Perturbation of microRNA signalling by doxorubicin in spermatogonial, Leydig and Sertoli cell lines in vitro.

We have previously shown that in addition to its widely recognised cardiotoxicity, the chemotherapeutic doxorubicin (DOX) is able to induce transcriptional, microRNA (miRNA) and DNA methylation changes in the mouse testis. These changes perturb pathways involved in stress/cell death and survival and testicular function and lead to germ cell loss and reproductive organ damage. Here, we further investigated the differential miRNA expression induced by DOX in mouse spermatogonial (GC1), Leydig (TM3) and Sertoli (TM4) cell lines in vitro. We began by performing cell cycle analysis of the three mouse testicular cell lines to evaluate their sensitivity to DOX and thus select suitable doses for miRNA profiling. In keeping with our in vivo data, the spermatogonial cell line was the most sensitive, and the Sertoli cell line the most resistant to DOX-induced cell cycle arrest. We then further demonstrated that each cell line has a distinct miRNA profile, which is perturbed upon treatment with DOX. Pathway analysis identified changes in the miRNA-mediated regulation of specialised signalling at germ-Sertoli and Sertoli-Sertoli cell junctions following treatment with DOX. Amongst the most significant disease categories associated with DOX-induced miRNA expression were organismal injury and abnormalities, and reproductive system disease. This suggests that miRNAs play significant roles in both normal testicular function and DOX-induced testicular toxicity. Comparison of our in vitro and in vivo data highlights that in vitro cell models can provide valuable mechanistic information, which may also help facilitate the development of biomarkers of testicular toxicity and high-throughput in vitro screening methods to identify potential testicular toxicants.

Perturbation of epigenetic processes by doxorubicin in the mouse testis.

Epigenetic processes play a major role in normal mammalian development, particularly during gametogenesis and early embryogenesis. Thus, perturbation of epigenetic processes in the testis by xenobiotics could have a major impact on testicular function and fertility, and potentially affect the development and health of subsequent generations. There has been substantial research into the epigenetic toxicity of environmental exposures over the last decade. However, few studies have focussed on pharmaceutical drugs, which due to the nature of their use are typically found at much higher concentrations within exposed individuals than environmental chemicals. Here, we investigated genome-wide changes in testicular mRNA transcription, microRNA expression and DNA methylation to assess the contribution of epigenetic mechanisms to the testicular toxicity induced by doxorubicin (DOX) as a representative, widely used and well-characterised anti-cancer drug. We demonstrated that DOX is able to induce transcriptional, microRNA and DNA methylation changes, which perturb pathways involved in stress/cell death and survival and testicular function and lead to germ cell loss and reproductive organ damage. This identified potential novel mechanisms of DOX-induced testicular toxicity for further focussed investigations. Such work is required to fully assess the role of epigenetics in toxicity, determine whether single and/or multigenerational epigenetic toxicity is a real public health concern, and begin to develop and incorporate relevant epigenetic endpoints into regulatory toxicology.