Isotope effects and their implications for the covalent binding of inhaled [ 3H]- and [ 14C]formaldehyde in the rat nasal mucosa

Abstract

DNA-protein crosslinks were formed in the nasal respiratory mucosa of Fischer-344 rats exposed for 3 hr to selected concentrations of [ 3H]- and [ 14C]formaldehyde ( 3HCHO and H 14CHO) ( M. Casanova and H. d'A. Heck (1987). Toxicol. Appl. Pharmacol. 89, 105–121). In rats depleted of glutathione (GSH) and exposed to 10 ppm of 3HCHO and H 14CHO, the 3H 14C ratio of the fraction of the DNA that was crosslinked to proteins was significantly (39 ± 6%) higher than that of the inhaled gas. This suggests an isotope effect, either on the formation of DNA-protein crosslinks by labeled HCHO or on the oxidation of labeled HCHO catalyzed by formaldehyde (FDH) or aldehyde dehydrogenase (AldDH). The possibility of an isotope effect on the formation of crosslinks was investigated using rat hepatic nuclei incubated with [ 3H]- and [ 14C]formaldehyde (0.1 m m, 37°C). A small (3.4 ± 0.9%) isotope effect was detected on this reaction, which slightly favored 3HCHO over H 14CHO in binding to DNA. The magnitude of this isotope effect cannot account for the high isotope ratio observed in the crosslinked DNA in vivo. The possibility of an isotope effect on the oxidation of 3HCHO and H 14CHO catalyzed by FDH was investigated using homogenates of the rat nasal mucosa incubated with [ 3H]- and [ 14C]formaldehyde at total formaldehyde concentrations ranging from 0.1 to 11 μ m, NAD + (1 m m), GSH (15 m m), and pyrazole (1 m m). The experiments showed that 3HCHO is oxidized significantly more slowly than H 14CHO under these conditions ( V max K m ( H 14CHO ) ÷ V max K m ( 3HCHO ) = 1.82 ± 0.11 ). A similar isotope effect was observed in the absence of GSH, presumably due to the oxidation of 3HCHO and H 14CHO catalyzed by AldDH. These results suggest that the residual (unoxidized) formaldehyde present in the nasal mucosa of rats exposed to [ 3H]- and [ 14C]formaldehyde may be “enriched” in 3HCHO relative to H 14CHO, which can bind to DNA resulting in an isotope ratio higher than that of the inhaled gas. The isotope effect on the oxidation of 3HCHO and H 14CHO suggests that previous estimates of the amount of HCHO covalently bound to nasal mucosal DNA ( M. Casanova-Schmitz, T. B. Starr, and H. d'A. Heck (1984). Toxicol. Appl. Pharmacol. 76, 26–44) may have been too large, especially at low airborne concentrations and that the shape of the concentration-response curve for DNA-protein crosslinking is more nonlinear than reported previously.