Identification of N-2 as a metabolite of acetylhydrazine in the rat

In the pathogenesis of isoniazid-induced hepatic injury, cytochrome P450-dependent metabolic activation of the metabolite, acetylhydrazine (AcHz), is the crucial step. Exhalation of [C-14]-carbon dioxide has previously been used to quantify indirectly this pathway. In contrast, according to the current concept of AcHz bioactivation, molecular nitrogen is produced directly, but has not yet been identified. Here, we measured [N-15]-nitrogen and (CO2)-C-14 exhalation, after the administration of [N-15(2)]-[C-14]-AcHz, in rats. Laser magnetic resonance (LMR) spectroscopy, a new sensitive and specific technique for the measurement of N-15 and N-14 in gas samples, was used. To demonstrate the involvement of cytochrome P450, rats were treated with phenobarbital (PB) or PB + cobalt(II) chloride (CoCl2) (n = 3 in each group). Time-dependent N-15(2) exhalation differed significantly between treatment groups (p < 0.001). At 240 min, cumulative exhalation of N-15 was 1.92 +/- 0.43% (mean +/- SE) of the dose in the control group, 2.53 +/- 0.23% in the PB group, and 1.00 +/- 0.15% in the PB + CoCl2 group (p < 0.05 compared to controls, p < 0.01 compared to PB). Cumulative exhalation of (CO2)-C-14 in 24 h ranged from 15.1 to 21.9%, with no significant difference between treatment groups. In conclusion, N-2 is a metabolite of AcHz. N-2 formation reflects the cytochrome P450-mediated activation of AcHz and can be used as an index of this pathway. Generally, LMR spectroscopy is valuable for monitoring any N-2-liberating process in vivo.