Substrate specificity and alkyl group selectivity in the metabolism of N-nitrosodialkylamines

Metabolic activation may be a key step in determining the tissue specificity of carcinogenic nitrosamines. In previous work, we characterized P450IIE1 (an acetone/ethanol-inducible form of cytochrome P-450) as the major enzyme for the metabolic activation of N-nitrosodimethylamine. In this work, we investigated the metabolism of other N-nitrosodialkylamines in rat liver microsomes and in reconstituted monooxygenase systems containing purified cytochrome P-450 isozymes. The enzyme specificities in the metabolism of N-nitrosoethylmethylamine and N-nitrosodiethylamine were similar to those of N-nitrosodimethylamine; i.e., these substrates were more efficiently metabolized by acetone- or ethanol-induced microsomes than by other types of microsomes. However, substituting one methyl group with a benzyl or butyl group, as in N-nitrosobenzylmethylamine or N-nitrosobutylmethylamine (NBMA), substantially changed the enzyme specificity. P450IIE1 efficiently catalyzed the demethylation but not the debutylation of NBMA, whereas P450IIB1 (a phenobarbital-inducible form) efficiently catalyzed both the debutylation and demethylation reactions. In the demethylation of NBMA by P450IIE1, the addition of cytochrome b5 markedly increased the activity at low but not at high substrate concentrations, suggesting a decrease in Km value. This effect, however, was not observed in the debutylation of NBMA by P450IIE1 or P450IIB1, and in the demethylation of NBMA by P450IIB1. These studies demonstrate the substrate specificity and alkyl group selectivity in the metabolism of nitrosamines by cytochrome P-450 isozymes.     

Metabolism of N-nitrosodialkylamines by human liver microsomes

The metabolism of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine, N-nitrosobenzylmethylamine, and N-nitrosobutylmethylamine was investigated in incubations with human liver microsomes. All of the 16 microsomal samples studied were able to oxidize NDMA to both formaldehyde and nitrite at NDMA concentrations as low as 0.2 mM; the rates of product formation of the samples ranged from 0.18 to 2.99 nmol formaldehyde/min/mg microsomal protein (median, 0.53 nmol). At a concentration of 0.2 mM NDMA, the rates of denitrosation (nitrite formation) were 5 to 10% (median, 6.3%) those of demethylation (formaldehyde formation); the ratio of denitrosation to demethylation increased with increases in NDMA concentration, in a similar manner to rat liver microsomes. Immunoblot analysis with antibodies prepared against rat P-450ac (an acetone-inducible form of cytochrome P-450) indicated that the P-450ac [P-450j (isoniazid-inducible form)] orthologue in human liver microsomes had a slightly higher molecular weight than rat P-450ac and the amounts of P-450ac orthologue in human liver microsomes were highly correlated with NDMA demethylase activities (r = 0.971; P less than 0.001). Analysis of four selected microsomal samples showed that human liver microsomes exhibited at least three apparent Km and corresponding Vmax values for NDMA demethylase. This result, suggesting the metabolism of NDMA by different P-450 enzymes, is similar to that obtained with rat liver microsomes, even though most of the human samples had lower activities than did the rat liver microsomes. The high affinity Km values of the four human samples ranged from 27 to 48 microM (median, 35 microM), which were similar to or slightly lower than those observed in rat liver microsomes, indicating that human liver microsomes are as efficient as rat liver microsomes in the metabolism of NDMA. The human liver microsomes also catalyzed the dealkylation and denitrosation of other nitrosamines examined. The rates of product formation and the ratios of denitrosation to dealkylation varied with the structures and concentrations of the substrates as well as with the microsomal samples tested. The results indicate that human liver microsomes are capable of metabolizing N-nitrosodialkylamines via the pathways that have been established with rat liver microsomes.     

Metabolism of nitrosamines by purified rabbit liver cytochrome P-450 isozymes

The metabolism of nitrosamines by microsomal cytochrome P-450 (P-450) isozymes was studied in a reconstituted monooxygenase system. P-450 LM2, LM3a, LM3b and LM3c, LM4, and LM6 were purified, respectively, from the livers of phenobarbital-treated, ethanol-treated, untreated, isosafrole-treated, and imidazole-treated rabbits. Of these isozymes, LM3a had the highest N-nitrosodimethylamine demethylase (NDMAd) activity with a Km of 2.9 mM and Vmax of 9.3 nmol/min/nmol. LM2, LM4, and LM6 exhibited NDMAd activity only at high N-nitrosodimethylamine concentrations, and isozymes LM3b and LM3c had poor activity even at the highest substrate concentrations examined. LM2, however, was more active than LM3a in the metabolism of N-nitrosomethylaniline. With each isozyme (LM3a or LM4), only one Km for NDMAd was observed, whereas with rabbit liver microsomes, multiple Km of 0.07, 0.27, and 36.8 mM were obtained. P-450 isozymes also catalyzed the denitrosation of nitrosamines at rates comparable to or lower than the demethylation, and the ratio of these two reactions was different with different nitrosamines. 2-Phenylethylamine and 3-amino-1,2,4-triazole, which were believed previously to affect NDMAd by mechanisms independent of P-450, were shown to be potent inhibitors of P-450-dependent NDMAd. These results further establish the role of P-450 isozymes in the metabolism of nitrosamines and indicate that LM3a is apparently responsible for the increased N-nitrosodimethylamine metabolism associated with ethanol treatment.     

Cytochrome P450 2E1 and 2A6 enzymes as major catalysts for metabolic activation of N-nitrosodialkylamines and tobacco-related nitrosamines in human liver microsomes.

An acetyltransferase-overexpressing strain of Salmonella typhimurium (NM2009) has been used to investigate roles of human liver microsomal cytochrome P450 (P450) enzymes in the activation of carcinogenic nitrosamine derivatives, including N-nitrosodialkylamines and tobacco-smoke-related nitrosamines, to genotoxic products. Studies employing correlation of activities with several P450-dependent monooxygenase reactions in different human liver samples, inhibition of microsomal activities by antibodies raised against human P450 enzymes and by specific P450 inhibitors, and reconstitution of activities with purified P450 enzymes suggest that the tobacco-smoke-related nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and N-nitrosonornicotine (NNN) as well as N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) are oxidized to genotoxic products by different P450 enzymes, particularly P450 2E1 and 2A6. The activation of NDMA and NNN by liver microsomes was suggested to be catalyzed more actively by P450 2E1 than by other P450 enzymes because the activities were well correlated with NDMA N-demethylation and aniline p-hydroxylation in different human samples, and purified P450 2E1 had the highest activities in reconstituted monooxygenase systems. The relatively high contribution of P450 2A6 to the activation of NDEA and NNK was supported by the correlation seen with coumarin 7-hydroxylation in human liver microsomes, and antibodies raised against P450 2A6 inhibited both activities by approximately 50%. P450 3A4, 2D6 and 2C enzymes appear not to be extensively involved in the activation of these nitrosamines as judged by several criteria examined. Thus, this work indicates that several P450 enzymes, particularly P450 2E1 and 2A6, catalyze metabolic activation of nitrosamine derivatives including N-nitrosodialkylamines and tobacco-smoke-related nitrosamines in human liver microsomes.     

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.     

Molecular basis for the sex-related difference in renal N-nitrosodimethylamine demethylase in C3H/HeJ mice

Previous work with rat and rabbit liver enzymes has demonstrated that cytochrome P450IIE1 is responsible for the metabolism of N-nitrosodimethylamine (NDMA), a widely occurring carcinogen. The present study demonstrated that a similar enzyme also exists in the mouse kidney and is regulated by testosterone. These results can account for the reported sex-related difference in the renal metabolism of NDMA in mouse strains such as C3H/HeJ. NDMA demethylase activities (expressed as pmol/min/mg protein) in kidney microsomes of female and male C3H/HeJ mice were 3.0 +/- 0.7 and 51.9 +/- 11.2, respectively. After testosterone treatment (500 mg/kg b.w. in olive oil, s.c.) for 2 days, the renal NDMA demethylase activity of the female mice was elevated 17-fold. The difference and change in NDMA demethylase activity were accompanied by corresponding differences and changes in P450IIE1 as quantified by immunoblot analysis (using antibodies prepared against rat P450IIE1) as well as in the mRNA level for P450IIE1 as determined by Northern and slot blot analyses (using a cDNA probe containing the coding sequence of rat P450IIE1 gene). Based on gel electrophoresis, the molecular weight of mouse renal P450IIE1 was 52,000 and the size of mouse renal P450IIE1 mRNA was approximately 1.8 kilobases; both were similar to those found in rat liver and kidney. Renal P450IIE1 mRNA levels in female, male, and testosterone-treated female mice were at a ratio of 1:22:20. On the other hand, this testosterone-related difference was not observed in hepatic P450IIE1. In liver microsomes, there were no significant differences in NDMA demethylase activity, P450IIE1 content, and P450IIE1 mRNA level between male and female mice or between untreated and testosterone-treated female mice. The apparent Km value of NDMA demethylase in mouse kidney microsomes (22 to 27 microM NDMA) were similar to that in rat liver microsomes. Renal NDMA demethylase activity was inhibited by a monoclonal antibody prepared against rat P450IIE1. These results suggest that mouse renal P450IIE1 is similar to rat P450IIE1 and is responsible for the low Km form of NDMA demethylase activity. Nevertheless, only the mouse renal enzyme is regulated by testosterone.     

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.     

Metabolism and activation of N-nitrosodimethylamine by hamster and rat microsomes: comparative study with weanling and adult animals

It has been reported that hamster liver preparations are more effective for the metabolic activation of N-nitrosodimethylamine (NDMA) to a mutagen than rat liver preparations. The enzymatic basis for this phenomenon, however, has not been clearly elucidated. The present study was undertaken to examine the enzymology of NDMA metabolism by different hepatic subcellular fractions prepared from hamsters and rats of two different ages, and to investigate the correlation between the metabolism and the activation of NDMA to a mutagen for Chinese hamster V79 cells. The content of cytochrome P-450 was approximately 1.5-fold higher in hamster microsomes than in rat microsomes from both ages (1.19-1.38 versus 0.73-0.83 nmol P-450/mg protein). Weanling hamster microsomes exhibited multiple apparent Km values for NDMA metabolism as did weanling rat microsomes. The apparent Km I value of NDMA demethylase (NDMAd) in hamster microsomes was about one-half that in rat microsomes (36 versus 83 microM) with corresponding Vmax values of 2.09 and 2.57 nmol/min/nmol P-450. The Km I values for denitrosation did not differ from the corresponding values for NDMAd with Vmax values of 0.17 and 0.22 nmol/min/nmol P-450 for hamster and rat microsomes, respectively. These apparent Km values were affected neither by sonication nor by the presence of cytosolic proteins in S9 fractions. Adult rat liver microsomes showed less than one-half the NDMAd activity in weanling rat liver microsomes, whereas such age difference was not observed in hamster liver microsomes. This result was confirmed by Western blotting showing the levels of P-450ac (an acetone-inducible form of P-450) of these microsomes at comparable levels to their NDMAd activities. NDMAd was highly correlated to the metabolic activation of NDMA to a mutagen for V79 cells in an activation system mediated by microsomes prepared from hamsters and rats of different ages. The results from this study clearly demonstrate the enzymatic basis for the more effective metabolism of NDMA in adult hamsters than in adult rats.     

Roles of cytochrome P450IIE1 in the dealkylation and denitrosation of N-nitrosodimethylamine and N-nitrosodiethylamine in rat liver microsomes

N-Nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) are widely occurring nitrosamines and require enzyme-catalyzed activation for their carcinogenic actions. The low Km forms of the enzyme are generally considered to be important in the activation of environmental carcinogens. In this work we examined the role of cytochrome P450IIE1--a constitutive enzyme that is also inducible by acetone, ethanol, fasting and other factors--in catalyzing the dealkylation and denitrosation of these two carcinogens. The experimentally determined Km value of NDMA demethylase depended upon the experimental conditions and was lower when lower protein concentrations were used. Low Km values of 15-20 microM were observed for NDMA demethylase with different preparations of microsomes. In the deethylation of NDEA, a low Km of approximately 40 microM was observed for both control and acetone-induced microsomes. Immunoinhibition studies indicated that P450IIE1 was responsible for almost all the low Km NDMA demethylase activity in acetone-induced microsomes and greater than 80% in control microsomes. This enzyme was also responsible for about three-quarters of the low Km NDEA deethylase activity in acetone-induced microsomes and about half in control microsomes. The denitrosation of NDMA and NDEA was inhibited to approximately the same extents as the dealkylation reactions under different experimental conditions, suggesting the involvement of the same enzyme and perhaps a common initial intermediate in these two types of reactions. The relevance of this work and its relationship to related information in the literature are discussed.     

Factors regulating activation and DNA alkylation by 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone and nitrosodimethylamine in rat lung and isolated lung cells, and the relationship to carcinogenicity

The molecular dosimetry for O6-methylguanine (O6MG) formation in DNA from rat lung and pulmonary cells was compared following treatment for 4 days with equimolar doses of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent pulmonary carcinogen or nitrosodimethylamine (NDMA), a weak carcinogen in rat lung. The dose response for O6MG formation from NNK was biphasic; the O6MG to dose ratio, an index of alkylation efficiency, increased dramatically as the dose of carcinogen was decreased. In contrast, the dose-response curve for methylation by NDMA appeared opposite of that for NNK with alkylation efficiency increasing as a function of dose. These results suggested that high and low Km pathways exist for the activation of NNK, whereas only high Km pathways may be involved in NDMA activation. Furthermore, DNA methylation by NNK was cell selective with the highest levels in the Clara cell, whereas methylation by NDMA was not. DNA methylation in the Clara cell was 50-fold greater by NNK than by NDMA at equimolar doses (0.005 mmol/kg). Thus, differences in O6MG formation, specifically the presence of a high affinity pathway in the Clara cell for activation of NNK, may explain why following low dose exposure, NNK is a potent pulmonary carcinogen while NDMA is not. Different cytochrome P-450 isozymes also appear to be involved in the activation of NNK and NDMA. Inhibition of in vitro methylation (with calf thymus DNA and lung microsomes) by antibodies to cytochrome P-450 isozymes provided evidence that a homolog of rabbit cytochrome P-450(2) (cytochrome P-450b) may be important in the activation of NNK in rat lung, whereas cytochrome P-450(5) may activate NDMA. A 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible cytochrome P-450 isozyme (P-450c) may also be involved in the activation of NNK but not NDMA. Treatment with TCDD increased both NNK activation by pulmonary microsomes and the formation of O6MG in Clara cells and type II cells incubated in vitro with NNK. alpha-Naphthoflavone (alpha-NF), a specific inhibitor of cytochrome P-450c reversed the increase in methylation by TCDD-induced microsomes but did not inhibit in vitro activation of NNK using microsomes from untreated rats. However, NNK mediated O6MG formation in Clara cells, but not in type II cells incubated with alpha-NF, was decreased by 21%. These data indicate that both cytochrome P-450b and P-450c are probably involved in the activation of NNK in Clara cells from untreated rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of carcinogenic nitrosamines by rat nasal mucosa and the effect of diallyl sulfide

Rat nasal cavity is one of the target organs for carcinogenesis induced by N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The present work investigated the metabolism of these nitrosamines by rat nasal microsomes, as well as the possible modulating factors. Microsomes prepared from rat nasal mucosa were efficient in metabolizing these nitrosamines. In general, the metabolism of the nitrosamines was slightly higher in 9-week-old rats than in 4-week-old animals, and there was no sex-related difference. Fasting of rats for 48 h, which is known to induce hepatic cytochrome P450IIE1 and NDMA metabolism, did not increase the nasal metabolism of NDMA, NDEA, or NNK. Pretreatment of rats with acetone, another inducer of hepatic P450IIE1, did not increase the metabolism of NDMA. Furthermore, it decreased the nasal metabolism of NDEA and NNK. Immunoinhibition studies suggest that, in the nasal mucosa, P450IIE1 is only partially responsible for the oxidation of NDMA and other P450 isozymes are responsible for the metabolism of NDEA. A single p.o. pretreatment of male rats with diallyl sulfide (DAS), a component of garlic oil, caused a significant decrease in the oxidative metabolism of NDEA and NNK in rat nasal mucosa. Whereas the nasal metabolism of NDMA was reduced by DAS pretreatment, there was no change in the amount of the nasal microsomal proteins immunoreactive with the antibodies against P450IIE1. The inhibitory effect of DAS on the nasal oxidative metabolism of NDMA, NDEA, and NNK was also observed in experiments in vitro. The results demonstrate the ability of nasal mucosa to metabolically activate these nitrosamines and the inhibition of this process by DAS, suggesting that DAS may be effective in inhibiting the related nasal tumorigenesis.     

High variability of nitrosamine metabolism among individuals: Role of cytochromes P450 2A6 and 2E1 in the dealkylation of N-nitrosodimethylamine and N-nitrosodiethylamine in mice and humans

We undertook this study to answer several questions regarding nitrosamine metabolism. Kinetics of nitrosamine metabolism showed the involvement of at least two enzymes in the dealkylation of N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA) in mouse liver microsomes. Coumarin inhibited both reactions competitively. On the other hand, microsomal coumarin 7-hydroxylase was inhibited by NDMA (K_i 2.7 mM) and NDEA (K_i 0.013 mM). The big difference in the K_i values suggests a higher affinity of NDEA than NDMA to Cyp2a-5 (mouse cytochrome P450_coh). A specific antibody against Cyp2a-5 inhibited more of the microsomal NDEA (up to 90%) than NDMA (up to 40%) dealkylation. The converse was true with anti-Cyp2e-1 antibody. These results suggest that the primary substrate for Cyp2a-5 is NDEA and for Cyp2e-1, NDMA. Western blot analysis of human liver microsomes showed a great interindividual variation in the amounts of CYP2A6 (human cytochrome P450_coh) and CYP2E1. Also, courmarin 7-hydroxylation and nitrosamine dealkylation varied greatly among individuals. A high correlation (r = 0.93, P < 0.001) was found between NDEA and coumarin metabolism. Both activities were associated with CYP2A6. On the other hand, little or no correlation was found between microsomal CYP2A6 and CYP2E1 or between CYP2E1 and NDEA dealkylation. Immunoinhibition of human microsomal NDEA metabolism by CYP2a-5 antibody varied greatly among individuals (10–90%), suggesting, as in the case of mice, that NDEA is metabolized primarily by CYP2A6, at least in some individuals. Taken together the data suggest that (1) the metabolic activation of nitrosamines in humans varies greatly among individuals; (2) different nitrosamines may partially be metabolized by different cytochrome P450 isozymes; and (3) because of similarities between nitrosamine metabolism in mice and humans, inbred strains of mice would be relevant experimental models for studying nitrosamine activation.     

The induction and competitive inhibition of a high affinity microsomal nitrosodimethylamine demethylase by ethanol

The effects of ethanol on the metabolism of nitrosamines byrat liver microsomes have been studied. Treatment of rats with10 or 15% ethanol in drinking water for 3 days causes a 4- to5-fold enhancement in microsomal N-nitrosodimethylamine demethylase(NDMAd) activity and a 40–60% increase in gross P-450content. The enhancement is mainly due to the induction of alow K_m form (K_m = 0.07 mM) of NDMAd. The treatment induces proteinspecies with molecular weights between 50 000 and 52 000, someof which are believed to be P-450 isozymes with high affinityto NDMA. In addition to NDMA, treatment with ethanol also enhancesthe metabolism of N-nitroso-N-methylethylamine, N-nitrosomethylamline,and N-nitroso-N-methylbenzylamine. When added to the incubationmixture, ethanol and its homologs inhibit the demethylationof these nitrosamines by microsomes. Ethanol is a competitiveinhibitor of the low K_m NDMAd with a K_i of 0.31 mM and is lesseffective in inhibiting the metabolism of more lipophilic nitrosamines.     

Effect of (+)-catechin, dimethyl sulfoxide and ethanol on the microsome-mediated metabolism of two hepatocarcinogens, N-nitrosodimethylamine and aflatoxin B1

Effects of catechin, a plant phenolic flavonoid, and of the commonly used organic solvents dimethyl sulfoxide (DMSO) and ethanol (EtOH) on the microsome-mediated metabolism of two hepatocarcinogens, N-nitrosodimethylamine (NDMA) and aflatoxin B1 (AFB1), are presented. Using hamster liver microsomes as a source of mixed-function oxidases, it was shown that catechin at 0.1-0.2 mM levels had no effect on the oxidation of either carcinogen. However, at 1-5 mM levels it caused a concentration-dependent inhibition (38-70%) of the formation of formaldehyde from NDMA, and at the 5 mM level it caused a 40% inhibition of AFB1-DNA binding. DMSO and EtOH totally inhibited NDMA demethylase activity but had little effect on the binding of AFB1 to DNA. These observations indicate that the mixed-function oxidases (cytochrome P450) essential for the metabolic activation of these carcinogens exhibit different sensitivities to different inhibitors.     

Alterations of microsomal monooxygenase system and carcinogen metabolism by streptozotocin-induced diabetes in rats

The effects of diabetes on the liver microsomai monooxy-genaseenzymes and carcinogen metabolism have been studied in rats.Treatment with streptozotocin causes a marked enhancement inmicrosomai N-nitrosodimethylamine (NDMA) demethylase activity.The enhancement is due mainly to the induction of a high affinityNDMA demethylase (K_m, {small tilde}0.05 mM) which is accompaniedby the induction of a protein species with mol. wt. of 50 000.The treatment also induces aniline hydroxylase whose activityis in parallel with NDMA demethylase. Streptozotocin-induceddiabetes also increases the metabolism of N-nitrosomethyl-ethylaminebut not that of N-nitrosomethylaniline or N-nitrosomethylbenzylamine.On the other hand, diabetes decreases the metabolism of benzo[a]pyrene,benzphetamine, and ethylmorphine. The results suggest that diabetescauses an alteration of the composition of cytochrome P-450isozymes; the forms efficient in metabolizing NDMA are increasedwhile certain other forms of cytochrome P-450 are decreased.     

Effects of ethanol on the DNA-damage induced by 2 carcinogenic nitrosamines

We studied the DNA single-strand breaks (DNA SSBs) induced by two nitrosamines using rat hepatocytelin situ nick translation assay. In the hepatocytes treated with 20 mu M of N-nitrosodimethylamine (NDMA), 100 mM ethanol enhanced DNA SSBs 3 times higher than those of control. However, there was no significant difference between the DNA SSBs with and without ethanol in 300 mu M of N-nitrosodiethylamine (NDEA) treated groups. Pretreatment of 100 mM ethanol increased P450IIE1 levels determined by Western blotting, whereas the amount of total P450 was not affected. Although NDMA is possibly activated by P450IIE1, there could be other isozymes responsible for the activation of NDEA. Phenobarbital inducible isozymes such as P450IIB1 and IIB2, or P450IIA3 may be primarily responsible.     

Characterization of xenobiotic-metabolizing enzymes and nitrosamine metabolism in the human esophagus

Esophageal cancer has been associated with tobacco smoking, and nitrosamines are possible causative agents for this cancer. The present study investigated the metabolism of the tobacco carcinogens N'- nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK), and N-nitrosodimethylamine (NDMA), as well as the presence of xenobiotic-metabolizing enzymes in human esophageal tissues from individuals in the United States and Huixian, Henan Province, China (a high-risk area for esophageal cancer). All esophageal microsomal samples activated NNN and the metabolic rate was 2-fold higher in the esophageal samples from China than the USA. All microsomal samples activated NDMA. However, most of the microsomal samples did not activate NNK. Troleandomycin (an inhibitor of cytochrome P450 3A) decreased the formation of NNN-derived keto acid by 20-26% in the esophageal microsomes. The activities for NADPH: cytochrome c reductase, ethoxycoumarin O-deethylase, NAD(P)H: quinone oxidoreductase and glutathione S-transferase were present in the esophageal samples. Coumarin 7-hydroxylase (a representative activity for P450 2A6) activity was not detected in the esophageal microsomal samples. The activities for nitrosamine metabolism and xenobiotic- metabolizing enzymes were decreased (by 30-50%) in the squamous cell carcinomas compared with their corresponding non-cancerous mucosa. The presence of activation and detoxification enzymes in the esophagus may play an important role in determining the susceptibility of the esophagus to the carcinogenic effect of nitrosamines. Our results suggest that P450s 3A4 and 2E1 are involved in the activation of NNN and NDMA, respectively, in the human esophagus.      

Enzyme kinetics of N-nitrosodimethylamine demethylase in rodents and humans

A variety of Km values have been reported for hepatic microsomal N-nitrosodimethylamine demethylase (NDMAd). We demonstrated previously that the biologically important, high affinity (KmI) form of microsomal NDMAd is manifested by cytochrome P450IIE1 (also known as P450ac and P450j). The KmI value of NDMAd was, however, affected greatly by assay conditions: the possible presence of inhibitors and the presence of cytochrome b5. We re-examined the KmI value by testing the effect of enzyme concentrations and of different types of enzyme preparations on the Km. The KmI value ranged from 15 to 22 microM, as estimated by the direct linear plot, using a microsomal protein concentration in the range of 0.1 to 0.8 mg/ml with correction for substrate utilization. A slight yet significant dependency of microsomal protein concentration on the Km (r = 0890; p less than 0.05) was seen. When five different microsomal preparations were compared, the KmI value ranged from 14 to 24 microM (median, 20 microM), as estimated by the direct linear plot. The Km estimated by the commonly used Eadie-Hofstee plot did not differ from that by the direct linear plot. These Km values are close to the values obtained in studies with isolated cells and tissue slices. The KmI form of NDMAd (P450IIE1 and its orthologues) is present in rats, mice, rabbits, hamsters and guinea-pigs. It is responsible for the age-dependent differences between rats and hamsters and for the sex-related differences in mouse kidneys, and for the bioactivation and toxicity of NDMA. This enzyme also exists in human liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alkylation of cellular macromolecules and target specificity of carcinogenic nitrosodialkylamines: metabolic activation by cytochromes P450 2B1 and 2E1

The alkylation of DNA, RNA and protein by labeled metabolites of [alpha- 14C]nitrosodimethylamine (NDMA), [alpha-14C]nitrosodipropylamine (NDPA) and [alpha-14C]nitrosodibutylamine (NDBA) was determined as a measure of the metabolic activation of these nitrosamine carcinogens in vitro using microsomes prepared from freshly isolated rat hepatocytes as well as in intact cells using primary cultured rat hepatocytes. The abilities of these nitrosodialkylamines to alkylate cellular macromolecules were significantly affected by pretreatment of rats with inducers of cytochrome P450 and were related to the specific activities of cytochrome P450 2B1 or 2E1 in rat hepatocytes. Pretreatment of rats with phenobarbital (PB) substantially increased the catalytic activity of pentoxyresorufin (PR) O-depentylase, an activity catalyzed by cytochrome P450 2B1, in rat hepatocytes. The increase in the PR O- depentylase activity was associated with a significant increase in the alkylation of DNA or RNA by NDPA, and in alkylation by NDBA, particularly of proteins. However, induction of cytochrome P450 2B1 resulted in a significant decrease in alkylation of cellular macromolecules by NDMA in all cases. In contrast, enhancement of the catalytic activity of the p-nitrophenol (pNP) hydroxylase (P450 2E1) due to pretreatment of rats with pyridine (PYR) resulted in a significant increase in the alkylation of cellular DNA by NDMA. The induction of cytochrome P450 2E1 also increased the alkylation of DNA and RNA by NDPA, but to a lesser extent. Inhibition studies using the chemical inhibitors orphenadrine (OP) and diethyldithiocarbamate (DDC), which are specific for cytochromes P450 2B1 and 2E1, respectively, indicated that cytochrome P450 2B1 was not involved in the metabolic activation of NDMA and that cytochrome P450 2E1 was not responsible for the bioactivation of NDBA. The results presented here demonstrate the substrate specificity and important role of cytochromes P450 2B1 and 2E1 in the bioactivation of nitrosodialkylamines, and suggest that multiple mechanisms may be involved in carcinogenesis induced by nitrosodialkylamines.