Iron-induced oxidative DNA damage and its repair in primary rat hepatocyte culture.
Iron-overload diseases frequently develop hepatocellular carcinoma. The genotoxic mechanism whereby iron is involved in hepatocarcinogenesis might involve an oxidative process via the intermediate production of reactive oxygen species. This was presently investigated by examining kinetics of formation and repair of DNA base lesions in primary rat hepatocyte cultures supplemented with the iron chelate, ferric nitrilotriacetate Fe-NTA (10 and 100 microM). Seven DNA base oxidation products have been identified in DNA extracts by gas chromatography-mass spectrometry, which showed a predominance 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 products revealed a significant dose-dependent increase at 4 to 48 h after Fe-NTA supplementation, among which fapy-adenine showed the highest increase and 5-OH-cytosine was the least prominent. Involvement of iron in this oxidative process was established by a correlation between extent in DNA oxidation and intracellular level of toxic low molecular weight iron. DNA excision-repair activity was estimated by release of DNA oxidation products in culture medium. All the seven DNA oxidation products were detected in the medium of control cultures and showed basal repair activity. This DNA repair activity was increased in a time- and dose-dependent fashion with Fe-NTA. Oxidized-pyrimidines, among which was 5-OHMe-Uracil, were preferentially repaired, which explains the low levels detected in oxidized DNA. Since oxidized bases substantially differed from one another in terms of excision rates from cellular DNA, specific excision-repair enzymes might be involved. Our findings, however, demonstrate that even though DNA repair pathways were activated in iron-loaded hepatocyte cultures, these processes were not stimulated enough to prevent an accumulation of highly mutagenic DNA oxidative products in genomic DNA. The resulting genotoxic effect of Fe-NTA might be relevant in understanding the hepatocarcinogenic evolution of iron-overload diseases.
Abalea
V
V
INSERM U456, Groupe Détoxification Réparation Tissulaire, and Laboratoire de biologie cellulaire, UFR des Sciences Pharmaceutiques et Biologiques, Rennes, France.
Cillard
J
J
Dubos
M P
MP
Anger
J P
JP
Cillard
P
P
Morel
I
I
D000818:Animals; D004249:DNA Damage; D004260:DNA Repair; D007501:Iron / Q000493:pharmacokinetics / Q000494:pharmacology*; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D009153:Mutagens / Q000493:pharmacokinetics / Q000494:pharmacology*; D018384:Oxidative Stress; D051381:Rats; D017207:Rats, Sprague-Dawley
D009153:Mutagens; D007501:Iron