Assessing the effect of N-oxidation on the mutagenicity of 1-pyrazolines using the Ames assay

Abstract

N-Nitrosamines are well known as environmental carcinogens. We have reported that N-nitroso-N-methylbutylamine was oxidized by Fe²⁺-Cu²⁺-H₂O₂ to 5-methyl-5-nitro-1-pyrazoline, a direct-acting N-oxide. 1-Pyrazolines have not been reported to exhibit genotoxicity. In this study, we investigated the effect of N-oxidation on the mutagenicity of 1-pyrazolines using the Ames assay. The mutagenicity of 5-alkyl-5-nitro-1-pyrazoline 1-oxide (1a; methyl, 1b; ethyl), the N-oxide isomer (3-alkyl-3-nitro-1-pyrazoline 1-oxide; 2a; methyl, 2b; ethyl), and the corresponding nonoxides (3-alkyl-3-nitro-1-pyrazoline; 3a; methyl, 3b; ethyl) was assayed in Salmonella typhimurium TA1535 and Escherichia coli WP2uvrA. The ratios of mutagenic potency in S. typhimurium TA1535 versus E. coli WP2uvrA were compared with those of N-alkylnitrosoureas. To predict the reaction site on the pyrazolines with nucleophiles, the electron density of the pyrazolines was obtained by theoretical calculations. The pyrazolines were mutagenic in S. typhimurium TA1535 and E. coli WP2uvrA. The ratio of S. typhimurium TA1535 to E. coli WP2uvrA 1a (87:13) or 1b (90:10) was similar to that of N-ethyl-N-nitrosourea (70:30). In contrast, the mutagenic ratio of 2a (22:78) or 2b (52:48) was similar to that of N-propyl-N-nitrosourea (48:52) or N-butyl-N-nitrosourea (14:86). The ratio of 3a (53:47) or 3b (54:46) was similar to that of N-propyl-N-nitrosourea or N-butyl-N-nitrosourea. The pyrazolines exhibit genotoxicity, and the mutagenic potency of the 1-pyrazolines is influenced by N-oxidation. We estimated that the mutagenicity of 1a or 1b was caused by DNA ethylation, and the isomers or the nonoxides were mutagenic via formation of alkylated DNA, which contains an alkyl chain longer than the propyl.