Graphene oxide (GO) is an common nanomaterial and has attracted unlimited interest in academia and industry due to its physical, chemical, and biological properties, as well as for its tremendous potential in applications in various fields, including nanomedicine. Whereas studies have evaluated the size-dependent cytotoxicity of GO in cancer cells, there have been no studies on the biological behavior of ultra-small graphene nanosheets in germ cells. To investigate, for the first time, the cyto- and geno- toxic effects of different sizes of GO in two different cell types, Leydig (TM3) and Sertoli (TM4) cells, we synthesized different sized GO nanosheets with an average size of 100 and 20 nm by a modification of Hummers’ method, and characterized them by various analytical techniques. Cell viability and proliferation assays showed significant size- and dose-dependent toxicity with GO-20 and GO-100. Interestingly, GO-20 induced significant loss of cell viability and cell proliferation, higher levels of leakage of lactate dehydrogenase (LDH) and reactive oxygen species (ROS) generation compared to GO-100. Both GO-100 and GO-20 induced significant loss of mitochondrial membrane potential (MMP) in TM3 and TM4 cells, which is a critical factor for ROS generation. Furthermore, GO-100 and GO-20 caused oxidative damage to DNA by increasing the levels of 8-oxo-dG, which is formed by direct attack of ROS on DNA; GO-100 and GO-20 upregulate various genes responsible for DNA damage and apoptosis. We found that phosphorylation levels of EGFR/AKT signaling molecules, which are related to cell survival and apoptosis, were significantly altered after GO-100 and GO-20 exposure. Our results showed that GO-20 has more potent toxic effects than GO-100, and that the loss of MMP and apoptosis are the main toxicity responses to GO-100 and GO-20 treatments, which likely occur due to EGFR/AKT pathway regulation. Collectively, our results suggest that both GO-100 and GO-20 exhibit size-dependent germ cell toxicity in male somatic cells, particularly TM3 cells, which seem to be more sensitive compared to TM4, which strongly suggests that applications of GO in commercial products must be carefully evaluated.
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