DNA adducts of formaldehyde as a biological monitoring marker

The purpose of this study was to demonstrate that modified deoxynucleosides from DNA adducts of human nasal epithelial cells (HNECs) can be utilized as selective markers of dose after exposure to formaldehyde (FA). The first task was to resolve the hydroxymethyl adducts of deoxyadenosine, deoxyguanosine, and deoxycytidine from their parent deoxynucleosides by high performance liquid chromatography (HPLC) and then to quantitate the modified deoxynucleosides with the appropriate detector (UV, electrochemical, or fluorescence) after incubating the pure deoxynucleosides with FA in vitro. The lower quantifiable limits (LQL) for UV detector at 254 nm for deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), thymidine (dT), N6-hydroxymethyl deoxyadenosine (N6-dA), N2-hydroxymethyl deoxyguanosine (N2-dG), and N4-hydroxymethyl deoxycytidine (N4-dC) were 11, 7.6, 12, 15, 10, 10, and 22 pmol, respectively. The electrochemical cell detector was specific for dG and N2-dG. The fluorescence detector allowed more sensitivity for the normal deoxynucleosides, but not for modified deoxynucleosides. The decomposition of N6-dA and N2-dG followed first order kinetics at room temperature and the half-life was 50.1+/-4.9 hours for the first 2 days and 21.0+/-1.1 hours for the first 20 hours, respectively after which equilibrium was attained. N6-dA and N2-dG were stable at 20°C for 2 and 1.5 weeks. The second task was to optimize the HPLC resolution after incubation of human placental DNA with FA relative to DNA and solvent blanks. Modified deoxynucleosides from human DNA were resolved from one another and enzyme interferences to produce a selective separation method for modified deoxynucleosides and normal deoxynucleosides for the first time. After 80 mug/mL human placental DNA reacted with 100 ppm FA, the abundance order of the modified deoxynucleosides was N6-dA (23 pmol) > N2-dG (10 pmol) > N4-dC (6.0 pmol). The final task was to detect and quantitate the adducts in human nasal epithelial cells (HNECs) exposed to FA. The optimized quantitative method was developed for hydroxymethyldeoxynucleosides of DNA in HNECs after exposure to FA. Viability of HNECs exposed to over 250 ppm FA for 24 hr decreased significantly. Amounts of 18 pmole N6-dA and 12 pmole N2-dG were quantified after 1.4 x 10 7 HNECs (106 mug DNA/200 muL) were exposed to 500 ppm (10 muL injection). The degree of N6-dA and N2-dG formation in HNECs show that DNA adducts are potential markers of FA dose for HNECs in vitro.