Up-regulation of cytochrome P450 and phase II enzyme systems in rat precision-cut rat lung slices by the intact glucosinolates, glucoraphanin and glucoerucin

It is believed that the chemopreventive activity of cruciferous vegetables in the lung and other tissues is exclusively the result of exposure to degradation products of glucosinolates, such as the isothiocyanates, and that the parent glucosinolates make no contribution. In the present study, evidence is presented for the first time that, in rat lung, the intact glucosinolates, glucoraphanin and glucoerucin, can modulate carcinogen-metabolising enzyme systems. The glucosinolates were isolated from cruciferous vegetables and incubated (1-25 μM) with precision-cut rat lung slices for 24 h. Both glucosinolates, at concentrations as low as 1 μM, up-regulated the O-deethylation of ethoxyresorufin and the apoprotein levels of CYP1A1 and CYP1B1; supplementation of the incubation medium with myrosinase, the enzyme that converts glucosinolates to their corresponding isothiocyanates, abolished the rise in ethoxyresorufin O-deethylase activity. In contrast, neither glucosinolate, at the concentrations studied, influenced quinone reductase activity in the lung slices, but addition of myrosinase to the glucosinolate incubations led to a marked rise in activity. Glutathione S-transferase activity, monitored using 1-chloro-2,4-dinitrobenzene as the accepting substrate, was elevated in lung slices exposed to glucoraphanin. GSTα protein levels were increased by glucoraphanin and, to a much lesser extent, glucoerucin. It may be concluded that intact glucosinolates can modulate the activity of pulmonary carcinogen-metabolising enzyme systems, and can thus contribute to the documented chemopreventive activity of cruciferous vegetables in the lung.     

Selective mutagenic activation by cytochrome P3-450 of carcinogenic arylamines found in foods

Heterocyclic arylamines found in cooked foods including fish and beef are potent mutagens and carcinogens. The purpose of this investigation was to determine the specificity of cytochromes P1-450 and P3-450 toward the metabolic activation of these arylamines. We used a novel mutagenicity test system which combined human cells expressing either recombinant cytochrome P1-450 or P3-450 with Salmonella typhimurium to score mutants. Cytochrome P3-450, a single isoform of the cytochrome P-450 supergene family, bioactivated these food mutagens. Cytochrome P1-450 showed little or no activation of these arylamines but was the isoform predominantly responsible for the activation of the aromatic hydrocarbon benzo[a]pyrene-7,8-diol. This assay system should serve to define the specificities of individual cytochromes P-450 in the metabolic activation of carcinogens.     

Modulation of hepatic cytochromes P450 and phase II enzymes by dietary doses of sulforaphane in rats: Implications for its chemopreventive activity

The principal objectives of our study were to ascertain whether sulforaphane, at dietary levels of intake, modulates rat hepatic cytochrome P450 and phase II enzyme systems and to evaluate the impact of such changes in the chemopreventive activity of this isothiocyanate. Animals were exposed to sulforaphane in their drinking water for 10 days, equivalent to daily doses of 3 and 12 mg/kg. Depentylation of pentoxyresorufin decreased and was paralleled by a decline in CYP2B apoprotein levels. At the higher dose, erythromycin N-demethylase activity declined and was accompanied by a similar decrease in CYP3A2 apoprotein levels. However, sulforaphane treatment upregulated CYP1A2 levels, determined immunologically, but the dealkylations of methoxy- and ethoxyresorufin were not similarly increased. Hepatic S9 preparations from sulforaphane-treated rats were less effective than control preparations in converting IQ (2-amino-3-methylimidazo-[4,5-f]quinoline) to mutagenic intermediates in the Ames test. To clarify the underlying mechanism, in vitro studies were undertaken. In beta-naphthoflavone-treated rats, the inhibition by sulforaphane of the O-dealkylations of methoxy- and ethoxyresorufin was enhanced if the isothiocyanate was preincubated in the presence of NADPH. It may be inferred that sulforaphane induces hepatic CYP1A2 but the enzyme is not catalytically competent because of bound sulforaphane metabolite(s). Finally, sulforaphane stimulated, in a dose-dependent fashion, quinone reductase but failed to influence glutathione S-transferase, epoxide hydrolase and glucuronosyl transferase activities. It is concluded that, even at dietary doses, sulforaphane can modulate the xenobiotic-metabolising enzyme systems, shifting the balance of carcinogen metabolism toward deactivation, and this may be an important mechanism of its chemopreventive activity.     

Non-specific inhibition of cytochrome P450 activities by chlorophyllin in human and rat liver microsomes

Chlorophyllin, a copper/sodium salt of chlorophyll used in thetreatment of geriatric patients, inhibits the mutagenicity of2-amino-3-methylimidazo[4, 5-f)quinoline (IQ), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), aflatoxin B₁ and benzo[a]pyrene (B[a]P).Recent in vitro and in vivo studies have shown that a molecularcomplex is formed between IQ and chlorophyllin, suggesting thatthis complex formation might be responsible for the antigenotoxiceffect of chlorophyllin observed. Cytochrome P450 (P450) enzymesappear to be the major catalysts in the bioactivation of thesecarcinogens. We have investigated the in vitro effects of chlorophyllinon several P450 activities including ethoxyresorufin O-deethylation,benzyloxyreso-rufin O-debenzylation, coumarin 7-hydroxylation,7-ethoxycoumarin O-deethylation, B[a]P 3-hydroxylation, andchlorzoxazone 6-hydroxylation. Chlorophyllin non-specificallyinhibited all of P450 activities observed. Spectrally detectableP450 was also destroyed in microsomes and purified P450 in areconstituted system in the presence of chlorophyllin and anNADPH-generating system. These results suggest that the antigenotoxiceffect of chlorophyllin might be due to inhibition of P450 enzymesinvolving bioactivation of carcinogens in addition to molecularcomplex formation between carcinogens and chlorophyllin. Comparisonof the apparent K₁ for P450 inactivation with previously estimatedconstants for chlorophyllin-IQ complexation suggest that P450inhibition should be the dominant mechanism of inhibition.     

Induction of cytochrome P-450 and 5-aminolevulinate synthase activities in cultured rat hepatocytes

Cytochromes P-450IIB1 and P-450IIB2 were recently shown to be inducible in rat hepatocyte cultures maintained on a reconstituted extracellular tumor matrix (Matrigel) as indicated by increases in P-450IIB1 and -IIB2 mRNAs and immunoreactive proteins (J. Cell. Physiol., 134: 309-323, 1988). Here we show that treatment of cultured rat hepatocytes with phenobarbital and other compounds known to induce P-450IIB1/2 in vivo increased spectral cytochrome P-450, immunoreactive proteins, and benzyloxy- and pentoxy-resorufin dealkylases, activities known to be specific for cytochrome P-450IIB1/2. These increases were observed when cells were cultured on either Matrigel or collagen matrix in Williams E medium. Cytochrome P-450III was also increased by phenobarbital and dexamethasone on either matrix. Propoxycoumarin depropylase activity, which has been proposed as a specific activity catalyzed by cytochrome P-450III, was increased 3-4-fold more by treatment with 3-methylcholanthrene than by phenobarbital or dexamethasone. The activity catalyzed by P-450III could be distinguished from that catalyzed by other P-450 forms using the specific inhibitor triacetyloleandomycin. Benzoyloxyresorufin dealkylase was also increased in these cells by treatment with 2,4,5,2',4',5'-hexachlorobiphenyl, glutethimide, or mephenytoin. Treatment with phenobarbital or 2-allyl-2-isopropylacetamide slightly induced 5-aminolevulinate synthase activity. 5-Aminolevulinate synthase activity was slightly increased in cells treated with phenobarbital or 2-allyl-2-isopropylacetamide. Succinyl acetone also induced 5-aminolevulinate synthase activity and, in combination with either of the other two drugs, synergistically increased the enzyme activity regardless of whether cells were cultured on collagen or Matrigel. These results indicate that with simple and economical enzyme assays for holocytochrome P-450 and 5-aminolevulinate synthase, the rat hepatocyte culture system can be used for studies of the interrelationships between phenobarbital induction of cytochrome P-450 and heme metabolism.     

Potent and non-specific inhibition of cytochrome P450 by JM216, a new oral platinum agent.

Bis-acetato-ammine-dichloro-cyclohexylamine-platinum (IV), JM216, is the first antineoplastic platinum compound that can be given to patients orally. Several phase II clinical trials of JM216 monotherapy have already been reported. However, no information on the potential drug interactions caused by JM216 is available. In this study, the capacity of JM216 to inhibit cytochrome P450 (CYP) in human liver microsomes was investigated by measuring the inhibition potential (IC50 and Ki) on prototype reactions. Specific substrates of CYP included testosterone (catalysed by CYP3A4), paclitaxel (CYP2C8), 7-ethoxyresorufin (CYP1A1, CYP1A2), coumarin (CYP2A6), aniline (CYP2E1) and (+/-)-bufuralol (CYP2D6). JM216 inhibited the catalytic activities of CYP isozymes. The IC50 values were between 0.3 microM and 10 microM, indicating strong and non-specific inhibitory effects of JM216. The inhibition occurred in a non-competitive manner, and the Ki value was 1.0 and 0.9 microM for metabolite formation of testosterone and paclitaxel respectively. Therefore, some in vivo studies should be conducted to determine whether or not there is a correlation between in vivo and in vitro results.     

Induction of cytochrome P450 enzymes and over-generation of oxygen radicals in beta-carotene supplemented rats

Effects of beta-carotene (betaCT) on microsomal CYP-linked monooxygenases were investigated using both the regio- and stereo-selective hydroxylation of testosterone (as multibiomarker) and highly specific substrates as probes of various isoenzymes. CYP-catalyzed reactions were studied in the liver, kidney, lung and intestine of Sprague-Dawley rats of both sexes supplemented with 250 or 500 mg/kg body wt betaCT (per os) in a single or repeated (daily for 5 days) fashion. Generalized boosting effects (2-15-fold increases) were observed in the various tissues for carcinogen metabolizing enzymes associated with CYP1A1/2, CYP3A1/2, CYP2E1, CYP2B1/2 and CYP2C11. Induction of the most affected CYPs was corroborated by western blot linked to densitometric analyses. Measurement of reactive oxygen species (ROS) produced by subcellular preparations from either control or betaCT supplemented rats was performed by EPR detection of the nitroxide radical yielded by the reaction with ROS of the hydroxylamine spin probe bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate. Marked ROS over-generation associated with CYP induction (up to 33-fold increase in the liver) was recorded in the various organs (liver > lung > intestine > kidney). CYP and ROS induction are substantially in keeping with the concentration of betaCT accumulated in the various tissues, the liver being the most affected organ. These findings are consistent with the concept that betaCT is a pro-oxidant and potentially co-carcinogenic pro-vitamin, and may help explain why, in large quantities, it can have harmful effects in humans.     

Induction of cytochrome P450IA1 in rat colon and liver by indole-3-carbinol and 5,6-benzoflavone

It is known that consumption of cruciferous vegetables protects against the chemical induction of cancer in many organs. It has been suggested that this protection is mediated through an effect on the cytochrome P450 monooxygenase system. This system is responsible for the activation of a number of chemical carcinogens to their ultimate forms. In the present study, the effect of indole-3-carbinol (I3C) and 5,6-benzoflavone (5,6BF) on the expression of cytochrome P450IA1 in rat colon and liver has been investigated. Cytochrome P450IA1 mRNA was induced in colon following a single oral administration of I3C or 5,6BF. A biphasic induction profile was obtained with maxima at 4 and 16 h post-administration. Both inducers caused an approximately 2-fold increase in P450IA1 mRNA at 4 h and a 10-fold increase at 16 h. In contrast, both cytochrome P450IA1 and IA2 mRNAs was increased over the control between 4 and 24 h. The total amount of P450IA mRNAs in liver at 4 and 16 h was increased about 2- and 4-fold respectively by I3C; 5,6BF induced the P450IA mRNAs 4- and 5-fold respectively. The expression of cytochrome P450IA1 and IA2 is induced by I3C and several flavones present in cruciferous vegetables. This suggests that one of the protective effects of cruciferous vegetables in the reduction of chemically induced cancer may be regulation of cytochrome P450s involved in the metabolism of the chemical carcinogens.     

Metabolism of azoxymethane, methylazoxymethanol and N-nitrosodimethylamine by cytochrome P450IIE1

The metabolism of azoxymethane (AOM), methylazoxymethanol (MAM) and N-nitrosodimethylamine (NDMA) by liver microsomes from acetone-induced rats as well as by a reconstituted system containing purified cytochrome P450IIE1 was examined. The products consisted of MAM from AOM; methanol and formic acid from MAM; and methylamine, formaldehyde, methanol, methylphosphate and formic acid from NDMA. Compared to liver microsomes from untreated rats, the metabolic activity of acetone-induced microsomes was approximately 4 times higher for all three carcinogens. Using the reconstituted system, the enzyme activities (nmol substrate metabolized/nmol P450/min) for AOM, MAM and NDMA were 2.88 +/- 1.14, 2.87 +/- 0.59 and 9.47 +/- 2.24 respectively. Incubations carried out in the presence of a monoclonal antibody to cytochrome P450IIE1 resulted in a 85-90% inhibition of all three reactions in this system. These results provide conclusive evidence that AOM, MAM and NDMA are metabolized by the same form of rat liver cytochrome P450. In addition, the stoichiometry of NDMA products formed in these reactions indicates that denitrosation, a presumed detoxication process, and alpha-hydroxylation, an activation reaction, are also catalyzed by the same cytochrome P450 isozyme.     

Metabolic activation of benzo[a]pyrene in vitro by hepatic cytochrome P450 contrasts with detoxification in vivo: experiments with hepatic cytochrome P450 reductase null mice

Many studies using mammalian cellular and subcellular systems have demonstrated that polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are metabolically activated by cytochrome P450s (CYPs). In order to evaluate the role of hepatic versus extra-hepatic metabolism of BaP and its pharmacokinetics, we used the hepatic cytochrome P450 reductase null (HRN) mouse model, in which cytochrome P450 oxidoreductase, the unique electron donor to CYPs, is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated intraperitoneally (i.p.) with 125 mg/kg body wt BaP daily for up to 5 days. Clearance of BaP from blood was analysed by high-performance liquid chromatography with fluorescence detection. DNA adduct levels were measured by (32)P-post-labelling analysis with structural confirmation of the formation of 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene by liquid chromatography-tandem mass spectrometry analysis. Hepatic microsomes isolated from BaP-treated and untreated mice were also incubated with BaP and DNA in vitro. BaP-DNA adduct formation was up to 7-fold lower with the microsomes from HRN mice than with that from WT mice. Most of the hepatic microsomal activation of BaP in vitro was attributable to CYP1A. Pharmacokinetic analysis of BaP in blood revealed no significant differences between HRN and WT mice. BaP-DNA adduct levels were higher in the livers (up to 13-fold) and elevated in several extra-hepatic tissues of HRN mice (by 1.7- to 2.6-fold) relative to WT mice. These data reveal an apparent paradox, whereby hepatic CYP enzymes appear to be more important for detoxification of BaP in vivo, despite being involved in its metabolic activation in vitro.     

Lenalidomide: in vitro evaluation of the metabolism and assessment of cytochrome P450 inhibition and induction

Purpose  To assess the potential for drug–drug interactions between lenalidomide and substrates and inhibitors of cytochrome P450 (CYP) isozymes. Methods  In vitro metabolism of lenalidomide by human liver microsomes, recombinant human CYPs and human hepatocytes was evaluated. The inhibitory and inductive effects of lenalidomide on the CYP activities were evaluated in human liver microsomes and cultured human hepatocytes, respectively. Results  In vitro incubation of lenalidomide with human liver microsomes, recombinant-CYP isozymes, and human hepatocytes did not result in Phase I or Phase II metabolism, confirming the low propensity of lenalidomide for metabolism in vivo in humans. In vitro, lenalidomide did not inhibit CYP isozymes in human liver microsomes and did not induce CYP activities in cultured human hepatocytes. Conclusions  Lenalidomide is not a substrate, inhibitor, or inducer of CYP group of enzymes; clinically relevant pharmacokinetic drug–drug interactions are unlikely to occur between lenalidomide and co-administered CYP substrates or inhibitors.     

Induction of cyclin-dependent kinase inhibitors and G(1) prolongation by the chemopreventive agent N-acetylcysteine.

Cyclin-dependent kinase (cdk) inhibitors, such as p16(INK4a) and p21(WAF1/CIP1), often inhibit G(1) cyclin kinases and result in G(1) arrest. It has been suggested that p21(WAF1/CIP1) may also play a role in other chemopreventive activities such as DNA repair, slowdown of DNA replication and induction of cellular differentiation. In this report we demonstrate that the antioxidant N-acetylcysteine (NAC), a well-known chemopreventive agent, induces p16(INK4a) and p21(WAF1/CIP1) gene expression and prolongs cell-cycle transition through G(1) phase. A portion of the G(1) arrest by NAC is governed by p16(INK4a); it is independent of p53. NAC's usual mechanism of increasing intracellular glutathione level is not required for the G(1) arrest. An antioxidant whose action is limited to scavenging radicals, Trolox, does not induce G(1) arrest. Taken together, these results suggest a potential novel molecular basis for chemoprevention by NAC.     

Human cytochrome P450IIA3: cDNA sequence, role of the enzyme in the metabolic activation of promutagens, comparison to nitrosamine activation by human cytochrome P450IIE1

We report that, in a human cell line, human cytochrome P450IIA3 is capable of metabolizing aflatoxin B1, benzo[a]-pyrene, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) to cytotoxic and mutagenic species. Cytochrome P450IIA3-mediated activation of NDMA and NDEA was compared with human cytochrome P450IIE1-mediated activation in the same cell system. P450IIE1 was more effective at activating NDMA than P450IIA3, while P450IIA3 was more effective at activating NDEA than P450IIE1. Whole cells and microsomal fractions obtained from control cells and from cells expressing the P450IIA3 cDNA were characterized for expression of P450IIA3. Microsomal coumarin 7-hydroxylase activity was some 40 times greater in the transfected cells than in the control cells and was catalyzed by a protein that was immunochemically related to the rat liver cytochrome P450IIA gene family. Immunoblot analysis demonstrated that this protein was readily detectable in transfected cells but barely detectable in control cells. We also report the DNA and deduced amino acid sequence of the P450IIA3 cDNA isolate used in this study. Our isolate encodes a protein 489 amino acids that is five amino acids shorter at the N terminus but otherwise identical to a previously reported human P450IIA3 cDNA sequence.     

Induction of p21/WAF1 and G1 cell-cycle arrest by the chemopreventive agent apigenin

Apigenin is a plant flavonoid that has been shown to significantly inhibit ultraviolet-induced mouse skin tumorigenesis when applied topically and may be an alternative sunscreen agent for humans. A long-term goal of our laboratory is to elucidate the molecular mechanism or mechanisms by which apigenin inhibits skin tumorigenesis. In a previous publication, we characterized the mechanism by which apigenin induced G₂/M arrest in keratinocytes. More recent studies in our laboratory have provided evidence that apigenin can induce G₁ arrest in addition to arresting cells at G₂/M. Here we describe the mechanism of the apigenin-induced G₁ arrest in human diploid fibroblasts (HDF). Treatment of asynchronous HDF for 24 h with 10–50 μM apigenin resulted in dose-dependent cell-cycle arrest at both the G₀/G₁ and G₂/M phases as measured by flow cytometry. The G₀/G₁ arrest was more clearly defined by using HDF that were synchronized in G₀ and then released from quiescence by replating at subconfluent densities in medium containing 10–70 μM apigenin. The cells were analyzed for cell-cycle progression or cyclin D1 expression 24 h later. A dose of apigenin as low as 10 μM reduced the percentage of cells in S phase by 20% compared with control cultures treated with solvent alone. Western blot analysis of apigenin-treated HDF indicated that cyclin D1 was expressed at higher levels than in untreated cells, which signifies that they were arrested in G₁ phase rather than in a G₀ quiescent state. The G₁ arrest was further studied by cyclin-dependent kinase 2 (cdk2) immune complex–kinase assays of apigenin-treated asynchronous HDF, which demonstrated a dose-dependent inhibition of cdk2 by apigenin. Inhibition of cdk2 kiase activity in apigenin-treated cells was associated with the accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein as measured by western blot analysis. The cdk inhibitor p21/WAF1 was also induced in a dose-dependent manner, with a 22-fold induction of p21/WAF1 in 70 μM apigenin-treated cells. In conclusion, apigenin treatment produced a G₁ cell-cycle arrest by inhibiting cdk2 kinase activity and the phosphorylation of Rb and inducing the cdk inhibitor p21/WAF1, all of which may mediate its chemopreventive activities in vivo. To our knowledge this is the first report of a chemopreventive agent inducing p21/WAF1, a known downstream effector of the p53 tumor suppressor protein. Mol. Carcinog. 19:74–82, 1997. © 1997 Wiley-Liss, Inc.     

Induction of oxidative stress and oxidative damage in rat glial cells by acrylonitrile.

Chronic treatment of rats with acrylonitrile (ACN) resulted in a dose-related increase in glial cell tumors (astrocytomas). While the exact mechanism(s) for ACN-induced carcinogenicity remains unresolved, non-genotoxic and possibly tumor promotion modes of action appear to be involved in the induction of glial tumors. Recent studies have shown that ACN induced oxidative stress selectively in rat brain in a dose-responsive manner. The present study examined the ability of ACN to induce oxidative stress in a rat glial cell line, a target tissue, and in cultured rat hepatocytes, a non-target tissue of ACN carcinogenicity. Glial cells and hepatocytes were treated for 1, 4 and 24 h with sublethal concentrations of ACN. ACN induced an increase in oxidative DNA damage, as evidenced by increased production of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in glial cells but not in rat hepatocytes. Hydroxyl radical formation following ACN treatment was also selectively increased in glial cells. Following 1 and 4 h of ACN exposure, the levels of the non-enzymatic antioxidant glutathione, as well as the activities of the enzymatic antioxidants catalase and superoxide dismutase were significantly decreased in the rat glial cells. Lipid peroxidation and the activity of glutathione peroxidase were not affected by ACN treatment in rat glial cells. No changes in any of these biomarkers of oxidative stress were observed in hepatocytes treated with ACN. These data indicate that ACN selectively induced oxidative stress in rat glial cells.     

Induction of cytochrome P450 1B1 in lung, liver and kidney of rats exposed to diesel exhaust.

We have shown previously that diesel exhaust particle (DEP) extracts (DEPE) and 1-nitropyrene were genotoxically activated by human cytochrome P450 1B1 in SOS/umu assay. In this study, the in vivo induction of P450 family 1 enzymes in rats by exposure to diesel exhaust was investigated with regard to mRNA levels, P450 enzyme content, drug oxidation activities in the microsomes and umu gene expression of typical P450 substrates and DEPE itself catalyzed by the microsomes. Male Fischer 344 rats (4 weeks old) were exposed to 0.3 and 3.0 mg/m(3) DEP for 12 h per day for 4 weeks; the former dose corresponded to the typical daily airborne particle concentration. The levels of mRNA of rat P450 1B1 and P450 1A1 in the lung and liver were significantly increased 1.1-1.4-fold by exposure to 0.3 mg/m(3) DEP. Diesel exhaust particle extracts induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of a functional P450 system and were further activated by human recombinant P450 1B1. Using an O-acetyltransferase overexpressing Salmonella strain, genotoxic activation of P450 1B1 marker chemicals (1-nitropyrene, 1-aminopyrene and DEPE) by lung, liver and kidney microsomes was increased 1.7-4.2-, 1.4-1.5- and 1.0-1.3-fold, respectively, by exposure to 0.3 mg/m(3) DEP. Activation of 3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole (Trp-P-1; marker for P450 1A1) by lung microsomes and the P450 1A2 content in liver microsomes were slightly increased by exposure to 3.0 mg/m(3) DEP. This is the first report to suggest that typical daily contaminant levels (0.3 mg particle/m(3)) of diesel exhaust can induce P450 1B1 in rats and that the induced P450 1B1 may catalyze the genotoxic activation of DEP.     

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.