The microbial PhIP metabolite 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3 ',2 ': 4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1) induces DNA damage, apoptosis and cell cycle arrest towards Caco-2 cells

Abstract 7-Hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3′,2′:4,5]imidazo[1,2- a ]pyrimidin-5-ium chloride (PhIP-M1) is a newly identified intestinal microbial metabolite from the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP). Although the mutagenic potential of the endogenous N-hydroxy PhIP derivate has been reported, the risks associated with PhIP-M1 have not yet been explored. In this work, the cytotoxic and genotoxic effects originating from PhIP-M1 were assessed in the epithelial intestinal Caco-2 cell line. PhIP-M1 significantly decreased in a time- and dose-dependent manner mitochondrial dehydrogenase activity and protein synthesis, with IC 50 values of, respectively, 180 ± 39.4 and 173 ± 20.3 μM after 24 h, and 33.8 ± 3.5 and 37.3 ± 10.9 μM after 72 h. Apoptosis within the concentration ranges of cytotoxicity was confirmed by morphological examination, DAPI nuclear staining and annexin V staining. PhIP-M1 provoked cell cycle arrest, characterized by a significant increase in the number of nucleoids in the G2/M phase. A dose-dependent increase in DNA damage, as quantified by the alkaline comet assay, was observed after 3 h in the 50–200 μM range. Because these PhIP-M1-induced genomic and cellular events may contribute to the carcinogenicity of PhIP, the potency of the colon microbiota to bioactivate PhIP must be included in future risk assessments.