An assay to detect chemically induced DNA repair in rat spermatocytes

Abstract

An in vivo/in vitro DNA repair assay has been developed to quantitate chemically induced unscheduled DNA synthesis (UDS) in rat spermatocytes utilizing autoradiography. Male Fischer-344 rats were treated by i.p. injection or gavage with a variety of genotoxic agents dissolved in dimethyl sulfoxide, corn oil, or water. At selected times after treatment, spermatocytes were isolated by trypsin digestion of testes and cultured for 24 hr in the presence of 3H-thymidine. The direct-acting genotoxicants methyl methanesulfonate (MMS) and ethyl methanesulfonate and the chemotherapeutic agent cyclophosphamide (CPA) produced positive UDS responses in spermatocytes isolated 1 hr after i.p. injection. The UDS response evoked by either CPA or MMS was maximal within 1 hr after injection and declined rapidly thereafter to control levels. Other known genotoxicants--including dimethylnitrosamine, aflatoxin B1, 2-acetylaminofluorene, 2,6-dinitrotoluene, and 1,6-dinitropyrene--failed to induce UDS, even with routes of administration and at times of exposure known to produce a positive response in hepatocytes. This negative response is consistent with these genotoxicants lack of mutagenic effect in rodent germ cells. These results demonstrate that the in vivo/in vitro spermatocyte DNA repair assay may be useful as a predictive screen for germ cell mutagens. Moreover, by its compatibility with similar assays which utilize other tissues from the same treated animal, this assay permits assessment of the organ specificity of the genotoxic response.

Unscheduled DNA synthesis assay in mammalian spermatogenic cells: An update

“…The most extensively used is the unscheduled DNA synthesis (UDS) assay [Sega, 1974;Sotomayor et al, 1978Sotomayor et al, , 1979Sega and Sotomayor, 1982;Working and Butterworth, 1984]. Earlier in vitro studies set up the basis for the development of this assay ; Kofman-Alfaro and Gledhill and Darzynkiewicz, 1973].…”

“…UDS has not been detected in mature spermatids or sperm [Sega, 1974;Sotomayor et al, 1978Sotomayor et al, , 1979Tanaka and Katoh, 1979;Zbinden, 1980;Sega and Sotomayor, 1982;Working and Butterworth, 1984;Bentley and Working, 1988b]. Recently, UDS has been demonstrated in stem and early differentiating spermatogonia of mice after treatment with 1-ethyl-1-nitrosourea (ENU) and 1-methyl-1-nitrosourea (MNU) [Sotomayor et al, 1999a].…”

Culture of pachytene spermatocytes for analysis of meiosis

“…Nonetheless, we demonstrate here that spermatocytes in short-term cultures retain chromosomal pairing and synapsis and other typical morphological features of meiotic prophase as well as the normal subnuclear distribution of transcriptional activity. Much remains to be done to develop the appropriate conditions for execution of meiotic prophase events in vitro; however, as we show here, cultured the "in vivoiin vitro" DNA repair assay [Working and Butterworth, 1984;Working, 19891. Recently, meiosis has been reported to occur in vitro in cultures of immortalized germ cells [Hofmann et al, 19941, or in cultures using immortalized Sertoli cells [Rassoulzadegan et al, 19931; however, the cytogenetic events of meiosis have not been investigated in detail in these studies.…”

Role of epididymal inflammation in the induction of dominant lethal mutations in Fischer 344 rat sperm by methyl chloride.

“…First, the pattern of dominant lethality induced by MeCl was not typical of that produced by germ-cell mutagens known to act via a direct, genotoxic mechanism (14). Second, MeCl was known to be an extremely weak direct-acting mutagen, producing genotoxic damage in vitro only at concentrations far in excess of those which produced dominant lethal effects in vivo-e.g., 50,000 ppm for bacteria, >10,000 ppm for human lymphoblasts, and 30,000-50,000 ppm for rat spermatocytes (17)(18)(19). Third, MeCl exposure conditions that caused epididymal inflammation and dominant lethality did not induce DNA repair in spermatogenic cells in vivo (18,19).…”

“…Second, MeCl was known to be an extremely weak direct-acting mutagen, producing genotoxic damage in vitro only at concentrations far in excess of those which produced dominant lethal effects in vivo-e.g., 50,000 ppm for bacteria, >10,000 ppm for human lymphoblasts, and 30,000-50,000 ppm for rat spermatocytes (17)(18)(19). Third, MeCl exposure conditions that caused epididymal inflammation and dominant lethality did not induce DNA repair in spermatogenic cells in vivo (18,19). Fourth, MeCl did not alkylate mammalian DNA after in vivo exposure (20,21).…”