Elevated 8-hydroxydeoxyguanosine in hepatic DNA of rats following exposure to peroxisome proliferators: relationship to mitochondrial alterations

Recent studies have indicated a lack of correlation betweenhepatic 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and thecarcinogenicity of peroxisome proliferators (PP) and suggestedthat DNA in intact hepatic nuclei may be insensitive to increasesin 8-OHdG resulting from PP exposure. The possibility that PP-inducedelevations in acyl CoA oxidase (ACO) activity might result inoxidative damage to mitochondrial DNA (mtDNA) was thereforeinvestigated by feeding male F344 rats the hepatocarcinogenicPP Wy-14,643 (Wy, 0.1% in the diet) for 3, 6, 11, or 22 weeks,or clofibric acid (CA, 0.5% in the diet) for 22 weeks. Followingthe respective PP exposures, hepatic peroxisomal acyl CoA oxidaseactivity was determined and DNA isolated from either mitochondriaor unfractionated liver homogenates and analysed for the presenceof 8-OHdG. PP treatment caused an increase in ACO activity (10-to 15-fold) at all time points examined and an increase of 8-OHdG(1.5- to 2-fold) in DNA isolated from unfractionated liver homogenatesfollowing PP treatment for 11 or 22 weeks. No increase of 8-OHdGin mtDNA was detected. However, quantitation of a PCR amplifiedregion from the D-loop of mtDNA demonstrated a 2- to 3-foldincrease in the relative amount of mtDNA in DNA isolated fromunfractionated liver homogenates following 3, 11, and 22 weeksexposure to Wy or CA (22 weeks only). In addition, a slightincrease in the mitochondrial volume density (1.4-fold) wasobserved in electron micrographs of liver samples from ratsexposed to Wy for 22 weeks. These results (i) demonstrate thatPP treatment, at levels which cause an increase in ACO activity,does not cause oxidative damage to mtDNA, and (ii) suggest thatone reason for the observed increase of 8-OHdG in DNA from unfractionatedliver homogenates may be an increase in the amount of mtDNApresent in these samples. Furthermore, these studies provideadditional evidence against a role of oxidative DNA damage,measured as 8-OHdG, in PP-induced rodent hepatocarcinogenesisand suggest that alterations in mitochondria or other effectsmay be more pertinent to PP-related carcinogenesis.