Iron-induced oxidative DNA damage and its repair in primary rat hepatocyte culture

Iron-overload diseases frequently develop hepatocellular carcinoma. Thegenotoxic mechanism whereby iron is involved in hepatocarcinogenesis mightinvolve an oxidative process via the intermediate production of reactiveoxygen species. This was presently investigated by examining kinetics offormation and repair of DNA base lesions in primary rat hepatocyte culturessupplemented with the iron chelate, ferric nitrilotriacetate Fe-NTA (10 and100 microM). Seven DNA base oxidation products have been identified in DNAextracts by gas chromatography- mass spectrometry, which showed apredominance of oxidized-purines (8- oxo-guanine, xanthine, fapy-adenine,2-oxo-adenine) above oxidized pyrimidines (5-OHMe-uracil, 5-OH-uracil,5-OH-cytosine) in control cultures. All these DNA oxidation productsrevealed a significant dose- dependent increase at 4 to 48 h after Fe-NTAsupplementation, among which fapy-adenine showed the highest increase and5-OH-cytosine was the least prominent. Involvement of iron in thisoxidative process was established by a correlation between extent in DNAoxidation and intracellular level of toxic low molecular weight iron. DNAexcision- repair activity was estimated by release of DNA oxidationproducts in culture medium. All the seven DNA oxidation products weredetected in the medium of control cultures and showed basal repairactivity. This DNA repair activity was increased in a time- anddose-dependent fashion with Fe-NTA. Oxidized-pyrimidines, among which was5-OHMe-Uracil, were preferentially repaired, which explains the low levelsdetected in oxidized DNA. Since oxidized bases substantially differed fromone another in terms of excision rates from cellular DNA, specificexcision- repair enzymes might be involved. Our findings, however,demonstrate that even though DNA repair pathways were activated iniron-loaded hepatocyte cultures, these processes were not stimulated enoughto prevent an accumulation of highly mutagenic DNA oxidative products ingenomic DNA. The resulting genotoxic effect of Fe-NTA might be relevant inunderstanding the hepatocarcinogenic evolution of iron-overload diseases.