Search

N-Nitroso-N-methylaniline (NMA) is a strong oesophageal carcinogen in rats but exhibits few overt genotoxic effects. Previous work from our laboratory established that NMA is readily metabolized by cytochrome P450-catalysed N-demethylation to produce the benzenediazonium ion (BDI), a relatively stable but reactive electrophilic agent. We have also shown that BDI reacts with DNA to form an acid-labile adduct. We have now shown that BDI, generated chemically or by the metabolism of NMA in vitro, reacts with DNA to form a triazene coupling product at the N6-position of adenine residues. This adduct has also been shown to be produced in the liver DNA of rats treated with NMA. Procedures for the isolation of DNA and for analysis of the adduct are presented.

N-Nitroso-N-methylaniline (NMA) is an esophageal carcinogen in F344 rats. Attempts to detect binding of NMA to DNA or RNA have not been successful. NMA is not mutagenic in the standard Ames bacterial assay, and it did not induce sister chromatid exchanges in mammalian cells. NMA forms the benzenediazonium ion (BDI) during metabolism. This ion has been known to react with aromatic amines, such as adenine, to form triazene coupling products. The purpose of this research was to demonstrate that a triazene adduct, which would be expected to be hydrolytically unstable, was formed by coupling with the adenine residues in DNA. Liver DNA from a rat treated with NMA or from in vitro reactions of BDI with DNA was treated with sodium borohydride. This reaction was shown to result in the reduction of 6-(1-phenyltriazeno)purine to 6-hydrazinopurine (N6-aminoadenine). The hydrolysate of the DNA, presumably containing the hydrazine, was treated with 4-(dimethylamino)naphthaldehyde, and the resulting hydrazone was isolated by reverse-phase HPLC using fluorescence detection. The identity of the adduct was demonstrated by high-resolution mass spectrometry. These data suggest strongly that NMA forms an unstable triazene adduct with adenine in DNA both in vitro and in vivo.

The principal pathway of nitrosamine metabolism has long been considered to be alpha-hydroxylation. For N-nitrosodialkylamines, this hypothesis requires that a molecule of molecular nitrogen be released for every molecule of nitrosamine that is alpha-hydroxylated. Thus, the quantitative determination of nitrogen formation should provide a measure of the importance of this pathway. This method was applied earlier to the doubly-labelled nitrogen-15 compounds, N-nitrosodimethylamine (NDMA), N-nitrosomethylphenylamine (NMPhA) and N-methyl-N-nitrosourea (MNU), using both a 9 000 X g supernatant fraction of liver and the intact animal as metabolic systems. The in-vitro results were quite different from those obtained in vivo. The majority of the NDMA (67%) and the MNU (88%) were converted to nitrogen in vivo, while NMPhA gave considerably less nitrogen (52%). These results differed by a factor of approximately two from those obtained in vitro (NDMA, 33%; NMPhA, 18.8% and MNU, 96%). Since such differences may be a result of the loss of cellular architecture, we have extended the work to include isolated hepatocytes. It had been shown previously that isolated hepatocytes constitute a practical alternative to in-vivo systems, even though the correlation with in-vivo metabolism appears to depend on the substrate analysed. The values obtained using this system (NDMA, 47%; NMPhA, 23%; and MNU 105%) reconfirm that metabolism may be substrate dependent. As in our previous studies, no mixed nitrogen (15N14N) or labelled nitrogen oxides were found. The data are all consistent with the hypothesis that at least one demethylase for each of the nitrosamine substrates is associated with a cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

The N-demethylation of 15N-labeled N-nitrosodimethylamine (DMN) and N-nitroso-N-methylaniline (NMA) by isolated rat hepatic cells has been investigated. The values obtained in this system for molecular nitrogen formed during metabolism, compared with substrate consumed, were DMN 47%, NMA 23%, and N-nitroso-N-methylurea (NMU) 105%. The results for DMN are roughly halfway between those previously determined with rat liver S-9 fraction in vitro (33%) and in vivo (67%). For NMA, the hepatocyte data are closer to those obtained from S-9 in vitro (19%), rather than the in vivo (52%). No mixed nitrogen ( 15N14N ) or labeled nitrogen oxides were found.

N-Nitroso-N-methylaniline (NMA) and N-nitroso-N-methyl-4-fluoroaniline (p-F-NMA), both non-mutagenic in Salmonella typhimurium and N-nitroso-N-methyl-4-nitroaniline (p-NO2-NMA), a potent mutagen, were tested for carcinogenicity in F344 rats. NMA was shown to induce a high level of tumors in the upper gastrointestinal tract, particularly in the esophagus. Male rats treated with NMA died with tumors at a slightly higher rate than females, although the final tumor yield was the same. Most of the rats treated with p-F-NMA also developed tumors of the esophagus, but they died less rapidly than the NMA treated rats, indicating that p-F-NMA is a slightly weaker carcinogen than NMA. The powerful, directly acting mutagen, p-NO2-NMA did not appear to induce tumors at all since its tumor spectrum was essentially identical to that of the untreated control rats. Thus, the carcinogenic activities of NMA and its substituted analogs do not appear to correlate with bacterial mutagenesis assays. Additionally, NMA, p-F-NMA and N-nitroso-N-methyl-4-bromoaniline, the last a strong mutagen in S. typhimurium, were shown not to induce sister chromatid exchanges in CHO cells and in a clone of a CHO:liver cell hybrid which had previously been shown to be sensitive to chemical agents which require metabolic activation.

Evolution of 15N2-labeled molecular nitrogen was used to gauge the extent of alpha-hydroxylation during rat liver homogenate metabolism of doubly 15N-labeled N-nitrosodimethylamine (DMN) and N-nitrosomethylaniline (NMA). These measurements were correlated with the extent of total metabolism as measured by the disappearance of the nitrosamines and by the formation of formaldehyde. The results indicate that approximately 34% of DMN and 19% of NMA were metabolized by the alpha-hydroxylation pathway. Positive controls utilizing doubly 15N-labeled N-nitroso-N-methylurea yielded 96% of labeled nitrogen. These results are in variance with previously published data which claimed that either less than 5% or about 100% of DMN is metabolized by that route in vitro. Formaldehyde formation was shown to be a poor measure of the extent of metabolism. Semicarbazide gave rise to both formaldehyde and nitrogen, which makes it an undesirable component of the in vitro metabolism mixtures, particularly when those two substances are being measured.

N-nitrosomethylaniline, an esophageal carcinogen in the rat, was inactive in the Ames mutagenicity assay under all conditions examined. However, various ring-substituted N-nitrosomethylanilines were found to be mutagenic; some of these did not require activation. No correlation could be found between the rate of demethylation or substituent effects of the ring-substituted N-nitrosomethylanilines and mutagenic potency. The protein concentration and levels of aryl hydrocarbon hydroxylase in the liver S9 fraction were determined to be poor indices of the ability of S9 to activate nitrosamines to their mutagenic metabolites.

beta-Hydroxynitrosamines appear to be refractory to alpha-oxidation, the common pathway of metabolism of simple dialkylnitrosamines. Some years ago, we postulated that nitrosamines bearing a hydroxyl in the beta position may be activated to alkylating agents by metabolic transformation to sulfate conjugates. Recent evidence has provided support for this hypothesis. A sulfate ester of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (NHPOPA) has been found in the urine of hamsters treated with the nitrosamine. It has also been found that inhibition of sulfotransferases inhibited the development of DNA single-strand breaks in livers of rats treated with several beta-hydroxy-nitrosamines. Alkylation of rat liver DNA in vivo by N-nitroso(2-hydroxyethyl)methylamine (NHEMA) favoured methylation over 2-hydroxyethylation by a factor of 10. The methylation reaction was inhibited by sulfotransferase inhibitors. Thus, sulfation appears to be an important pathway for activation of beta-hydroxy-nitrosamines. There are, however, other pathways, such as the oxidation of the beta-hydroxyl group to a carbonyl, which may also result in the formation of electrophilic species capable of modifying cellular macromolecules.

The metabolism of the non-carcinogenic N-nitrosoproline (NPRO) was investigated in vitro using both S9 preparations and isolated hepatocytes from F344 rats. The studies were performed using 15N-labeled nitrosamine and the reaction mixtures were examined mass spectrometrically for the presence of 15N2 or other 15N-labeled gaseous products. In addition, the metabolism of NPRO was monitored by capillary gas chromatography. The results indicated no 15N2 production from either the hepatocyte or S9 preparations, as well as no detectable loss of substrate from the reaction mixtures. Mass spectrometric analysis failed to reveal any metabolites of NPRO. The results suggest that NPRO may be refractory to the normal nitrosamine activating enzymes, confirming its suitability for use in human epidemiological studies of endogenous nitrosation.

The in vivo alkylation of DNA by N-nitrosomethyl-(2-hydroxyethyl)amine (NMHEA) was examined in male and female F-344/N rats. NMHEA is a strong hepatocarcinogen in female rats when administered by gavage but a weaker hepatocarcinogen in male rats. Groups of 5 rats of each sex were treated by gavage with various doses of NMHEA dissolved in corn oil. After 4 h the animals were sacrificed and the livers, lungs, and kidneys were removed. The DNA from each liver was isolated and the neutral thermal and mild acid hydrolysates were separated by high-performance liquid chromatography. The alkylated guanines were quantified by fluorescence spectroscopy. NMHEA gives rise to four fluorescent alkylated guanines, 7- and O6-methylguanines, and 7- and O6-hydroxyethylguanines. The dose-response data revealed that all four lesions increased with dose. There was approximately 10x more methylation than hydroxyethylation at the 7 position of guanine. There was less O6 alkylation, but both methylation and hydroxyethylation were observed at all of the doses studied. The overall alkylation was the same in males and females at the 10- and 20-mg/kg doses, but at higher doses the females exhibited significantly higher levels of alkylation than males. The level of alkylation of DNA isolated from non-target tissues, lung, and kidney was low. The persistence of these lesions in vivo was studied at a dose of 25 mg/kg. Groups of five animals each were sacrificed at various times from 0 to 96 h. There was no significant difference between the sexes in persistence of any of the lesions in the liver. The 7-alkylguanines disappeared slowly over the observation period. 7-Methylguanine was present at 30% of the maximum level after 96 h, while 7-hydroxyethylguanine appeared to be more stable. The O6-alkylguanines were removed rapidly from the liver, being at base level by 48 h. The rapid removal of O6-hydroxyethylguanine suggests a repair process independent of O6-alkylguanine-DNA guanine alkyl transferase: an excision repair is postulated. In vitro alkylation of calf thymus DNA by N-nitrosomethyl-(2-tosyloxyethyl)amine, a surrogate for the putative O-sulfate conjugate of NMHEA, resulted in exclusive methylation of DNA-guanine at both the 7 and O6 positions; no hydroxyethylation was detected. In vitro alkylation of calf thymus DNA with 2-hydroxyethyl-ethylnitrosourea resulted in exclusive hydroxyethylation of DNA-guanine at the 7 and O6 positions.(ABSTRACT TRUNCATED AT 400 WORDS)

The steady state kinetics and isotope effects were examined for demethylation of dimethylnitrosamine and phenylmethylnitrosamine, as well as their deuterated analogs, using the S-9 fraction, the microsomal pellet, and postmicrosomal supernatant from rat livers. The isotope effect (ratio of maximal rates for the deuterated and light substrates) using the S-9 from Long-Evans rat livers was found to be 1.82 for dimethylnitrosamine and 5.38 for phenylmethylnitrosamine. Phenobarbital was shown to induce dimethylnitrosamine demethylase activity in the microsomal pellet of both Long-Evans and Sprague-Dawley rats but to repress this activity in the postmicrosomal supernatant in the Long-Evans rats, while markedly increasing it in the Sprague-Dawley rats. It was also found that there was nitrosamine demethylase activity in the so-called "pH 5 enzymes" and in the supernatant from that preparation. The latter activity shows substantially different characteristics from that found in the other fractions.

The carcinogenic effects of sequential treatment of female F344 rats with two nitrosamines were studied. The animals received either methylethylnitrosamine (NMEA), a strong liver carcinogen, N-nitrosomethylaniline (NMA), a moderately strong esophageal carcinogen, or N-nitrosopyrrolidine, (NPyr), a weaker liver carcinogen. The sequentially treated groups were given NMEA followed by NMA and vice versa, NPyr followed by NMEA and vice versa. The dose and duration for each chemical in the sequentially treated groups were identical for the individual treatments. The animals were allowed to die or were killed when moribund. The animals surviving longer than 110 weeks were sacrificed. The NMEA-NPyr and NPyr-NMEA groups had a tumor spectrum characteristic for NMEA alone (a mixture of hepatic carcinomas and sarcomas with extensive metastases to the lungs). The survival was reduced in the NMEA-NPyr group compared to the NMEA alone group. The time to death of the NMA-NMEA group was not affected by the NMA treatment, but many of the animals had esophageal neoplasms. The NMEA-NMA group survival was reduced when compared to the NMEA alone group but the tumor spectrum was dominated by NMEA. The data indicate that when the target organ is the same, the effect of two nitrosamines is additive with the stronger carcinogen dominating the tumor spectrum. When the target organs are different, the initial exposure influences the tumor spectrum, although the treatment with the second nitrosamine enhances the tumorigenicity of the initial nitrosamine.