Purification and characterization of cytochrome P-450 induced by benz(a)anthracene in mouse skin microsomes

Topical application of benz(a)anthracene to mouse skin elicited a 2-fold increase in cytochrome P-450 content, with accompanying increases in monooxygenase activities such as benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and acetanilide 4-hydroxylation, in the microsomes. A major form of cytochrome P-450 was purified from skin microsomes of mice treated with polycyclic aromatic hydrocarbon. A specific content of 1.95 nmol/mg of protein, which corresponded to 48-fold purification from the microsomes, was observed. The purified protein produced a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a molecular weight of 55,000. Using Western blotting, the band immunochemically cross-reacted with antibody which had been raised against rat liver cytochrome P-450MC-1. The purified preparation efficiently catalyzed benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation when reconstituted with NADPH-cytochrome P-450 reductase. These activities were inhibited by 7,8-benzoflavone as well as anti-cytochrome P-450MC-1 antibody, but not by P-450PB-1 antibody. The results indicate that, in mouse skin microsomes, a cytochrome P-450 induced by benz(a)anthracene is enzymatically and immunochemically similar to rat liver cytochrome P-450MC-1. It is suggested that this enzyme plays an important role in the activation of carcinogenic polycyclic aromatic hydrocarbons.     

Microsomal cytochrome P-450-linked monooxygenase systems and lipid composition of human hepatocellular carcinoma

The tissues of hepatocellular carcinoma were operatively resected from six patients. All four components of the systems of microsomal cytochrome P-450-linked monooxygenase of the tissues were investigated and compared to those of normal liver tissue. The concentrations of cytochromes P-450, P-420 and b5 were measured optically and the concentrations of all components except cytochrome P-450 were measured by the Western blotting method followed by immunochemical staining. In microsomes of hepatocellular carcinoma tissues, there was as much cytochrome P-450 and other redox components as in the normal liver tissues, but cytochrome P-450 in liver cancer tissues was unstable and easily converted to cytochrome P-420. The specific activities of NADPH- and NADH-ferricyanide and cytochrome c reductase of each sample were also measured. In the microsomes of the cancer tissues, the specific activities were remarkably reduced compared with those of normal liver tissues. The lipid compositions of the microsomes and the phospholipid/cholesterol ratios (w/w) were 13.1 +/- 3.13 in the cancer tissues and 43.0 +/- 6.74 in normal liver tissues. This difference of the lipid composition elucidates the instability of cytochrome P-450 molecules and the inefficiency of the electron transport of cytochrome P-450-linked monooxygenase systems.     

Effect of dietary butylated hydroxyanisole on the mouse hepatic monooxygenase system of nuclear and microsomal fractions

The effect of butylated hydroxyanisole (BHA) administrationon the hepatic monooxygenase system of nuclear and microsomalfraction was investigated in male mice. Addition of BHA to thediet significantly lowered the content of cytochrome P-450 inliver nuclei and increased the specific activity of NADPH-cytochromec reductase and the content of cytochrome b₅ in liver microsomes.Incubation of benzo[a]pyrene (BP) with liver nuclei from BHA-fedmice resulted in inhibition of binding of BP metabolites tonuclear macromolecules by 50% compared with control. However,there was no effect of BHA on the binding of BP metabolitesto macromolecules when BP was incubated with added DNA and livermicrosomes from BHA-fed mice. It has been postulated that modificationof nuclear monooxygenases by BHA may play a role in the inhibitoryeffect of BHA on BP carcinogenesis.     

Specificity of hepatic cytochrome P-450 isoenzymes from PCB-treated rats and participation of cytochrome b5 in the activation of aflatoxin B1

Employing six forms of cytochrome P-450s fractionated from thehepatic microsomes of PCB-treated rats, the activation of aflatoxinB₁ (AFB₁) was examined in the reconstituted cytochrome P-450system. AFB₁ was specifically activated into DNA-binding formby cytochrome P-450 I-a, which is one of P-450 type cytochromesand possesses an absorption peak at 450.0 nm in its carbon monoxidedifference spectrum. This activation was enhanced by cytochromeb₅ and the maximal enhancement (1.6-fold of the control) wasobserved with the molar ratio of 0.25 cytochrome b₅:1.0 cytochromeP-450.     

Characterization of cytochrome P450-dependent dimethylhydrazine metabolism in human colon microsomes

Polyclonal antibodies to components of the rat liver cytochrome P450 system were used to examine the composition and function of the microsomal cytochrome P450-dependent monooxygenase system of human colonic mucosal cells. Anticytochrome P450 reductase antibody gave a strong band of immunocross-reactivity in human colon microsomes at the same molecular weight level as purified cytochrome P450 reductase from rat liver, as well as hepatic microsomes isolated from untreated or phenobarbital-treated rats. These results demonstrate the presence of cytochrome P450 reductase in human colon cells. Similarly, cytochromes P450 IIB1 and IIA1 also appear to be present in Western blots of human colon microsomes. These antibodies, as well as antibodies to reductase and cytochrome b5, inhibit dimethylhydrazine metabolism in human colon microsomes to varying degrees. These data argue for a functional P450-dependent drug metabolism system in colon capable of activating/metabolizing the colon-specific model carcinogen, 1,2-dimethylhydrazine.     

Increases in cytochrome P-450 mediated 17{beta}-estradiol 2-hydroxylase activity in rat liver microsomes after both acute administration and subchronic administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin in a two-stage hepatocarcinogenesis model

Administration of single doses of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin(TCDD) (10 µ/kg) increased estradiol 2-hydroxylase (E₂OHase)activity 2-fold in liver microsomes of female rats but hadno effect on E₂OHase activity in hepatic microsomes of malerats. In contrast, TCDD increased P-450d (an enzyme which hasa high turnover number for E₂OHase in a reconstituted enzymesystem) 10- to 20-fold in livers of both male and female rats.The discrepancy between the increases in P-450d and E₂OHaseactivity in liver microsomes of TCDD-induced rats was abolishedby the addition of exogenous purified P-450 reductase to themicrosomal assays for E₂OHase, suggesting that reductase waslimiting in the in vitro assays. When E₂OHase activity was assayedin the presence of exogenous reductase, TCDD increased E₂OHase2-fold and 4-fold respectively in liver microsomes of male andfemale rates. Antibody to P-450d completely inhibited the increasein E₂OHase activity in liver microsomes of TCDD-treated maleand female rats, but had little effect on E₂OHase activity inliver microsomes of untreated male or female rats. These dataindicate that P-450d is responsible for the increase in E₂OHaseactivity in TCDD-treated rats, but other P-450 isozymes areresponsible for constitutive E₂OHase activity. Biweekly administrationof 1.4 µg/kg of TCDD for 30 weeks as a potential promoterof hepatocarcinogenesis increased the volume of the liver occupiedby -glutamyl transpeptidase (GGT)-positive foci in livers offemale rats given a necrogenic dose of diethylnitrosamine (DEN)(200 mg/kg) as the initiator. Biweekly doses of 0.14–1.4µg/kg TCDD in this model also increased P-450d (7-fold)and E₂OHase activity maximally (4-fold) in DEN-initiated rats.Moreover, initiation with DEN substantially enhanced the effectsof the low dose of TCDD on both hepatic microsomal P-450d andE₂OHase activity.     

Participation of cytochrome P-450 in reductive metabolism of 1-nitropyrene by rat liver microsomes

Reductive metabolism of carcinogenic 1-nitropyrene by rat liver microsomes and reconstituted cytochrome P-450 systems was investigated. Under the nitrogen atmosphere, 1-aminopyrene was the only detected metabolite of 1-nitropyrene. The reductase activity in liver 105,000 X g supernatant fraction was ascribed to DT-diaphorase, aldehyde oxidase, and other unknown enzyme(s) from the results of cofactor requirements and inhibition experiments. The microsomal reductase activity was inhibited by oxygen, carbon monoxide, 2,4-dichloro-6-phenylphenoxyethylamine, and n-octylamine. Flavin mononucleotide markedly enhanced the activity, and 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride also enhanced it, but slightly. The microsomal activity was induced by the pretreatment of rats with 3-methylcholanthrene, sodium phenobarbital, or polychlorinated biphenyl, and the increments of the activity correlated well with those of the specific contents of cytochrome P-450 in microsomes. The reductase activity could be reconstituted by NADPH-cytochrome P-450 reductase and forms of cytochrome P-450 purified from liver microsomes of polychlorinated biphenyl-induced rats. Among four forms of cytochrome P-450 examined, an isozyme P-448-IId which showed high activity in hydroxylation of benzo(a)pyrene catalyzed most efficiently the reduction of 1-nitropyrene. The results of this study indicate the central role of cytochrome P-450 in the reductive metabolism of 1-nitropyrene in liver microsomes.     

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.     

Metabolism of N-nitroso-2,6-dimethylmorpholine by isozymes of rabbit liver microsomal cytochrome P-450

The cis isomer of N-nitroso-2,6-dimethylmorpholine (NNDM), a pancreatic carcinogen for the Syrian golden hamster, is metabolized by hamster liver microsomes to yield N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) as the major product. Rabbit liver microsomes catalyze the metabolism of cis-NNDM to HPOP at a rate slower than that observed with hamster microsomes, but significantly faster than that obtained with rat microsomes. Pretreatment of rabbits with phenobarbital results in a 6-fold increase in the cis-NNDM hydroxylase activity of the rabbit microsomes to levels equal to that observed with the hamster; pretreatment of rabbits with other xenobiotics had no effect on the hydroxylation of cis-NNDM. The role of rabbit liver microsomal cytochrome P-450 in the metabolism of the cis isomer of NNDM was studied in the reconstituted system consisting of NADPH:cytochrome P-450 reductase, phospholipid, and cytochrome P-450. Cytochrome P-450LM2, which is induced by pretreatment with phenobarbital, exhibited the highest activity for the metabolism of cis-NNDM. The Vmax for the formation of HPOP was 1.78 nmol/min/nmol cytochrome P-450LM2, and the apparent Km was 360 microM. Cytochrome P-450LM3a also catalyzed the metabolism of NNDM to HPOP at a significant rate (0.25 nmol/min/nmol cytochrome P-450). Of the four other isozymes of cytochrome P-450 (forms 3b, 3c, 4, and 6) tested in the reconstituted system, only forms 3b and 3c exhibited measurable activities (approximately 0.04 nmol of HPOP formed/min/nmol cytochrome P-450). The addition of antibodies to isozyme 2 to microsomes from phenobarbital-treated rabbits resulted in approximately 95% inhibition of the metabolism of NNDM, while the addition of antibodies to LM3a inhibited NNDM metabolism by only 7%. In microsomes from untreated rabbits, inhibition by anti-LM2 and anti-LM3a antibodies was 50 and 64%, respectively. The addition of antibodies to isozyme 3a to microsomes isolated from ethanol-treated rabbits caused approximately 90% inhibition of the metabolism of NNDM. These data conclusively demonstrate that several forms of cytochrome P-450 can catalyze the metabolism of cis-NNDM and that isozymes 2 and 3a play important roles in the rabbit hepatic metabolism of NNDM to HPOP, the proximate carcinogenic metabolite.     

High-affinity nitrosamine dealkylase system in rat liver microsomes and its induction by fasting

In order to elucidate the enzymic basis of nitrosamine metabolism, the in vitro metabolism of nitrosamines by rat liver microsomes and the effects of fasting on the microsomal enzymes have been studied. Fasting for 1 to 3 days causes a 2- to 3-fold enhancement of the reduced nicotinamide adenine dinucleotide phosphate-dependent nitrosodimethylamine demethylase (NDMAD) activity. The cytochrome P-450 content and the activities of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase and benzphetamine demethylase, however, are only modestly increased. Gel electrophoretic analysis reveals the induction of a 50,000-dalton protein band during fasting. The induction of this protein band as well as the enhancement of NDMAD activity are inhibited by CoCl2 and inhibitors of protein and RNA biosynthesis. The involvement of cytochrome P-450 in the NDMAD is supported by the fact that microsomal reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase is required for the demethylase activity. Kinetic analysis indicates that a low-Km form of NDMAD (apparent Km, 0.07 mM) is markedly induced by fasting. With microsomes of control rats, there are at least three apparent Km values (0.07, 0.38, and 38.6 mM) for NDMAD; but with microsomes of fasting rats, the low-Km (0.07 mM) form is predominant. These results suggest that rat liver microsomes contain a cytochrome P-450 isozyme which has high affinity for nitrosodimethylamine, and this isozyme is induced by fasting. In addition to nitrosodimethylamine, the oxidative demethylation of N-nitroso-N-methylethylamine, N-nitroso-N-methylbutylamine, N-nitroso-N-methylaniline, and N-nitroso-N-methylbenzylamine is also enhanced by fasting. The extent of enhancement and substrate dependency of these reactions, however, is different from that of NDMAD.     

Aflatoxin B1 hydroxylation by the pregnenolone-16{alpha}-carbonitrile-inducible form of rat liver microsomal cytochrome P-450

The effects of treating rats with various pregnenolone-16-carbonitrile(PCN)-type inducers of cytochrome P-450p on the liver microsomalmetabolism of aflatoxin B₁ (AFB₁) were investigated. Treatmentof male rats with PCN resulted in a 6-fold increase in the 9-hydroxylationof AFB₁ to aflatoxin Q₁ (AFQ₁). Treatment of female rats withPCN resulted in a 16-fold increase in the formation of AFQ₁.The age-dependent decline in constitutive cytochrome P-450plevels in female but not male rats resulted in a sex differencein the formation of AFQ₁ in liver microsomes from untreatedrats (male: female 3: 1). The formation of AFQ₁ was stimulatedup to 5.4-fold when liver microsomes from triacetyloleandomycin(TAO)-treated rats were treated with potassium ferricyanide,which dissociates the complex between cytochrome P-450p andTAO. Treatment of male rats with the cytochrome P-450p inducer,dexamethasone, increased ( 7-fold) the 9-hydroxylation of AFB₁to AFQ₁ by liver microsomes, and also enhanced ( 2-fold) themicrosomal activation of AFB₁ to metabolites that were mutagenicto Salmonella typhimurium TA98 and TA100. These results indicatethat the 9-hydroxylation of AFB₁ to AFQ₁ is catalyzed by ratliver microsomal cytochrome P-450p.     

Inactivation of 1,3-, 1,6-, and 1,8-dinitropyrene by cytochrome P-450 enzymes in human and rat liver microsomes

NADPH-fortified human liver microsomes were examined with regard to ability to detoxicate several chemicals that do not require enzymatic oxidation to elicit a genotoxic response in a Salmonella typhimurium TA1535/pSK1002 system where umu response is used as an indicator of DNA damage. Microsomes did not affect the response seen with daunomycin, mitomycin C, 2,4,7-trinitro-9-fluorene, 1-nitropyrene, doxorubicin, 1-methyl-3-nitro-1-nitrosoguanidine, 2-nitrofluorene, or 1-ethyl-3-nitro-1-nitrosoguanidine (cited in order of decreasing umu response per mol). Human and rat liver microsomes did inactivate 1,3-, 1,6-, and 1,8-dinitropyrene; with human liver microsomes the activity of 1,3-dinitropyrene was most strongly inhibited, while with rat liver microsomes the genotoxicities of all three dinitropyrenes were inhibited to a similar extent. NADPH-cytochrome P-450 reductase was demonstrated to inactivate 1,6- and 1,8-dinitropyrene but not 1,3-dinitropyrene. Both rat cytochrome P-450 beta NF-B (P-450 IA1) and P-450ISF-G (P-450 IA2) inactivated 1,3-dinitropyrene, with the former being more effective. Correlation studies done with liver microsomes prepared from variously treated rats and immunoinhibition studies suggest that cytochrome P-450 beta NF-B and P-450ISF-G are both involved in the detoxication of all three of the dinitropyrenes in rat liver microsomes. In a series of assays done with various human liver microsomal preparations, the inactivation of the three dinitropyrenes was not correlated to each other at all. Correlation analysis and inhibition studies with 7,8-benzoflavone and antibodies indicate that human cytochrome P-450 enzymes in the IA family are most effective in detoxicating this compound; the contribution of cytochrome P-450PA (P-450 IA2, the phenacetin O-deethylase) is deemed more important, but a role for the small amount of cytochrome P1-450 (P-450 IA1) in the liver cannot be ruled out. In contrast to the case of 1,3-dinitropyrene, the inactivation of 1,6-dinitropyrene is well correlated with levels of cytochrome P-450NF (P-450 IIIA4, nifedipine oxidase) and its catalytic activities. The inactivation of 1,8-dinitropyrene was not correlated with any of the above parameters and only correlated with the conversion of benzo(a)pyrene to its 3-hydroxy and 4,5-dihydrodiol products, for which the principal enzymes involved in human liver are unknown. Thus, distinct human cytochrome P-450 enzymes are involved in the detoxication of different dinitropyrene congeners, and the situation appears to contrast with that in rat liver.     

On the identity of the xenobiotic-metabolizing form(s) of cytochrome P-450 in endocrine organs

The adrenal cortex, the testes and the ovary metabolize polycyclic aromatic hydrocarbons (PAHs), e.g., 7,12-dimethylbenz[a]anthracene (DMBA). These activities have previously been shown to involve the cytochrome P-450 monooxygenase system [18-20]. In attempt to identify the form(s) of cytochrome P-450 involved, microsomes from these endocrine organs were subjected to SDS-gel electrophoresis, followed by immunochemical analysis using the Western blot technique. Antisera raised against the purified rat hepatic cytochrome P-450 isozymes a, b + e, c, d and PB-PCNE were tested. It was concluded that the electrophoretic mobilities of the immunoreactive bands obtained were not identical to the mobilities of the purified isozymes cytochromes P-450 a, b, c, d, e or PB-PCNE. These results indicate that the PAH-metabolizing monooxygenase(s) in these endocrine organs may involve a novel form(s) of cytochrome P-450.     

Immunochemical study on the contributions of two molecular species of cytochrome P-450 in mutagenesis by selected aminoazo dyes

The relative contributions of two species of cytochrome P-450,the major cytochrome P-450 components of liver microsomes ofphenobarbital-treated rats (PB-P-450) and of 3-methylcholanthrene-treatedrats (MC-P-448), in the mutagenic activation of 3'-methyl-N,N-dimethyl-4-aminoazobenzene(3'-Me-DAB) and its eight metabolites were studied in the Salmonellaassay system using specific antibodies and inhibitors. The antibodyagainst MC-P-448 considerably inhibited the mutagenicities of3'-Me-DAB, 3'-CH₂OH-DAB, their three N-demethylated compounds,3'-CHO-DAB, and 3'-Me-4'-OH-DAB in strain TA98, whereas theantibody against PB-P-450 inhibited their mutagenicities <29%.In contrast, the antibody against MC-P-448 caused no or slight(29%) inhibition of the mutagenicities of 3'-hydoxymethyl-N-methyl-4-aminoazobenzene(3'-CH₂OH-MAB) and 3'-COOH-DAB. However, the mutagenicitiesof both compounds were considerably inhibited by 7,8-benzo-flavone,like those of other seven aminoazo dyes. These results demonstratethat rat liver cytochrome P-450, especially MC-P-448, is involvedin mutagenic activation of the aminoazo dyes. Participationof a second form of cytochrome P-448 in mutagenic activationof 3'-CH₂OH-MAB and 3'-COOH-DAB is discussed.     

Xenobiotic metabolism in human alveolar type II cells isolated by centrifugal elutriation and density gradient centrifugation

Alveolar type II cells were isolated from five human lung specimens obtained during resection or lobectomy and enriched to 63-85% purity. Digestion with Sigma protease type XIV followed by centrifugal elutriation and Percoll density gradient centrifugation yielded 1.2 +/- 0.4 X 10(6) cells/g lung in the type II cell fractions. The activities of some enzymes involved in the metabolism of xenobiotics were determined in these freshly isolated type II cells and compared with activities in alveolar macrophages and fractions of unseparated cells from the same tissue samples. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity was similar in the three cell fractions from all five patients (18-29 nmol/mg protein/min). An antibody to rabbit reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase inhibited reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reduction as much as 70% in microsomal preparations of the isolated human pulmonary cells, although this same antibody barely reacted with microsomes of the human cells in a Western blot assay. Epoxide hydrolase activity was highest in the alveolar type II cells (1.08 +/- 0.17 nmol/mg protein/min). This activity was 6 times higher than in the alveolar macrophage or unseparated cell fractions. 7-Ethoxycoumarin deethylase activity, a cytochrome P-450-dependent pathway, was low or undetectable in the three cell fractions. Trace amounts of 7-ethoxyresorufin O-deethylase activity (0.5-1.5 pmol/mg protein/min) were detected in microsomes of the isolated human cells, even though a polycyclic hydrocarbon-inducible cytochrome P-450 which metabolizes 7-ethoxyresorufin (form 6 in rabbits) was not detected immunochemically.     

Phosphorylation of carcinogen metabolizing enzymes: regulation of the phosphorylation status of the major phenobarbital inducible cytochromes P-450 in hepatocytes

We present data showing that the major phenobarbital inducible cytochromes P-450 (cytochrome P-450IIB1 and cytochrome P-450IIB2) were phosphorylated in intact hepatocytes. This phosphorylation was greatly increased by the cAMP derivatives N6-dibutyryl-cAMP and 8-thiomethyl-cAMP mediated by a cAMP-dependent protein kinase. Most importantly the phosphorylation status of cytochromes P-450 was shown to change in the hepatocytes after treatment with glucagon, which is known to increase the level of cAMP in hepatocytes. The observed impact of the hormone glucagon on the phosphorylation of distinct cytochrome P-450 forms in intact hepatocytes reveals the possibility that the enzyme activity of cytochromes P-450 could be rapidly and differentially regulated by their phosphorylation and therefore dependent on the hormonal status of the organism.     

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.     

Metabolism of aflatoxin B1 in the bovine olfactory mucosa

Carcinomas of the ethmoidal region of the nose are observedrelatively frequently in cattle in several countries in tropicaland subtropical latitudes. Viruses have been implicated as causativeagents, but it has been observed that affected animals sometimessuffer from aflatoxicosis, and a role of aflatoxin B₁ (AFB₁)in the aetiology has also been proposed. We have examined whetherthe bovine nasal olfactory mucosa has a capacity to metabolizeAFB₁. The contents of cytochrome P–450 and cytochromeb₅, and the NADPH cytochrome c reductase activity in the nasalolfactory mucosa have also been determined. Comparative experimentshave been performed with the liver. Incubations with ³H-labelledAFB₁ showed that the nasal olfactory mucosa has a much highercapacity than the liver to form lipid-soluble, water-solubleand tissue-bound AFB₁-metabolites. High-resolution microautoradiographyshowed a strong localization of tissue-bound metabolites inthe sustentacular cells in the apical portion of the olfactorysurface epithelium and in Bowman's glands in the olfactory laminapropria mucosae. Especially in the sustentacular cells the labellingwas preferentially located in the nuclei of the cells. Liquidchromatography of chloroform extracts of the nasal olfactorymucosa and the liver incubated with ³H-AFB₁ showed formationof several metabolites. The dominating peak in both tissueswas aflatoxin M₁ (AFM₁). However, the amount of AFM₁ was higherin the nasal olfactory mucosa than in the liver, and the amountsand proportions of several other metabolites also differed markedlybetween the two tissues. The level of cytochrome P-450 in thenasal olfactory mucosa was found to be about one quarter ofthat in the liver, but the NADPH cytochrome c reductase activitywas much higher in the nasal olfactory mucosa than in the liver.In addition, the cytochrome b₅: cytochrome P-450 ratio was higherin the nasal olfactory mucosa than in the liver. The highermetabolism of AFB₁ in the nasal olfactory mucosa than in theliver may be related to differences in the cytochrome P-450isoenzyme profile. In addition, the microsomal electron transportto cytochrome P-450 may be facilitated by the high reductase:cytochrome P–450 ratio and the high cytochrome b₅: cytochromeP–450 ratio in the nasal olfactory mucosa. It is consideredthat the results of the present study strengthen the hypothesisthat exposure of AFB₁-contaminated feed may be an importantaetiological factor in the development of nasal tumours in cattle.     

Alterations in expression of CYPlA1 and NADPH-cytochrome P450 reductase during lung tumor development in SWR/J mice

We investigated the expression of the cytochrome P450 isozyme,CYP1A1, during the course of tumor development and examinedthe distribution of the CYP1A1 protein in hyperplastic foci,adenomas and carcinomas. The expression of NADPH-cytochromeP450 reductase, a flavoprotein that mediates the reduction ofcytochrome P450, was also determined. Mice were administeredurethane (1 mg/g body wt) and were killed at 10, 22 and 52 weeksto coincide with the time at which hyperplastic foci, adenomasand carcinomas were established, respectively. Protein immuno-blottingrevealed that the antibody for CYP1A1 detected a protein bandof {small tilde}M_r 56 000 in microsomes from mice treated withßP-naphthoflavone. The antibody for NADPH-cytochromeP450 reductase detected a protein band of     

Geno- and cytotoxicity of nitrosamines, aflatoxin B1, and benzo[a]-pyrene in continuous cultures of rat hepatoma cells

Two closely related hepatoma cell lines were examined for theirresponse to carcinogens requiring metabolic activation: H₅,a dedifferentiated line expressing cytochrome P-448-dependentmonooxygenase(s); and HF_1–4, a differentiated line whichalso expresses cytochrome P-450-dependent monooxygenase(s).The hepatocarcinogens dimethyl- and diethylnitrosamine and aflatoxinB₁, preferred substrates for cytochrome P-450-dependent monooxygenase(s),and the non-hepatocarcinogen benzo[a]pyrene, which is preferentiallymetabolized by cytochrome P-448-dependent monooxygenase forms,were used as test agents. Their effects were compared to thoseof the directly alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) and N-ethyl-N-nitrosourea (ENU). The cytotoxicity wasevaluated by plating efficiency, the genotoxicity by the appearanceof alkaline labile DNA sites. The nitrosamines had a cytotoxicand genotoxic effect on the differentiated HF_1–4 cells,but had no effect on H₅ cells. Aflatoxin B₁ affected both celllines, but was 10-times more potent in the HF_1–4 thanin the H₅ cells. In contrast to the nitrosamines and the mycotoxin,benzo[a]-pyrene exerted a stronger effect on the dedifferentiatedcell line. Pretreatment of cultures with dexamethasone increasedboth the cytotoxicity and genotoxicity of the hepatotoxic agents.MNNG and ENU induced a similar degree of DNA-damage after short-term(2 h) exposure in the two cell lines. When cells were allowedto recover for 16 h HF_1–4 cells, but not H₅ cells, regainedtheir full growth potential suggesting a marked capacity forthe repair of MNNG- and ENU-induced lesions in the HF_1–4cells. The results indicate that continuous lines of mammaliancells may retain a considerable degree of organ-specific responseto chemical carcinogens. Hepatoma cells of the type describedabove may be useful for screening the wide spectrum of chemicalswhich are potentially genotoxic in liver and in extrahepatictissues and for analyzing their metabolic activation and mechanismof action.