Denitrification of the genotoxicant 2-nitropropane : relationship to its mechanism of toxicity

Abstract

1. The stabilities of the industrial chemical and constituent of cigarette smoke 2- nitropropane (2-NP) and its aci tautomer propane 2-nitronate (P2N) towards hepatic enzymes and proteins such as serum proteins and oxyhaemoglobin were investigated in vitro in biological (hepatocytes and subcellular liver fractions) and model systems (serum proteins, oxyhaemoglobin, methylene blue). 2. Denitrification of 2-NP, P2N and 2-deutero 2-nitropropane (²H-NP) occurred in murine hepatocytes significantly faster than in rat cells.For 2-NP the rates were 1271 ± 167 versus 820 ± 125 pmol nitrite × min⁻¹ × 10⁶ cells⁻¹. 3. A similar observation was made in microsomes, where 2-NP denitrification was 1460 ± 110 (mouse) versus 480 ± 80 pmol nitrite × min⁻¹ × mg protein⁻¹ (rat). 4. The major NO₂⁻-forming activity was found to be localized in the microsomal fraction. 5. Conversionof 2-NP into P2N, either chemically or enzymatically,was a prerequisite for rapid denitrification. 6. Serum proteins and oxyhaemoglobin proved to be capable of denitrifying P2N (198 ± 24 pmol nitrite × min⁻¹ × mg protein⁻¹ and 7·1 ± 1·0 nmol nitrite × min⁻¹ × nmol HbO₂⁻¹ respectively), but were much less active towards 2-NP (24 ± 2 pmol nitrite × min⁻¹ × mg protein⁻¹ and none respectively). 7. Methylene blue decomposed 2-NP and P2N at rates of 11 ± 3 and 192 ± 4 pmol nitrite × min⁻¹ × nmol methylene blue⁻¹ respectively. The dye also enhanced NO₂⁻ formation from P2N and 2-NP in the presence of hepatocytes or serum proteins, with a concomitant enhancement of both 2-NP and P2N toxicity. 8. The results presented report species differences in the denitrification rate of 2-NP and highlight the crucial nitro-aci tautomerism of 2-NP as a pivotal determinant of 2-NP toxicity.