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Whereas nitrosation of secondary amines produces nitrosamines, amino acids with primary amino groups and glycine ethyl ester were reported to react with nitrite to give unidentified agents that alkylated 4-(p-nitrobenzyl)pyridine to produce purple dyes and be direct mutagens in the Ames test. We report here that treatment of glycine ethyl ester at 37 °C with excess nitrite acidified with HCl, followed by ether extraction, gave 30−40% yields of a product identified as ethyl chloro(hydroximino)acetate [ClC(&dbd;NOH)COOEt, ECHA] and a 9% yield of ethyl chloroacetate. The ECHA was identical to that synthesized by a known method from ethyl acetoacetate, strongly alkylated nitrobenzylpyridine, and may have arisen by N-nitrosation of glycine ethyl ester to give ethyl diazoacetate, which was C-nitrosated and reacted with chloride to give ECHA. Nitrosation of ethyl diazoacetate also yielded ECHA. Ethyl nitroacetate was not an intermediate as its nitrosation did not produce ECHA. ECHA reacted with aniline to give ethyl (hydroxamino)(phenylimino)acetate [PhN&dbd;C(NHOH)CO2Et]. This product was different from ethyl [(phenylamino)carbonyl]carbamate [PhNHC(&dbd;O)NHCO2Et], which was synthesized by reacting ethyl isocyanatoformate (OCN·CO2Et) with aniline. ECHA reacted with guanosine to give a derivative, which may have been a guanine-C(&dbd;NOH)CO2Et derivative. ECHA showed moderate toxicity and weak but significant mutagenicity without activation in Salmonella typhimurium TA-100 (mean, 1.31 × control value for 12−18 μg/plats) and for V79 mammalian cells (1.5−1.7 × control value for 60−100 μM). In conclusion, gastric nitrosation of glycine derivatives such as peptides with a N-terminal glycine might produce ECHA analogues that alkylate bases of gastric mucosal DNA and thereby initiate gastric cancer.

Arylhydrazides, arylhydrazines, and N-alkyl-N-arylnitrosamines are metabolized to arenediazonium ions which yield C⁸-arylpurine adducts in calf thymus and cellular DNA. The mechanism of adduct formation has not been fully elucidated. C⁸-Arylguanine adducts likely form from direct aryl radical (Ar^•) addition to the C⁸ position of guanine. However, the amounts of C⁸-aryladenine adducts measured here are inconsistent with direct radical attack at the C⁸ position of adenine. An intermediate product, an aryltriazene, is likely formed which then decomposes to the C⁸-aryladenine adduct. We have demonstrated that N¹-aryl-N³-purinyltriazene adducts are formed from a variety of para-substituted arenediazonium ions with adenine. Decomposition of the N¹-aryl-N³-purinyltriazene, at high pH and elevated temperatures, has been shown to give C⁸-aryladenine derivatives, and a free radical mechanism for this process has been proposed. Here we show that this process can occur under physiological conditions and that the C⁸-aryladenine adduct can be quantitated by HPLC. ESR studies, in which DMPO was used as a spin trap, have been used to demonstrate the intermediacy of aryl radicals during the decomposition of the N¹-aryl-N³-purinyltriazenes and to demonstrate that this process also occurs in calf thymus (ct) DNA treated with arenediazonium ions. These results suggest the involvement of an aryl radical in the formation of the observed DNA adducts. Finally, we have found that the treatment of ct DNA with arenediazonium ions produces a significant amount of depurination. Both the formation of C⁸-arylguanine and C⁸-aryladenine adducts and the generation of apurinic sites may contribute to the genotoxicity of arylhydrazides, arylhydrazines, N-alkyl-N-arylnitrosamines, and arenediazonium ions.

Many arylhydrazines are genotoxins, although the mechanism of their genotoxicity is unknown. Previous studies have shown that arylhydrazines are metabolized to arenediazonium ions, which produce C8-arylguanine adducts in DNA suggesting the intermediacy of an aryl radical. Here we have looked for the formation of aryl radicals from arylhydrazines and microsomes by ESR spin trapping. Only hydroxyl radicals are trapped upon incubation of p-methylphenylhydrazine with rat liver microsomes and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, hydroxyl and aryl radicals were trapped upon incubation of p-(methoxymethyl)phenylhydrazine with rat liver microsomes. Evidence for hydroperoxyl radical formation was also obtained. In contrast, when either of these substrates was incubated with microsomes from C5O cells, aryl and hydroxyl radicals were trapped. The ESR signal intensity of the spin-trapped aryl radicals parallels the extent of C8-arylguanine formation in DNA, and therefore, the aryl radical is likely the intermediate responsible for C8-arylguanine adduct formation. Aryl radicals and C8-arylguanine adducts may be related to the genotoxicity of arylhydrazines and related compounds that are oxidatively metabolized to arenediazonium ions, the precursor to aryl radicals, including arylalkyl nitrosamines, arylazo compounds, and triazenes.

The activation of 3H-labeled N-nitrosobis(2-oxopropyl)amine ([3H]BOP) by pancreas acinar and duct tissue from Syrian hamsters and MRC-Wistar rats in vitro was measured as DNA alkylation. Hamster tissue was incubated with [3H]BOP (0.1 mM; 20 µCi/ml) for 2 h. Initial levels of alkylation were similar, 41.7±3.7 (acinar) and 51.5±7.8 (duct) dpm/µg DNA. Alkylation persisted for longer in duct (t/2>46 h) than in acinar tissue (t/2=6 h). The faster repair of alkylation in acinar tissue was not due to acinar cell death. In rat duct tissue the level of alkylation 2 h after incubation (38.9±4.5 dpm/µg DNA) was similar to that in hamster ducts but declined more rapidly (t/2=27 h). Hamster and rat acinar and duct tissue was incubated with BOP followed by [3H]thymidine to measure DNA synthesis. BOP stimulated DNA synthesis in hamster but not in rat duct tissue or hamster acinar tissue. These data support the hypothesis that the duct tissue is the target tissue for BOP in Syrian hamsters.