Covalent binding of inhaled formaldehyde to DNA in the nasal mucosa of Fischer 344 rats: analysis of formaldehyde and DNA by high-performance liquid chromatography and provisional pharmacokinetic interpretation

Abstract

Inhalation of 3HCHO and H14CHO (6 ppm, 6 hr) resulted in the formation of DNA-protein crosslinks in the rat nasal respiratory mucosa. The DNA was extracted and was fractionated into aqueous (AQ) and interfacial (IF) portions. AQ DNA and IF DNA were enzymatically hydrolyzed to deoxyribonucleosides in Tris buffer and analyzed by HPLC with liquid scintillation counting (LSC). HCHO was bound exclusively to the IF DNA, indicating that the HCHO was bound as DNA-protein crosslinks. Hydrolysis of the DNA quantitatively released the HCHO; no evidence was obtained for the formation of hydroxymethyl adducts. An adduct detected previously following incubation of mammalian cells with HCHO, N6-hydroxymethyldeoxyadenosine (hm6dA) [Beland, F.A., Fullerton, N.F., and Heflich, R.H. (1984) J. Chromatogr. 308, 121-131], was shown to be produced by reaction of HCHO with deoxyadenosine (dA) in bis-Tris buffer under conditions similar to those used for hydrolysis of the DNA. This reaction does not occur in Tris buffer. Evidence was obtained that most or all of the hm6dA observed can be explained by this reaction. Based on these results, an improved method to determine the amount of H14CHO bound to DNA was developed: the DNA is hydrolyzed in Tris buffer and analyzed by HPLC, and the released H14CHO is derivatized with dimedone and quantitated by LSC. Rats were exposed to a wide range of H14CHO concentrations (0.3, 0.7, 2, 6, or 10 ppm; 6 hr). DNA-protein crosslinking occurred at all concentrations. The formation of crosslinks was interpreted in terms of a nonlinear pharmacokinetic model incorporating oxidation of inhaled HCHO as a defense mechanism. The slope of the fitted concentration-response curve at 10 ppm is 7.3-fold greater than at 0.3 ppm, and the detoxication pathway is half-saturated at an airborne concentration of 2.6 ppm.