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ToxSci Advance Access published online on August 3, 2007
Toxicological Sciences, doi:10.1093/toxsci/kfm191
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© The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

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

E Cordelli*, AM Fresegna, A D'Alessio, P Eleuteri, M Spanò, F Pacchierotti and P Villani

Section of Toxicology and Biomedical Sciences, ENEA, 00123 Rome, Italy

* Corresponding author: Dr Eugenia Cordelli, BAS- Section of Toxicology and Biomedical Sciences, ENEA CR Casaccia, Via Anguillarese 301 - 00123 Rome, Italy, cordelli{at}casaccia.enea.it, tel +39 06 3048 4784, fax +39 06 3048 6559

Received May 23, 2007; revision received July 16, 2007; accepted July 23, 2007


  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 or melphalan and comet assay was conducted both with and without protein supplementation. No effect of methyl-methanesulfonate or melphalan 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.

Key Words: Comet assay; reproductive toxicology; bull sperm; ReProTect; in vitro toxicology; DNA damage.


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