Low and High Resolution GC-MS Detection of N2, 3-Ethenoguanine in Livers of Vinyl Chloride Exposed Sprague-Dawley Rats and of Unexposed Mammals

Vinyl chloride (VC) is a known human and animal carcinogen requiring metabolic activation by cytochrome P450-dependent monooxygenases. The metabolic products of vinyl chloride, chloroethylene oxide and chloroacetaldehyde, react with deoxyribonucleic acids to form DNA adducts. The formation and persistence of DNA adducts have been previously reported in preweanling Sprague-Dawley rats and dams exposed to vinyl chloride by inhalation. In the present study, adult male Sprague-Dawley rats were exposed to 500 ppm VC by inhalation for 1, 2, 4, or 8 weeks (4 hrs/day, 5 days/week). The quantitation of N^2,3-ethenoguanine (ɛG) was accomplished by low resolution gas chromatography-negative ion chemical ionization-mass spectrometry (GC-MS) monitoring of the [M-181]" fragment of ɛG's dipentafluorobenzyl derivative, 3,5-PFB[2]-ɛG. ɛG concentrations, expressed as μmol adduct/μmol unmodified guanine, were 0.57±0.06, 1.3±0.54, 1.4±0.21, and 2.0±0.91 following 1, 2, 4, or 8 weeks of VC exposure, respectively. To verify the low resolution GC-MS, a high resolution GC-MS method was applied to provide both a highly sensitive and highly specific method for quantitating ɛG. Whereas low resolution could selectively monitor single ion masses of m/z 354±0.5 for PFB-ɛG and m/z 358±0.5 for PFB-[13Cc]ɛG, the high resolution method could detect exact masses (10,000 resolution power) of the ɛG analyte and its 13C-labeled analog, [13C4]-ɛG. The relative response ratios of the ɛG analyte to its internal standard, [13C4-ɛG, were comparable between low resolution and high resolution GC-MS. The concentrations of ɛG using high resolution GC-MS were 0.45±0.07, 1.1±0.61, 1.5±0.43, and 1.7±0.79 following 1, 2, 4, or 8 weeks of VC exposure, respectively. In addition, the amounts of endogenous ɛG were analyzed in liver DNA from untreated Sprague-Dawley rats (53-week old), Sprague-Dawley rats (8-week old), Fischer 344 rats, and human samples. The ɛG concentrations detected by low resolution were 0.27±0.07, 0.57±0.48, 0.16±0.02, and 0.56±0. 35 μmol eɛ/μmol guanine for Sprague-Dawley rats, Fischer 344 rats, and humans. Comparable levels (0.02[n=l], 0.32±0.17, 0.06±0.07, and 0.81±0.97μmol ɛG/μmolG) were measured by high resolution. While the sources of endogenous ɛG are presently unknown, the application of high resolution GC-MS to its detection left little doubt as to its identity. Since ɛG is one of the most promutagenic DNA adducts formed by exogenous alkylating agents, this endogenous DNA adduct may be of importance in human cancer. Our findings that ɛG increased in an exposure-related manner in DNA from rats exposed to vinyl chloride, and was also present in unexposed animals present important new data for VC-induced carcinogenesis.Master of Science in Public Healt