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Formation of 7-(2-oxoethyl) guanine from lipid peroxidation and vinyl chloride exposure in male sprague dawley rats.
Citation:
Mutlu, E., Y. Jeong, L. Colllins, A. L. Ham, P. Upton, G. E. Hatch, D. W. WINSETT, P. A. EVANSKY, AND J. A. SWENBERG. Formation of 7-(2-oxoethyl) guanine from lipid peroxidation and vinyl chloride exposure in male sprague dawley rats. Presented at North Carolina Society of Toxicology Meeting Presidents Award (PARC), RTP, NC, September 22, 2011.
Impact/Purpose:
This abstract represents pre-EPA work of a postdoctoral fellow. Its contents are relevant to EPa efforts in carcinogenesis.
Description:
With a development of a new sensitive LC-MS/MS method to analyze 7-(2-oxoethylguanine) (7OEG), we confirmed and differentiated 7-0EG DNA adduct formation endogenously from lipid peroxidation and exogenously from Vinyl Chloride (VC) exposure. VC is an industrial chemical that is known to be carcinogenic to animals and humans. VC primarily induces hepatic angiosarcomas following high exposures (≥50 ppm). Other than occupational exposure and smoking, VC is also found in Superfund sites at ppb concentrations as a result of microbial metabolism of trichloroethylene and perchloroethylene. VC is metabolically activated to chloroethylene oxide (CEO) by cytochrome P450 2E1, then covalently binds to DNA and induces four major DNA adducts. We developed a new sensitive LC-MS/MS method to analyze 7-(2-oxoethylguanine) (7-0EG). We used this method to analyze tissue DNAs from both adult and weanling rats exposed to 1100 ppm [13C2]-VC for 5 days. After neutral thermal hydrolysis of sample DNA, the 7-0EG was derivatized with o-τtbutylhydroxylamine to an oxime adduct, followed by LC-MS/MS analysis. The limit of detection is 1 fmol and limit of quantitation is 1.5 fmol on column. The use of stable isotope VC allowed us to discern VC-induced 7-0EG from endogenous DNA adducts. In addition, we report for the first time that endogenous 7-0EG was present in tissue DNA [Figure 1]. The preliminary data for the formation of [13C2]-7-0EG from the reaction of calf thymus DNA (CtDNA) with [13C18]-ethyl linoleate (EtLa) under peroxidizing conditions supports our hypothesis that endogenous 7-0EG occurs from lipid peroxidation. The reaction was carried out according to previously published methods with minor modifications [1]. The amounts of endogenous 7-0EG in liver, lung, kidney, spleen, testis and brain DNA from adult and weanling rats typically ranged from 0.1-1.0 adducts/IO5 guanine [Table 1]. The number of exogenous 7-0EG in liver DNA of adult rats exposed to 1100 ppm [13C2]-VC for 5 days was 10.4±2.3 adducts/105 guanine (n=4), while exogenous [13C2]-7-0EG concentrations in other tissues ranged from 0.1-3.9 adducts/l05 guanine (n=4). Due to reactivity and short half life of CEO, we hypothesize that VC is metabolized to CEO in each organ by P450, rather than arising via the circulation of CEO in the body. The lower exogenous adduct level in brain, spleen and testis support this hypothesis. While number of endogenous 7-0EG adducts in these [13C2]-7-0EG tissues of weanling rats were similar to those of adult rats, exogenous concentrations were higher in weanlings, with liver averaging 30 adducts/105 guanine. Studies on the persistence of [13C2]-7-0EG in adult rats sacrificed 2, 4, or 8 wks post exposure to [13C2]-VC accurately determined the half-life of 7-0EG in liver and lung to be 4 days for both tissues. The data in this study suggest that not only liver but other organs such as lung and kidney might be considered target organs for VC-induced genotoxicities following inhalation exposure. (Abstract does not represent EPA policy).