An automated method was developed to measure excised methyl adenine (mA) and methyl guanine (mG) DNA adducts in urine obtained from workers exposed to nitrosamines, such as nitrosodimethylamine. Workers were categorized into 5 groups, I through V, with category I working in closest proximity as described in a recent NIOSH Health Hazard Evaluation Report (HETA 94-0072-2648). First void urine taken on the morning after exposure was frozen. A Benchmate robotic workstation was used for Solid Phase Extraction (SPE) processing of urine. Deuterated 3-mA ([(13) C-d3]-N3mA) for use as an internal standard, and radiolabeled [(14)C]-N3mA for use as a tracer, were synthesized. Urine was filtered to remove exfoliated cells, adjusted to pH 8, and refiltered. The Benchmate workstation was programmed to tare sample tubes, add internal standards (2.5 ng [(13)C-d3]N3mAC-d) to 3-mL urine, mix and determine sample density. Bond Elute 500 mg C8 SPE columns were conditioned with 3 mL MeOH:NH4OH (10:0.1 conc.) and 3 mL H2O pH 8. Urine was loaded onto the SPE columns, rinsed , sequentially with 1 mL H2O pH 8 and 1mL HC1 0.1 N, and >97% of mA and mG adducts were eluted in a l-mL fraction of 20% MeOH in HCI 0.1 N. Samples dried under vacuum were derivatized in 100 ul pyridine and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. Gas Chromatographic-Mass Spectrometric (GC-MS) analysis using a 25 meter X 0.2 mm I.D. Hewlett-Packard Ultra 2 column was accomplished using a Hewlett-Packard quadruple GC-MS system in the selected ion mode (SIM). Limits of quantitation (LOQ) for mA adducts ranged from 10-100 ng/mL and from 10-250 ng/mL for mG adducts. Levels of 2-, 3-, 6-, and 7-mA and 2-, 6-, and 7- mG were detected for workers tested, but intra-category variation was high. When only exposure category was considered, no significant difference was noted for the adducts tested. The results of the study showed that mA or mG urinary adducts can be rapidly quantified in a single assay using an automated system, but additional sample numbers may be required for further evaluation.
The Toxicologist. Society of Toxicology 37th Annual Meeting, March 1-5,1998, Seattle, Washington