Hydroxylation and nitroreduction are required to activate dimethylnitramine into alkylating and mutagenic agents.

Dimethylnitramine (DMNO) was shown to undergo hydroxylation in the presence of 9000 g supernatant from rat liver (S9) to yield hydroxymethyl-methylnitramine (OH-MNO). OH-MNO displayed a 100-fold higher mutagenic activity in Salmonella typhimurium TA100 strain than DMNO, when compared on a molar basis. The mutagenicity of DMNO in TA100 strain in the presence of S9 paralleled the production of OH-MNO. Acetoxymethyl-methylnitramine (Ac-MNO) and methylnitramine (MNO), two synthetic derivatives of DMNO, were also investigated. Ac-MNO was found to be mutagenic in TA100 strain only in the presence of S9, probably through the release of OH-MNO catalysed by esterase(s); under similar conditions, MNO showed no mutagenicity or toxicity to TA100 strain. OH-MNO showed no alkylating activity towards nicotinamide. These findings implicate OH-MNO as a proximate mutagenic metabolite of DMNO. DMNO and Ac-MNO were found to be more mutagenic in a nitroreductase(s)-proficient (TA100) than in a deficient (TA100 NR) strain. After reduction of OH-MNO with Zn/NH4Cl, it yielded an agent(s) which alkylated nicotinamide. The latter results imply a reduction of the nitro group in OH-MNO to yield a hydroxylamino derivative as the ultimate (or penultimate) mutagenic metabolite. The enzymes and reactive intermediates that may be involved in the activation of DMNO are discussed.