Assessment of in vivo genotoxicity of the rodent carcinogen furan: evaluation of DNA damage and induction of micronuclei in mouse splenocytes

Abstract

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.

Evidence of the in vitro genotoxicity of methyl-pyrazole pesticides in human cells

“…The above data confirm that the H2AX assay may be more sensitive than the genotoxicity tests currently used [7,18,19,[43][44][45]. Moreover, we confirm that this assay is well adapted to screening of the genotoxicity of compounds in different human cell lines; potentially it could be used for high-throughput screening purposes [25,26].…”

Comparative potency approach based on H2AX assay for estimating the genotoxicity of polycyclic aromatic hydrocarbons

“…It was established recently by different teams that the γ-H2AX assay was more sensitive than the comet test, notably in vivo (Trouiller et al, 2009) but also in vitro (Ismail et al, 2007;Watters et al, 2009;Leopardi et al, 2010). Moreover, this assay was found to be more sensitive than the comet assay when we compare our results regarding PAHs genotoxicity with published data on the same cell line (Plazar et al, 2007;Winter et al, 2008;Tarantini et al, 2009).…”

Mutagenicity of furan in female Big Blue B6C3F1 mice

“…Furan induced chromosomal aberrations but no sister chromatid exchange in bone marrow of mice following intraperitoneal administration [2]. The detection of DNA strand-breaks in splenocytes following oral exposure of mice to carcinogenic doses of furan required cell division [26]. We observed atypical mitotic figures with some degree of abnormal arrangement of chromosomes and spindle fibers, such as lag-type mitosis with non-attached condensed chromatin, multipolar mitoses with atypical configuration of the equatorial plate, and hollow metaphase mitoses, as described previously [53,54].…”

“…Genotoxicity tests with furan have given mixed results, with many studies providing negative data [2,20,23–26]. However, furan exposure is associated with unique Hras1 mutations in liver tumors [27,28], strengthening support for at least a partial genotoxic mechanism for furan’s carcinogenic properties.…”