Detection of phase specificity of in vivo germ cell mutagens in an in vitro germ cell system

Abstract

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...

In vitro responses to known in vivo genotoxic agents in mouse germ cells

“…For spermatocyte fractions, it was 88–90% and 88–92% for the spermatid fractions. The results also were confirmed by Western blotting for phase‐specific markers exactly as described previously [Habas et al, ].…”

“…MMS has also been found to induce a high incidence of dominant lethal mutations in spermatids [Ehling, ]. Our previous study [Habas et al, ], using the Comet assay in rat, also showed that ENU produced a high level of DNA damage in spermatogonia incurring significantly greater DNA damage. Spermatocytes were most sensitive to 6‐MP while spermatids were particularly susceptible to MMS [Habas et al, ].…”

“…Mutant frequencies in male rat germ cells were determined after exposure to ENU, MNU, 6MP, 5BrdU, MMS, and EMS and thus have been reported to cause specific DNA damage in isolated germ cells from adult rat testis [Ehling et al, ; Anderson et al, ; Russell et al, ; Levkoff et al, ; Kanemitsu et al, ; Habas et al, ]. To examine species differences in the DNA damage between rats and mice three male germ cell mutagens (ENU, 6‐MP and MMS) were selected for testing.…”

“…Approximately 2 × 10 5 germ cells were suspended in Eppendorf® tubes and incubated with ENU, 6‐MP and MMS at final concentrations of 0, 0.05, 0.5, and 1 mM, and grown in 5% CO 2 and 95% air at 37°C. Cell debris was removed and cells remaining in the plates from each treatment were harvested by centrifugation and then used for the examination of DNA damage using the Comet assay as described previously [Habas et al, ]. Olive tail moment (OTM) and % tail DNA were measured, calculated, quantified and expressed (fold of control) in mean ± S.D ( n = 3) for isolated germ cells using the (Comet 6.0; Andor Technology, formerly Kinetic Imaging) software image analysis system, Belfast, UK.…”

“…Our previous study [Habas et al, ], using the Comet assay in rat, also showed that ENU produced a high level of DNA damage in spermatogonia incurring significantly greater DNA damage. Spermatocytes were most sensitive to 6‐MP while spermatids were particularly susceptible to MMS [Habas et al, ]. This present study aimed to assess the phase specificity of the susceptibility of spermiogenic germ cells in mice to genetic damage induced by ENU, 6‐MP and MMS in vitro .…”