22In vivo tests for male reproductive genotoxicity are time consuming, resource-23 intensive and their use should be minimised according to the principles of the 3Rs.
24Accordingly, we investigated the effects in vitro, of a variety of known, phase-specific 25 germ cell mutagens, i.e. pre-meiotic, meiotic, and post-meiotic genotoxins, on rat 26 spermatogenic cell types separated using Staput unit-gravity velocity sedimentation, 27 evaluating DNA damage using the Comet assay. N-ethyl-N-nitrosourea (ENU), N-28 methyl-N-nitrosourea (MNU) (spermatogenic phase), 6-mercaptopurine (6-MP) and 29 5-bromo-2'-deoxy-uridine (5-BrdU) (meiotic phase), methyl methanesulphonate
30(MMS) and ethyl methanesulphonate (EMS) (post-meiotic phase) were selected for 31 use as they are potent male rodent, germ cell mutagens in vivo. DNA damage was 32 detected directly using the Comet assay and indirectly using the TUNEL assay.
33Treatment of the isolated cells with ENU and MNU produced the greatest 34 concentration-related increase in DNA damage in spermatogonia. Spermatocytes
35were most sensitive to 6-MP and 5-BrdU while spermatids were particularly 36 susceptible to MMS and EMS. Increases were found when measuring both Olive tail 37 moment (OTM) and % tail DNA, but the greatest changes were in OTM. Parallel 38 results were found with the TUNEL assay, which showed highly significant, (Parodi et al., 2015; Tralau et al., 2012). A particular problem in germ 51 cell mutagenicity studies is the relative lack of suitable tools for detecting mutation 52 induction (Yauk et al., 2015). Historically, studies have utilized huge numbers of 53 animals in assays such as the morphological specific locus (MSL) test (Russell et al., 54 1979) and dominant lethal assay (Anderson et al., 1977), to reveal valuable 55 information about the relative sensitivities male germ cells at different phases of 56 development (phase-specificity). Nevertheless, there is still a general paucity of 57 information on how endogenous factors, for example genetic polymorphisms and 58 exogenous factors such as environmental-toxin exposure, affect the type of germ cell 59 mutations induced and the risk of their induction (Beal et al., 2012).
60In animal tests the rules of the 3 R's: Reduction, Refinement and Replacement 61 (Russell and Burch, 1959), should be applied in planning and performing 62 experiments (Flecknell, 2002). Currently, a variety of alternative animal techniques 63 for assessing the toxicity/genotoxicity of compounds have been developed (Jung et 64 al., 2015; Kandarova and Letasiova, 2011). STAPUT methods require far fewer 65 animals compared with traditional methods thus aiding reduction efforts. Since the 66 animals are not treated, this refines toxicological approaches. Therefore, because 67 the uses of STAPUT combine these advantages, its use in a novel toxicity testing 68 strategy could potentially replace some in vivo testing (Habas et al., 2014). The 69 present study is a first step in testing this idea.
4There is a growing cons...
