Combined application of comprehensive analysis for DNA modification and reporter gene mutation assay to evaluate kidneys of gpt delta rats given madder color or its constituents

Abstract

DNA adductome analysis using liquid chromatography-tandem mass spectrometry is a promising tool to exhaustively search DNA modifications. Given that the molecular weight of chemical-specific adducts is determined by the total molecular weights of the active form and nucleotide bases, we developed a new method of comprehensive analysis for chemical-specific DNA adducts based on the principle of adductome analysis. The actual analytical mass range was 50 mass units up or down from the average molecular weight of the four DNA bases plus the molecular weight of the expected active form of the chemical. Using lucidin-3-O-primeveroside (LuP), lucidin-modified bases formed by its active form were exhaustively searched using this new method. Various DNA adducts, including Luc-N (2)-dG and Luc-N (6)-dA, were identified in the kidneys of rats given LuP. Together with measurement of 8-hydroxydeoxyguanosine (8-OHdG) levels, the combined application of this new method with a reporter gene mutation assay was performed to clarify renal carcinogenesis induced by madder color (MC) that includes LuP and alizarin (Alz) as constituent agents. A DNA adductome map derived from MC-treated rats was almost identical to that of LuP-treated rats, but not Alz-treated rats. Although 8-OHdG levels were elevated in MC- and Alz-treated rats, significant increases in gpt and Spi(-) mutant frequencies were observed only in MC- and LuP-treated rats. In addition, the spectrum of gpt mutants in MC-treated rats showed almost the same pattern as those in LuP-treated rats. The overall data suggest that LuP may be responsible for MC-induced carcinogenicity and that the proposed methodology is appropriate for exploring and understanding mechanisms of chemical carcinogenesis.