Oxidative DNA damage and apoptosis induced by metabolites of butylated hydroxytoluene

DNA damage by metabolites of a food additive, butylated hydroxytoluene (BHT), was investigated as a potential mechanism of carcinogenicity. The mechanism of DNA damage by 2,6-di-tert-butyl-p-benzoquinone (BHT-quinone), 2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-2,5-cyclohexadienone (BHT-OOH), and 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHT-CHO) in the presence of metal ions was investigated by using 32P-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. BHT-OOH caused DNA damage in the presence of Cu(II), whereas BHT-quinone and BHT-CHO did not. However, BHT-quinone did induce DNA damage in the presence of NADH and Cu(II). Bathocuproine inhibited Cu(II)-mediated DNA damage, indicating the participation of Cu(I) in the process. Catalase also inhibited DNA damage induced by BHT-quinone, but not that induced by BHT-OOH. The DNA cleavage pattern observed with BHT-quinone plus NADH was different from that seen with BHT-OOH. With BHT-quinone plus NADH, piperidine-labile sites could be generated at nucleotides other than adenine residue. BHT-OOH caused cleavage specifically at guanine residues. Pulsed field gel electrophoresis showed that BHT-OOH and BHT-quinone induced DNA strand breaks in cultured cells, whereas BHT-CHO did not. Both BHT-quinone and BHT-OOH induced internucleosomal DNA fragmentation, which is the characteristic of apoptosis. Furthermore, flow cytometry analysis revealed an increase of peroxides in cultured cells treated with BHT-OOH or BHT-quinone. These results suggest that BHT-OOH participates in oxidative DNA damage directly, whereas BHT-quinone causes DNA damage through H2O2 generation, which leads to internucleosomal DNA fragmentation.