Assessment of in vivo genotoxicity of the rodent carcinogen furan: evaluation of DNA damage and induction of micronuclei in mouse splenocytes.
In recent years, several surveys have highlighted the presence of the rodent carcinogen furan in a variety of food items. Even though the evidence of carcinogenicity of furan is unequivocal, the underlying mechanism has not been fully elucidated. In particular, the role of genotoxicity in furan carcinogenicity is still not clear, even though this information is considered pivotal for the assessment of the risk posed by the presence of low doses of furan in food. In this work, the genotoxic potential of furan in vivo has been investigated in mice, under exposure conditions similar to those associated with cancer onset in the National Toxicology Program long-term bioassay. To this aim, male B6C3F1 mice were treated by gavage for 4 weeks with 2, 4, 8 and 15 mg furan/kg b.w./day. Spleen was selected as the target organ for genotoxicity assessment, in view of the capability of quiescent splenocytes to accumulate DNA damage induced by repeat dose exposure. The induction of primary DNA damage in splenocytes was evaluated by alkaline single-cell gel electrophoresis (comet assay) and by the immunofluorescence detection of foci of phosphorylated histone H2AX (gamma-H2AX). The presence of cross-links was probed in a modified comet assay, in which cells were irradiated in vitro with gamma-rays before electrophoresis. Chromosome damage was quantitated through the detection of micronuclei in mitogen-stimulated splenocytes using the cytokinesis-block method. Micronucleus induction was also assessed with a modified protocol, using the repair inhibitor 1-beta-arabinofuranosyl-cytosine to convert single-strand breaks in micronuclei. The results obtained show a significant (P < 0.01) increase of gamma-H2AX foci in mitogen-stimulated splenocytes of mice treated with 8 and 15 mg furan/kg b.w. and a statistically significant (P < 0.001) increases of micronuclei in binucleated splenocytes cultured in vitro. Conversely, no effect of in vivo exposure to furan was observed when freshly isolated quiescent splenocytes were analysed by immunofluorescence and in comet assays, both with standard and radiation-modified protocols. These results indicate that the in vivo exposure to furan gives rise to pre-mutagenic DNA damage in resting splenocytes, which remains undetectable until it is converted in frank lesions during the S-phase upon mitogen stimulation. The resulting DNA strand breaks are visualized by the increase in gamma-H2AX foci and may originate micronuclei at the subsequent mitosis.
Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy.
Paola
P
Leopardi
Eugenia
E
Cordelli
Paola
P
Villani
Tiziana Patrizia
TP
Cremona
Luigi
L
Conti
Gabriele
G
De Luca
Riccardo
R
Crebelli
D000818:Animals; D002273:Carcinogens / Q000633:toxicity*; D020552:Comet Assay; D004249:DNA Damage / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D005663:Furans / Q000633:toxicity*; D006657:Histones / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D008297:Male; D051379:Mice; D048629:Micronuclei, Chromosome-Defective / Q000187:drug effects; D013154:Spleen / Q000166:cytology / Q000187:drug effects
D002273:Carcinogens; D005663:Furans; D006657:Histones; C039281:furan; C475961:gamma-H2AX protein, mouse