-hydroxylation in TCDD microsomes,whereas the form(s) responsible for the metabolic capacity ofthe low affinity component were not identified. Each of the1-, 3-, 5- and N-hydroxylations were best defined by a singleenzyme system in both control and TCDD microsomes (3- and 5-hydroxylationscould not be defined in TCDD microsomes). Close agreements werefound between the apparent Km for N-hydroxylation in control,TCDD induced microsomes and with form 4. -Naphthoflavone inhibitedAAF N-hydroxylation to a similar extent in control and TCDDmicrosomes and in form 4. These date indicate that: (i) a subpopulationof cytochrome P-450 isoenzymes, which includes all the purifiedP-450 forms tested in the present study, is solely involvedin detoxification (i.e., 7-hydroxylation) of AM, and as suchprobably behave as a functional unit in vivo; (ii) modulationof cytochrome P-450 content by inducers such as TCDD resultsin emergence of relatively few cytochrome P-450 isoenzymes thatcan account for most of the oxidative metabolism of AAF; and(iii) a single cytochrome P-450 isoenzyme (i.e., form 4) isresponsible for catalyzing N-hydroxylation of AAF, the firstand the obligatory step in the metabolic activation of thiscarcinogen.     

Metabolic activation of 4-ipomeanol by complementary DNA-expressed human cytochromes P-450: evidence for species-specific metabolism.

4-Ipomeanol is a pulmonary toxin in cattle and rodents that is metabolically activated by cytochromes P-450 (P-450s). P-450-mediated activation of 4-ipomeanol to DNA binding metabolites was evaluated using a vaccinia virus complementary DNA expression system and an in situ DNA-binding assay. Twelve human P-450s and two rodent P-450s were expressed in human hepatoma Hep G2 cells and examined for their abilities to metabolically activate this toxin. Three forms, designated CYP1A2, CYP3A3, and CYP3A4, were able to catalyze significant production of DNA-bound metabolites of 20-, 8-, and 5-fold, respectively, above binding catalyzed by Hep G2 cells infected with wild-type vaccinia virus. These enzymes, with highest activities, are not known to be expressed in human or rodent lung. CYP2F1 and CYP4B1, two enzymes that are expressed in lung, display only modest 3- and 2-fold respective increased abilities to metabolically activate 4-ipomeanol. Two human forms were inactive and seven other human forms showed activities ranging from 0.5- to 2-fold above control level. Surprisingly, rabbit complementary DNA-expressed CYP4B1 was the most active enzyme (180-fold above control) among all P-450s tested in producing DNA-binding metabolites from this mycotoxin. These studies demonstrate a species difference in 4-ipomeanol metabolism and suggest caution when attempting to extrapolate rodent data to humans.     

Immunohistochemical localization of cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase in the rat ventral prostate

Rabbit antibodies raised against the major isozymes of cytochrome P-450 isolated from hepatic microsomes of beta-naphthoflavone- (BNF) and phenobarbital-treated rats (cytochrome P-450 BNF-B2 and cytochrome P-450 PB-B2, respectively) and against rat liver NADPH-cytochrome P-450 reductase were used to localize these enzymes immunohistochemically in the rat ventral prostate. Using the unlabeled antibody peroxidase-antiperoxidase technique, NADPH-cytochrome P-450 reductase was detected exclusively in the epithelial cells of the gland to the same magnitude in untreated, phenobarbital-, and BNF-treated rats. Cytochrome P-450 BNF-B2-like immunoreactivity was exclusively present in the glandular epithelium in BNF-treated rats, whereas staining could not be visualized in untreated or in phenobarbital-treated rats. The staining for NADPH-cytochrome P-450 reductase was more uniformly distributed within the epithelium than was the cytochrome P-450 BNF-B2-like immunoreactivity. Cytochrome P-450 PB-B2-like immunoreactivity was not found, regardless of animal pretreatment. These findings support our previous results (Haaparanta, T., Halpert, J., Glaumann, H., and Gustafsson, J-A., Cancer Res. 43: 5131-5137, 1983) demonstrating the presence of constitutive NADPH-cytochrome P-450 reductase in the prostate and that an isozyme of cytochrome P-450 is highly inducible by BNF in this gland. The significance of these findings are discussed in view of the essentially unknown etiology of human prostatic cancer.     

Induction of microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450IVA1 (P-450LA omega) by dehydroepiandrosterone in rats: a possible peroxisomal proliferator

Dehydroepiandrosterone (DHEA) is a naturally occurring C19-steroid that is found in the peripheral circulation of mammals, including humans. The feeding of DHEA to rodents has been shown to inhibit chemical carcinogenesis in colon, liver, and lung. Therefore, the effect of DHEA on hepatic enzyme activities that are associated with carcinogen metabolism was assessed. Microsomal NADPH-cytochrome P-450 reductase activity and the content of cytochrome b5 were induced 1.8- and 1.4-fold, respectively, upon feeding male Sprague-Dawley rats a synthetic diet containing 0.45% DHEA (w/w). No significant changes in total content of microsomal cytochrome P-450 or the activities of microsomal NADH-cytochrome b5 reductase and cytosolic or microsomal NAD(P)H-quinone oxidoreductase were noted at day 7 of feeding. Cytosolic glutathione S-transferase activity was decreased to 68% of control activity. Administration of DHEA p.o. or by i.p. injection for 5 days led to the same extent of induction of NADPH-cytochrome P-450 reductase activity. Maximal induction of this flavoprotein reductase was noted between days 3 and 4 of feeding or at a dose of 80-120 mg/kg i.p. A small but statistically significant increase in total microsomal cytochrome P-450 was observed after DHEA administration i.p. Rats fed DHEA had a slower growth rate compared with rats fed control diet, whereas rats treated with DHEA i.p. had growth rates identical to those of controls. The liver weights of rats given DHEA by p.o. or i.p. routes were increased significantly compared to those of control rats. Pair feeding of rats with DHA-containing or control diets served to demonstrate that the levels of induction of hepatic microsomal NADPH-cytochrome P-450 reductase and at least one form of cytochrome P450 (P-450IVA1) were the same as those seen in livers of rats fed DHEA ad libitum. This finding suggested that the induction of the flavoprotein and at least one form of the cytochrome was not due to caloric restriction. The increase in NADPH-cytochrome P-450 reductase content of liver microsomes prepared from rats either fed or treated i.p. with DHEA was also observed by Western blotting techniques. DHEA did not appear to induce any of the major forms of rat liver microsomal cytochrome P-450 that are normally increased by either phenobarbital, beta-naphthoflavone, or dexamethasone pretreatment of rats in vivo. However, the measurement of androstenedione and testosterone metabolism in vitro showed pronounced decreases in the 16 alpha-hydroxylase activities of liver microsomes following DHEA feeding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of mitomycin C by NADPH:cytochrome P-450 reductase

Mitomycin C is an alkylating agent used in cancer chemotherapy that shows some specificity towards hypoxic cells. The therapeutic effects of this compound are thought to result from its metabolic activation by enzymes such as NADPH:cytochrome P-450 reductase. In a previous report we described a Chinese hamster ovary cell line resistant to mitomycin C, which had a decreased NADPH:cytochrome P-450 reductase activity coupled with a lower rate of mitomycin C metabolism. In order to provide further evidence that the lower reductase activity is a factor in the resistance mechanism, we incorporated NADPH:cytochrome P-450 reductase into cytotoxicity assays and showed that it significantly sensitizes cells to mitomycin C. Also, the difference in drug sensitivity between the wild-type and drug-resistant Chinese hamster ovary cells was no longer observed. In addition to these studies, we expressed a rat liver NADPH:cytochrome P-450 reductase cDNA in a Salmonella typhimurium strain, LR5000. The bacteria expressing the rat NADPH: cytochrome P-450 reductase showed increased sensitivity to mitomycin C when incubated with this compound under aerobic conditions. However, under hypoxic conditions increased sensitivity was not observed. This parallels the previous finding with mitomycin C-resistant Chinese hamster ovary cells. These data provide direct evidence for the role of NADPH:cytochrome P-450 reductase in the cytotoxic action of this mitomycin C under aerobic but not hypoxic conditions and suggest that reduced levels of this enzyme can lead to drug resistance. P-450 reductase expressed in S. typhimurium may provide a valuable tool for evaluating the role of this enzyme in the toxicity of drugs activated through a one electron reduction pathway.     

Chloroethanes: their metabolism by hepatic cytochrome P-450 in vitro

1,1,1- and 1,1,2-trichloroethane and 1,1,2,2-tetra-chloroethanestimulate CO- inhibitable NADPH oxidation by hepatic microsomes.Incubation of the chloroethanes, hepatic microsomes, an NADPH-generatingsystem and EDTA results in the production of chlorinated metabolites.Both NADPH oxidation and metabolite production are inhibitedby CO, SKF 525A and/or metyrapone. The induction of cytochromeP-450 with phenobarbital enhances the binding and metabolismof the chloroalkanes, while the induction of cytochrome P-448with ß-napthoflavone does not. 1,1,1-trichloroethaneis converted to 2,2,2-trichloroethanol by hepatic microsomalcytochrome P-450, while the major metabolites of 1,1,2-trichloroethaneand 1,1,2,2-tetrachloroethane from this enzyme system are mono-and dichloroacetate, respectively. Mechanisms for the productionof these metabolites are suggested. The relative mutagenic andcarcinogenic potentials of the three chloroethanes are consideredwith respect to their relative extents of metabolism and theirproposed metabolic pathways.     

Microsomal P-450 induction by some secondary products from thermal oxidation of dietary lipids: epidermal hyperplasia, mutagenicity and cytochrome P-450 activities

Distillable secondary products from roasted fowl were found to be cytotoxic but not mutagenic when assayed with Salmonella typhimurium strains TA98, TA100 and TA1537. A crudely separated fraction of the volatiles produced focal hyperplasia and damage to the epidermis of the backs of mice. The volatiles also caused an apparent synthesis of non-constitutive forms of rat hepatic cytochromes P-450 which metabolize benzo[a]pyrene B [a]P differently from the constitutive P-450.     

Metabolic activation of phenacetin and phenetidine by several forms of cytochrome P-450 purified from liver microsomes of rats and hamsters

Metabolic activation of phenacetin by liver microsomes proceeds via both phenetidine and N-hydroxyphenacetin to direct-acting mutagens, i.e., N-hydroxyphenetidine and p-nitrosophenetole. Five different molecular species of cytochrome P-450 have been purified from liver microsomes of drug-pretreated Wistar rats or Syrian hamsters and their abilities to activate phenetidine and phenacetin were compared using reconstituted microsome systems. High-spin forms of cytochrome P-450 purified from 3-methylcholanthrene-pretreated rats (MC-P-448-H) or hamsters (P-488 ham-II) showed higher catalytic activity for N-hydroxylation of phenetidine than three other low-spin forms of cytochrome P-450 purified from the same animals or from phenobarbital-pretreated rats. MC-P-448-H and P-488 ham-II required the presence of cytochrome b5 for their maximum activities in the reconstituted system. The five forms of cytochrome P-450, however, exhibited no measurable activity for N-hydroxylation of phenacetin either with or without cytochrome b5. The mutagenicity of phenacetin and phenetidine toward Salmonella typhimurium TA100 was generated when the reconstituted microsomes containing MC-P-488-H or P-488 ham-II were used as activating enzymes. From these results, it was suggested that high-spin forms of cytochrome P-450 (MC-P-448-H and P-448 ham-II) played an important role in the metabolic activation of phenacetin to the direct-acting mutagens.     

Formation of hydroxy derivatives, aldehydes, and nitrite from N-nitrosomethyl-n-amylamine by rat liver microsomes and by purified cytochrome P-450 IIB1

The metabolism was examined of the esophageal carcinogen N-nitrosomethylamylamine (NMAA) by liver microsomes and slices from adult male Sprague-Dawley rats. Hydroxylation at C-2 to C-5 of the amyl group to give stable hydroxy-NMAAs was studied by gas chromatography-thermal energy analysis to determine the products. Microsomal metabolism produced mainly 4-hydroxy-NMAA, proceeded optimally in 100 mM phosphate at pH 7.4, and showed no sex differences. Induction by phenobarbital (PB) and 3-methylcholanthrene produced effects which were similar in slices and microsomes, with PB inducing hydroxylation at all positions and 3-methylcholanthrene specifically inducing 3-hydroxylation by factors of 2- and 6-fold. Clofibrate and isoniazid treatments did not affect NMAA metabolism by liver slices. Aroclor-1254 strongly induced microsomal 2- and 3-hydroxylation. For 2- to 5-hydroxylation, Km values for uninduced microsomes were, respectively, 1.6, 1.2, 0.3, and 1.1 mM, with Vmax of 0.08, 0.26, 1.06, and 0.15 nmol/min/mg protein. With PB-induced microsomes, all 4 Km values were 0.4-0.7 mM. Liver microsomal production of nitrite and aldehydes from NMAA was determined colormetrically or (for pentaldehyde) by high-pressure liquid chromatography of the 2,4-dinitrophenylhydrazone. Uninduced microsomes produced nitrite, formaldehyde and pentaldehyde from 0.6 mM NMAA at rates that were, respectively, 0.15, 0.72, and 1.15 times that for 4-hydroxylation. PB especially induced depentylation, whereas 3-methylcholanthrene induced depentylation and denitrosation, but suppressed demethylation. A reconstituted system containing cytochrome P-450 IIB1 gave metabolite ratios similar to those in PB-induced microsomes. The results account for most of the possible primary metabolites of NMAA and demonstrate the selectivity for metabolism at each position.     

Particular cytochrome P-450-dependent steroid metabolism: a new class of mouse liver tumor markers

An altered pattern of cytochrome P-450-dependent microsomal steroid metabolism was identified in female mouse liver tumors induced by 5,9-dimethyldibenzo[c,g]carbazole, a potent organo-specific liver carcinogen. These tumor tissues were compared to extratumoral liver parenchyme, to normal, fetal and neonatal livers and to spontaneous liver tumors, the frequency of which is very low in the highly hybridized mouse strain (XVIInc/Z) used for liver tumorigenesis. Cytochrome P-450-dependent steroid hydroxylase activities were measured by the identification and quantification of four monohydroxyprogesterone and eight monohydroxytestosterone metabolites. In contrast to a general decrease (50%) of total P-450 in tumor microsomes, the individual steroid hydroxylases were regulated differently. Progesterone 16 alpha- and testosterone 6 alpha-, 6 beta-, 7 alpha- and 16 alpha-hydroxylase activities were decreased 50%, and more, whereas progesterone and testosterone 15 alpha-hydroxylase activities were raised 3-4 times with regard to microsomal protein content and 6-7 times with regard to total P-450. Consequently the most prominent feature of the steroid metabolism by tumor-borne microsomes is the hydroxylation at the 15 alpha-position. Furthermore, minor testosterone 2- and 15 beta-hydroxylase activities showed equally an increase of approximately 4 times (8 times with regard to total P-450). The observed new tumoral pattern of P-450-dependent microsomal steroid metabolism appearing characteristically in spontaneous and chemically induced liver tumors indicates that particular P-450 enzymes are strongly expressed in mouse liver tumors. These enzymes may be used as markers for early stages in liver tumorigenesis.     

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.     

Age dependent expression of cytochrome P-450b and metabolism of the potent carcinogen 2-nirofluorene in the rat lung

Northern and Western blot analyses, and analyses of microsomalmetabolism of the carcinogen 2-nitrofluorene (NF) were conductedwith the aim of studying age dependent cytochrome P-450_b levelsin the rat lung. The level of P-450_b homologous mRNA and correspondingprotein is very low in lungs from fetal and newborn rats. Thelevels then increase between 3 and 4 weeks of age, and reachadult levels at 6–8 weeks. No sex differences were detectedwith regard to lung P-450_b mRNA levels or catalytical activities.Lung microsomal metabolism of NF increased in parallel withthe accumulation of P-450_b homologous mRNA and microsomal cytochromeP-450_b protein concentration. Formation of the major metabolite,and potent mutagen, 9-hydroxy-2-nitrofluorene (9-OHNF) was significantlyinhibited by addition of polyclonal anti-P-450_b-IgG, and byaddition of the inhibitor proadifen to incubations with lungmicrosomal protein. It is postulated that the observed, profoundage-related differences in level and activity of lung cytochromeP-450_b are likely to affect both availability and the ratioof metabolic detoxification and activation of chemical carcinogensdeposited in the lung.     

P-glycoprotein and cytochrome P-450 3A inhibition: dissociation of inhibitory potencies.

Many P-glycoprotein (P-gp) inhibitors studied in vitro and in vivo are also known or suspected to be substrates and/or inhibitors of cytochrome P-450 3A (CYP3A). Such overlap raises the question of whether CYP3A inhibition is an intrinsic characteristic of P-gp inhibitors, a matter of concern in the development and rational use of such agents. Thus, the purpose of the present study was to determine whether the ability to inhibit P-gp and CYP3A is, in fact, linked and whether specific P-gp inhibitors with limited ability to inhibit CYP3A can be identified. Therefore, the potency of a series of 14 P-gp inhibitors was assessed by measuring their inhibition of the transepithelial flux across Caco-2 cells of digoxin, a prototypical P-gp substrate. CYP3A inhibition was determined from the impairment of nifedipine oxidation by human liver microsomes. Determination of the apparent Ki values for CYP3A inhibition and the IC50s for P-gp and CYP3A inhibition allowed comparison of the relative inhibitory potency of the compounds on the two proteins' function. The IC50s for P-gp inhibition ranged from 0.04 to 3.8 microM. All compounds inhibited CYP3A with apparent Ki values of between 0.3 and 76 microM and IC50s between 1.5 and 50 microM. However, no correlation was found between the extent of P-gp inhibition and CYP3A inhibition, and the ratio of the IC50 for CYP3A inhibition to the IC50 for P-gp inhibition varied from 1.1 to 125. These results demonstrate that, although many P-gp inhibitors are potent inhibitors of CYP3A, a varying degree of selectivity is present. The development and use of P-gp inhibitors with minimal or absent CYP3A inhibitory effects should decrease the impact of drug interactions on the therapeutic use of such compounds.