Cytochrome P450 2a of nasal epithelium: Regulation and role in carcinogen metabolism

In this study, we found that rat nasal coumarin-7–hydroxylase (a) activity was two orders of magnitude higher than rat hepatic COH activity and could be induced by adding coumarin to the rats' drinking water. In western blot analysis, an anti-cytochrome P450 (a) 2a-5 (mouse liver COH) antibody recognized a sharp band in the microsomal fraction of rat nasal epithelium but not of the liver; the band comigrated with Cyp2a-5. The intensity of the band was increased by the coumarin treatment. Similarly, in northern blot analysis, a cDNA probe specific for Cyp2a-5 recognized an mRNA in the nasal epithelium having the same size as mouse liver Cyp2a-5 mRNA; however, no hybridizable mRNA was recognized in liver preparations. Unlike the protein level, the level of the mRNA was not increased by coumarin. When northern blot analyses were performed with two oligoprobes specific for rat lung CYP2A3, an mRNA of similar size to Cyp2a-5 mRNA was recognized. In immunoinhibition analysis, anti-Cyp2a-5 antibody inhibited rat nasal COH activity and aflatoxin B₁ (AFB₁) metabolism completely. It inhibited N-nitrosodiethylamine (a) and 4- (a) -1-(3-pyridyl)-1-butanone (a) metabolism by 80–90%. In contrast, the hepatic metabolism of the four compounds was not affected by the antibody. When coumarin instead of anti-Cyp2a-5 antibody was used, a strong but variable inhibition of the nasal metabolism of AFB₁, NDEA, and NNK was seen. The results suggest that an enzyme or enzymes similar to mouse liver Cyp2a-5, one of which may be CYP2A3, is expressed at high levels in rat nasal epithelium but not in the liver and that its expression is increased by coumarin, an odorant and a substrate of Cyp2a-5. The increase probably occurs by protein stabilization or stimulation of translation. The results also show that the enzyme has a key role in the nasal metabolism of three well-known carcinogens, AFB₁, NDEA, and NNK and may therefore be an important contributing factor in nasal carcinogenesis.©1995 Wiley-Liss, Inc.     

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 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.     

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.      

Rat oesophageal cytochrome P450 (CYP) monooxygenase system: comparison to the liver and relevance in N-nitrosodiethylamine carcinogenesis.

N-nitrosodiethylamine (NDEA) is able to induce tumours in the rat oesophagus. It has been suggested that this could be due to tissue specific expression of NDEA activating cytochrome P450 enzymes. We investigated this by characterizing the oesophageal monooxygenase complex of male Wistar rats and comparing it with that of the liver. Total amount of cytochrome P450, NADPH P450 reductase, cytochrome b5 and cytochrome b5 reductase of the oesophageal mucosa was approximately 7% of what was found in the liver. In addition, major differences were found in the cytochrome P450 isoenzyme composition between these organs: CYP 2B1/2B2 and CYP3A were found only in the liver, whereas CYP1A1 was constitutively expressed only in the oesophagus. Of the two well-known nitrosamine metabolizing enzymes, CYP2A3 was found only in the oesophagus whereas CYP2E1 was exclusively expressed in the liver. Catalytic studies, western blotting and RT-PCR analyses confirmed the expression of CYP2A3 in the oesophagus. CYP2A enzymes are known to be good catalysts of NDEA metabolism. Oesophageal microsomes had a K(m) for NDEA metabolism, which was about one-third of that of hepatic microsomes, but they showed similar activities when compared per nmol of total P450. NDEA activity in the oesophagus was significantly increased by coumarin (CO), which also induced oesophageal CYP2A3. Immunoinhibition of the microsomal NDEA activity showed that up to 70% of this reaction is catalysed by CYP2A3 in the oesophagus, whereas no inhibition of the hepatic NDEA activity could be achieved by the anti-CYP2A5 antibody. NDEA, but not N-nitrosodimethylamine (NDMA) inhibited the oesophageal metabolism of CO. The results of the present investigation show major differences in the enzyme composition of the oesophageal and hepatic monooxygenase complexes, and are in accordance with the hypothesis that the NDEA organotropism could, to a large extent, be due to the tissue specific expression of the activating enzymes.     

Different effects of chemicals on metabolism of N-nitrosamines in rat liver

The effects of phenobarbital (PB), 3-methylcholanthrene (MC), pyrazole (PY) and ethanol (EtOH) pretreatment on N-nitrosodimethylamine (NDMA), N-nitrosobutylmethylamine (NBMA) and N-nitrosomethylbenzylamine (NMBzA) metabolism were examined in rats. In isolated hepatocytes, PB increased the metabolic decomposition of NBMA and NMBzA, and MC increased that of NBMA; PY and EtOH increased only that of NDMA. In studies of hepatic microsomal dealkylation, PB increased NBMA debutylation and NMBzA debenzylation, and MC increased NBMA debutylation; PY and EtOH increased NDMA demethylation selectively. Several cytochrome P450 (P450) species were active in dealkylating nitrosamines, indicating that the organ-specific carcinogenicity of nitrosamines might be changed by various P450 inducers.     

The effects of ascorbic acid deficiency and excess on the metabolism and toxicity of N-nitrosodimethylamine and N-nitrosodiethylamine in the guinea pig

The influence of ascorbate deficiency and megadosage on themetabolism of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine(NDEA) was investigated in the guinea pig. After 21 days ona scorbutogenic diet, microsomal cytochrome P-450 and cytochromeb₅ levels fell by 51 and 32%, respectively, while cytochromec reductase activity remained constant. The activities of NDMAand NDEA dealkylase I were also depressed significantly. TheV_max of NDMA demethylase I and NDEA deethylase I was significantlydepressed. Also, ascorbate deficiency significantly decreasedthe plasma clearance of both nitrosamines though the LD₅₀ ofneither were altered by ascorbate nutrition. Covalent bindingof ¹⁴C from [¹⁴C]NDMA and [¹⁴C]NDEA to DNA obtained from liverslices was significantly lower in the deficient than in thecontrol samples; megadosage appeared to have the opposite effect.     

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.     

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.     

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.     

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.     

Metabolic activation of N-alkylnitrosamines in genetically engineered Salmonella typhimurium expressing CYP2E1 or CYP2A6 together with human NADPH-cytochrome P450 reductase

A Salmonella typhimurium tester strain YG7108 2E1/OR co-expressing human CYP2E1 together with human NADPH-cytochrome P450 reductase (OR) was established. The mutagen-activating capacity of human CYP2E1 for N-alkylnitrosamines was compared with that of CYP2A6 using the YG7108 2E1/OR and the YG7108 2A6/OR strains of SALMONELLA: Salmonella YG7108 2A6/OR is a derivative of YG7108 co-expressing CYP2A6 together with OR. Eight N-alkylnitrosamines, including N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosomethylphenylamine (NMPhA), N-nitrosopyrrolidine (NPYR), N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were examined. CYP2E1 expressed in the YG7108 2E1/OR cells showed mutagen-activating capacity, as indicated by induced revertants/min/pmol cytochrome P450, for NDMA, NDEA, NDPA, NDBA, NPYR and NNK, but not NMPhA and NNN. CYP2A6 activated NDMA, NDEA, NDPA, NDBA, NMPhA, NPYR, NNN and NNK. The ratio of the mutagen-activating capacity seen with CYP2A6 to that seen with CYP2E1 was calculated for each N-alkylnitrosamine. In the case of NDMA, NPYR and NDEA, the ratio was under 1.0, while the ratio was over 1.0 with NDPA, NDBA, NNK, NMPhA and NNN. We conclude that human CYP2E1 is mainly responsible for the metabolic activation of N-nitrosamines with a relatively short alkyl chain(s), whereas CYP2A6 was predominantly responsible for the metabolic activation of N-alkylnitrosamines possessing a relatively bulky alkyl chain(s).     

Enzymatic mechanisms in the metabolic activation of N-nitrosodialkylamines

The metabolism of several N-nitrosodialkylamines was studied using rat liver microsomes and purified cytochrome P450 isozymes in a reconstituted monooxygenase system. With purified acetone/ethanol-inducible cytochrome P450 (P450ac), high N-nitrosodimethylamine (NDMA) demethylase activity was observed. Cytochrome b5 was also involved in NDMA metabolism by decreasing the Km of NDMA demethylase. A close relationship between the demethylation and denitrosation of this substrate was observed. P450ac was also active in the metabolism of N-nitrosoethylmethylamine (NEMA), but was less active than phenobarbital-inducible cytochrome P450 (P450b) in the metabolism of N-nitrosobutylmethylamine (NBMA), especially in catalysing the debutylation reaction. Similar substrate specificity was demonstrated with liver microsomes from rats treated with other inducers. With different P450 isozymes and microsomes, a close relationship between metabolism and activation of nitrosamines to mutagens to V79 cells was demonstrated. DNA alkylation by NDMA in vitro was correlated with the rate of metabolism of these compounds, whereas DNA alkylation in vivo was more complex and was dose-dependent. The work demonstrates the importance of knowledge of the substrate specificity of cytochrome P450 isozymes in understanding the mechanisms of the metabolic activation of nitrosamines.     

Intake of volatile nitrosamines from consumption of alcohols.

Volatile nitrosamines were determined in alcoholic drinks during epidemiologic studies on the relationship between esophageal cancer incidence and alcohol consumption in Normandy, France. Nitrosodimethylamine (NDMA) was found commonly in most alcoholic drinks tested, with the exception of wine. The average level, about 2 micrograms/liter in beers, was higher than that for other drinks; the range was 0.2--8.6 micrograms/liter. Traces of nitrosodiethylamine (NDEA) were also detected in spirits and ciders. No significant increases in levels were found after nitrosation. Calculation of daily intake in the study region showed that the main intake of volatile nitrosamine is from NDMA in beer. The intake of NDEA through consumption of cider is about one-third that of NDMA from all sources.     

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.     

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.     

Presystemic intestinal metabolism of N-nitrosodimethylamine in mouse intestine

N-Nitrosodimethylamine (NDMA), a common food contaminant, is a potent liver carcinogen in rodents. A high presystemic intestinal metabolism has been shown for several nitrosamines including environmentally important compounds. We determined the metabolism of 1 micron [14C]-NDMA in isolated perfused mouse intestinal segments. We found NDMA to be equally distributed between the absorbed fluid and the perfusate. During a 2-h perfusion period, 0.13% of the radioactivity was converted to CO2. The formation of CO2 was decreased by pretreatment with diallylsulfide or addition of SKF 525A, and slightly increased by phenobarbital. Hydrophilic metabolites were found in the absorbate (0.9%) and perfusate (3.8%) of untreated mice. The amount of metabolites in the absorbate was increased by treatment with acetone or phenobarbital (8-fold), but not after starvation, with formaldehyde being present only in phenobarbital-treated animals. Treatment with diallylsulfide or addition of SKF 525A reduced the amount of metabolites in acetone-treated animals to control values. In conclusion, intestinal turnover does not significantly reduce the body burden of orally ingested NDMA and thus is not a first-line defense against this carcinogenic nitrosamine. NDMA metabolism has been attributed to the presence of cytochrome P450IIE1, which has not been detected in the intestine of untreated animals. The low turnover of NDMA, the induction by acetone and phenobarbital treatment, and the inhibition by diallylsulfide suggest the presence of low amounts of this or related cytochrome P450 isozyme(s) in mouse intestine.     

Nitrosamines in cured meat products.

One hundred samples of specially selected spiced meat products (sausages, salami, bologna, wieners, meat loaf, canned luncheon mean, etc) were analysed for nitrate, nitrite and volatile nitrosamines. None of the samples contained high levels of nitrosamines, but many contained traces, generally in the range from 2-50 mug/kg. Some contained as many as four nitrosamines, namely, NDMA, NDEA, NPip and NPy. In a few cases the samples were reanalysed after two weeks' storage at 4 or -20 degrees C, but no significant change in the nitrosamine levels could be detected. The identity of the nitrosamines was confirmed by GLC-high-resolution-MS.     

Effects of fluorination on in-vitro metabolism and biological activity of N-nitrosodialkylamines

Substitution of N-nitrosodialkylamines with fluorine at specific sites inhibits oxidative metabolism at the respective carbon atoms. The results of in-vitro metabolism studies with N-nitrosodiethylamine (NDEA), N-nitrosodibutylamine (NDBA) and their fluorinated analogues, N-nitroso-2,2,2-trifluoroethyl-ethylamine (NDEA-F3), N-nitroso-bis(2,2,2-trifluoroethyl)amine (NDEA-F6), N-nitroso-4,4,4-trifluorobutyl-butylamine (NDBA-F3), N-nitroso-bis(4,4,4-trifluo-robutyl)amine (NDBA-F6) and N-nitroso-bis(2,2,3,3,4,4,4-heptafluorobutyl)amine (NDBA-F14), showed effects of fluorination on biotransformation which can explain results of carcinogenicity and mutagenicity experiments; NDEA-F6 and NDBA-F14 were practically not metabolized by microsomal fractions, even though no decrease in binding affinity to cytochrome P450 was observed. Both compounds were not biologically active and were exhaled unchanged in high proportions after oral administration to the rat. Biologically active analogues, NDEA, NDBA, NDBA-F3 and NDBA-F6, were found to be dealkylated at the unfluorinated alkyl chains and, to a lesser extent, at the omega-fluorinated alkyl chains. Detection of corresponding alcohols as hydrolysis products confirmed the generation of electrophilic intermediates by alpha-C-hydroxylation. However, trifluorethanol was detected only in very small proportions from dealkylation of NDEA-F3, although dealkylation occurred almost exclusively at the unfluorinated site.     

Cell type-specific differences in metabolic activation of N-nitrosodiethylamine by human lung cancer cell lines

The metabolism of N-nitrosodiethylamine (NDEA) and its modulation by inhibitors of cytochrome P450 and prostaglandin H synthetase enzymes was investigated in seven well-differentiated early-passage human lung cancer cell lines. NDEA metabolism was assessed by covalent binding and evolution of carbon dioxide. Morphological diagnosis of cell lines was done by light and electron microscopy. Two cell lines (NCI-H69, NCI-H128) with characteristics of small-cell cancer did not metabolize NDEA. Two cells lines (NCI-H322) with features of adenocarcinoma, comprised of Clara cells, and (NCI-H727), with features of pulmonary endocrine cells, were more potent than all other cell lines in metabolizing NDEA. A cell line divided from an adenocarcinoma but comprised of alveolar type-II cells (NCI-H358) metabolized NDEA predominantly via prostaglandin H synthetase. Similarly, several cell lines with features of well-differentiated pulmonary endocrine cells (NCI-H727, NCI-H460) metabolized NDEA via prostaglandin H synthetase, while the cell line comprised of Clara cells (NCI-H322) activated the nitrosamine by cytochrome P450 but not by prostaglandin H synthetase. Although cancer cells may react differently from normal cells to xenobiotics, our data provide substantial evidence for the hypothesis that--as in the hamster--Clara cells and pulmonary endocrine cells are potential major targets of NDEA carcinogenesis in human lung. It is of particular interest that different cell types activate the nitrosamine via different enzyme systems.