Revealing DNA damage levels in rat testicular germ cells in vivo using an adapted version of the alkaline comet assay

Heritable mutations in male germ cells pose a critical risk to human health and future generations, however standardized methods for assessing germ cell genotoxicity remain limited. We refined the in vivo alkaline comet assay (proof-of-concept (Dirven et al. 2023); protocol (Olsen et al. 2024)) to detect DNA damage in testicular germ cells, with selective addressment of haploid spermatids and primary spermatocytes. Measurements of DNA damage (% Tail DNA) and DNA content (total fluorescence intensity) in individual comets were combined with visual comet identification to distinguish testicular comet populations based on differences in DNA content and appearance. To verify the method's functionality and reliability, DNA damage was assessed in rats exposed to the direct-acting, well-characterized genotoxicants X-rays and ethyl methanesulfonate across distinct testicular cell populations, alongside liver and blood. To minimize experimental variation, the protocol included stringent standardization of animal handling, tissue processing, and comet assay procedures. Both X-rays and EMS induced significant DNA damage in testicular germ cells, with comparable responses across testicular cell types and similar (X-rays) or higher levels observed in somatic tissues. The low inter-animal variability observed supports the robustness of the method. Importantly, inclusion of testicular germ cells in OECD test guideline 489 would provide a valuable tool for hazard identification and mutagenicity classification of chemicals under the Globally Harmonized System of Classification and Labelling of Chemicals. This versatile, sensitive, and resource-efficient assay enhances the assessment of male-mediated genetic risks and supports regulatory efforts to protect reproductive health and safeguard the genetic integrity of future generations through the use of safer chemicals.