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ReProComet: a new in vitro method to assess DNA damage in mammalian sperm.

TitoloReProComet: a new in vitro method to assess DNA damage in mammalian sperm.
Tipo di pubblicazioneArticolo su Rivista peer-reviewed
Anno di Pubblicazione2007
AutoriCordelli, Eugenia, Fresegna Anna Maria, D'Alessio Alessia, Eleuteri Patrizia, Spanò M., Pacchierotti Francesca, and Villani Paola
RivistaToxicol Sci
Volume99
Issue2
Paginazione545-52
Data di pubblicazione2007 Oct
Parole chiaveAnimals, Cattle, Comet Assay, DNA damage, Hela Cells, Humans, male, Methyl Methanesulfonate, Spermatozoa
Abstract

The increasing request of chemical safety assessment demands for the validation of alternative methods to reduce the resort to animal experimentation. Methods that evaluate reproductive toxicity are among those requiring the largest use of animals. Presently, no validated in vitro alternative exists for the assessment of reproductive toxicity. Mammalian sperm are sensitive targets of DNA-reactive chemicals, which form premutagenic adducts. Here, we propose a new method based on comet assay to detect DNA damage induced by potential germ cell mutagens in bull sperm available from assisted reproduction practices. In somatic cells, chemical-induced adducts can be revealed by comet assay that detects DNA breaks produced during adduct repair. Mature sperm, however, are devoid of repair enzymes, and adducts are processed only after fertilization. For this reason, comet assay is not sensitive to detect DNA lesions induced in sperm by most chemicals. To overcome such limitation, we developed a modified comet assay based on the addition of a protein extract from HeLa cells to agarose-embedded sperm on microscopic slides. To test the method, sperm were treated in vitro with methyl methanesulfonate (MMS) or melphalan (MLP) and comet assay was conducted both with and without protein supplementation. No effect of MMS or MLP was detected without protein supplementation; on the contrary, a clear-cut dose-dependent effect was measured after addition of the cell extract. These results represent a proof of concept of a novel in vitro mutagenicity test on sperm that could offer a promising approach to complement previously validated in vivo germ cell genotoxicity assays.

Note

cited By 18

URLhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-38449086588&doi=10.1093%2ftoxsci%2fkfm191&partnerID=40&md5=3ed77ad3230d88f77ac2b53f9a507a25
DOI10.1093/toxsci/kfm191
Alternate JournalToxicol. Sci.
Citation Key4916
PubMed ID17675332