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

Covalent Binding of Inhaled Formaldehyde to DNA in the NasalMucosa of Fischer 344 Rats: Analysis of Formaldehyde and DNAby High-Performance Liquid Chromatography and Provisional PharmacokineticInterpretation. CASANOVA, M., DEYO, D. F., AND HECK, H. D'A.(1989). Fundam Appl. Toxicol. 12, 397–417. Inhalationof ³HCHO and H¹⁴CHO(6 ppm, 6 hr) resulted in the formation ofDNA-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 enzymaticallyhydrolyzed to deoxyribonucleosides in Tris buffer and analyzedby HPLC with liquid scintillation counting (LSC). HCHO was boundexclusively to the IF DNA, indicating that the HCHO was boundas DNA-protein crosslinks. Hydrolysis of the DNA quantitativelyreleased the HCHO; no evidence was obtained for the formationof hydroxymethyl adducts. An adduct detected previously followingincubation of mammalian cells with HCHO, N⁶-hydroxymethyldeoxyadenosine(hm6dA)Beland F. A., Fullerton, N. F., and Heflich, R. H. (1984) J.Chromartogr. 308 121–131], was shown to be produced byreaction of HCHO with deoxyadenosine (dA) in bis-Tris bufferunder conditions similar to those used for hydrolysis of theDNA. This reaction does not occur in Tris buffer. Evidence wasobtained that most or all of the hm6dA observed can be explainedby this reaction. Based on these results, an improved methodto determine the amount of H¹⁴CHO bound to DNA was developed:the DNA is hydrolyzed in Tris buffer and analyzed by HPLC, andthe released H¹⁴CHO is derivatized with dimedone and quantitatedby LSC. Rats were exposed to a wide range of H¹⁴CHO concentrations(0.3, 0.7, 2, 6, or 10 ppm; 6 hr). DNA-protein crosslinkingoccurred at all concentrations. The formation of crosslinkswas interpreted in terms of a nonlinear pharmacokinetic modelincorporating oxidation of inhaled HCHO as a defense mechanism.The slope of the fitted concentration-response curve at 10 ppmis 7.3-fold greater than at 0.3 ppm, and the fitted detoxicationpathway is half-saturated at an airborne concentration of 2.6ppm.