Abstract
Several strategies for the assessment of reproductive toxicity of chemical compounds has have been proposed. In the present work, we devised experimental in vitro assays to test the effect of potential toxicants on proliferating primordial germ cells (PGCs) in vitro using recently developed methods for isolation and culture of mouse PGCs. Primordial germ cells are the embryonic precursors of gametes of the adult that carry the genome from generation to generation. Any damage or mutations caused to these cells by potential toxicants might impair normal reproduction and be transmitted to next generation. Three representative compounds, N-ethyl-N-nitrosourea (ENU), adriamycin (ADR), and mono-(2-ethylhexyl)phthalate (MEHP), toxic to different targets and known to affect germ cell development and impair fertility, were tested on PGCs in culture using three different experimental protocols. Survival and growth of PGCs and their ability to adhere to cell monolayers, were taken as endpoints for drug effects. For each compound, sublethal and acute toxicity doses were determined. In addition, information about the mechanisms of action of these compounds on PGCs was obtained. Whereas the effects of ENU and ADR on PGCs were attributable to growth inhibition and apoptosis induction, MEHP affected PGC adhesion to somatic cells without significantly altering their growth and survival. The results of our in vitro tests were not always exactly predictive of the effects of the tested compounds on PGCs in vivo, determined in parallel experiments in which pregnant mice were exposed to the same compounds. Nevertheless, they can provide information on the sensitivity of PGCs to the direct action of drugs or the mechanisms of action of such agents.
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References
Abercombie M.Estimation of nuclear population from microtome sections.Anat Rec. 1946:94:239–47.
Anas IMK, Suzuki C, Iwamura S, Yoshioka K.Effect ofmono-(2-ethylhexyl)phthalate on bovine oocyte maturation in vitro.Biol Reprod.2001;64 (supplement l):139.
Au WW, Hsu TC.The genotoxic effects of adriamycin in somatic and germinal cells of the mouse.Mut Res. 1980; 79:351–61.
Boekelheide K.Sertoli cell toxicants.In: Russell LD and Griswold MD, eds.The sertoli cell.Clearwater, FL: Cache River Press; 1993:551-75.
Buher M.The primordial germ cells of mammals: some current perspectives.Exp Cell Res. 1997:232:194–6.
Cai Z, Stancou R, Körner M, Chouaib S.Impairment of Fas-antigen expression in adriamycin-resistant but not TNF-resistant MCF7 tumor cells.Int J Cancer.1996:68:535–46.
Dees JH, Gazouli M, Papadopoulos V.Effect of mono-ethyl phthalate on MA-IO Leydig tumor cells.Reprod Toxicol. 2001:15:171–87.
De Felici M, McLaren A.In vitroculture of mouse primordial germ cells.Exp Cell Res. 1983:144:417–27.
De Felici M.Isolation and culture of germ cells from the mouse embryo.In: Celis, JE, ed.Cell biology: a laboratory hand-book, 2nd ed. Vol.1.London: Academic Press; 1998:73–85.
De Felici M.Regulation of primordial germ cell development in the mouse.Int J Dev Biol. 2000:44:575–80.
Fukuoka M, Kobayashi T, Zhou Y, Hayakawa T.Mechanism of testicular atrophy induced by cis-n-butyl phthalate in rats.4.Changes in the activity of succinate dehydrogenase and the levels of tansferrin and ferritin in the Sertoli and germ cells.J AppI Toxicol. 1993:13:241–6.
Gartenhaus RB, Wang P, Hoffmann P.Induction of the WAFI /CPI protein and apoptosis in human T-cell leukemia virus type I-transformed lymphocytes after treatment with adria-mycin by using a p53-independent pathway.Proc Nati Acad Sci USA.1996:93:265–8.
Gray TJ, Beamand JA.Effect of some phthalate esters and other testicular toxins on primary cultures of testicular cells.Food Chem Toxicol. 1984:22:123–31.
Grasso P, Heindel JJ, Powell CJ, Reichert LE Jr.Effects of mono(2-ethylhexyl) phthalate, a testicular toxicant, on folli-cle-stimulating hormone binding to membranes from cultured rat Setoli cells.Biol Reprod. 1993:48:434–59.
Hernsworth BN, Jackson H.Effect of busulphan on the developing gonad of the male rats.J Reprod Fertil. 1962:5:187–94.
Hernsworth BN, Jackson H.Effect of busulphan on the developing ovary in the rats.J Reprod Fertil. 1963:6:229-33.
Lamb JC, Chapin RE, Teague J, Lawton AD, Reel JR.Reproductive effects of four phthalic acid esters in the mouse.Toxicol Appi Pharmacol. 1987:88:255–69.
Lee J, Richburg JH, Younkin SC, Boekelheide K.The fas system is a key regulator of germ cell apoptosis in the testis.Endocrinology.1997:138:2081–8.
Li LH, Heindel JJ.Sertoli cell toxicants.In: Koracn K, ed.Reproductive and developmental toxicology.New York: Marcel Dekker; 1998:657–93.
Li LH, Jester, WL, Orth JM.Effects of relatively low levels of mono-(2-ethylhexyl) phthalate on cocultured Sertoli cells and gonocytes from neonatal rats.Toxicol Appi Pharmacol. 1998:153:258–65.
Li LH, Jester WF Jr, Laslett AL, Orth JM.A single dose of di-(2-ethylhexyl) phthalate in neonatal rats alters gonocytes, reduces Sertoli cell proliferation and decreases cyclin D2 expression.Toxicol Appi Pharmacol. 2000:166:222–9.
Lovekamp TM, Davis BJ.Mono-(2-ethylhexyl) phthalate suppresses aromatase transcript levels and estradiol produc-tion in cultured rat granulosa cells.Toxicol Appi Pharmacol. 2001:172:217–24.
Lu CC, Meistrich ML.Genotoxic effects of chemotherapeutic drugs on mouse testis cells.Cancer Res. 1979:89:3755–82.
Martelli A, Robbiano L, Gazzaniga GM, Brambilla G.Comparative study of DNA damage and repair induced by ten N-nitroso compounds in primary cultures of human and rat hepatocytes.Cancer Res 1988:48:4144–52.
Meistrich ML, van Beek ME, Liang JC, Johonson SL, Lu J.Low levels of chromosomal mutations in germ cells derived from doxorubicin-treated stem spermatogonia in the mouse.Cancer Res. 1990;50:370–4.
Merchant H.Rat gonadal and ovarian organogenesis with and without germ cells: an ultrastructural study.Dev Biol. 1975:44:1–21.
Müller I, Niethammer D, Brucheft G.Anthracycline-derived chemoterapeutics in apoptosis and free radical cytotocity.Int J Mol Med. 1998:1:491–4.
Nagao T, Sato M, Marurno H, Shibuya T, Imai K.Testicular development and fertility of mice treated prenatally wth N-nitroso-/N-ethylurea at various gestational stages.Teratog Carcinog Mutagens.1996:16:183–98.
Oishi S.Testicular atrophy induced by di(2-ethylhexyl)phthalate: changes in histology, cell specific enzyme activities and zinc concentrations in rat testis.Arch Toxicol. 1986:59:290–5.
Parvinen LM, Parvinen M.Biochemical studies of the rat seminiferous epithelial wave: DNA and RNA synthesis and effects of adriamycin.Ann Biol Anim Biochem Biophys. 1978:18:585–94.
Pesce M, Farace MG, Piacentini M, Doici S, De Felici M.Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis).Development.1993:118:1089–94.
Pesce M, Di Carlo A, De Felici, M.The c-kit receptor is involved in the adhesion of mouse primordial germ cells to somatic cells in culture.Mech Dev. 1997:68:37~44.
Richburg JH, Boekelheide K.Mono-2-ethylhexyl phthalate rapidly alters both Sertoli cell vimentin filaments and germ cell apoptosis in young rat testes.Toxicol Appi Pharmacol. 1996:137:42–50.
Richburg JH, Nanez A, Gao H.Participation of the Fas-signaling system in the initiation of germ cell apoptosis in young rat testes after exposure to mono-(2-ethylhexyl) phthalate.Toxicol Appi Pharmacol. 1999:160:271–8.
Shibuya T, Murota T, Tutikawa K.Mouse spot test with alkyinitrosoureas.Mutat Res. 1982:104:311-5.
Shibuya T, Horiya N, Matsuda H, Sakamoto K, Hara T.Dose-dependent induction of recessive mutations with N-ethyl-N-nitrosourea in primordial germ cells of male mice.Mutat Res. 1996:357:219–24.
Shinoda K, Mitsumori K, Yasuhara K, et al.Doxorubicin induces male germ cell apoptosis in rats.Arch Toxicol. 1999:73:274–81.
Sjoblom T, West A, Lahdetie J.Apoptotic response of spermatogenic cells to the germ cell mutagens etoposide, adriamycin, and diepoxybutane.Environ Mol Mutagen. 1998;31: 133–48.
Straughan DW, Fentem JH, Balla, M.In: Castell JV, Gornez-Lechon MJ, eds.In vitromethods in pharmaceutical research.New York: Academic Press.1997:1–13.
Tarn PPL, Snow MHL.Proliferation and migration of primordial germ cells during compensatory growth in mouse embryo.J Embryol Exp Morphol. 1981:64:133–47.
Tanaka T.A study of sex differentiation of the mice treated with mitomycin C in the embryonic stage.Acta Anat Nippon. 1966:41:306–12.
Toppari J, Larsen JC, Christiansen P, et al.Male reproductive and environmental xenoestrogens.Environ Health Perspect. 1996:104:741–03.
Zhou Y, Fukuoka M, Tanaka A.Mechanisms of testicular atrophy induced by di-n-butyl phthalate in rats.3.Changes in the activity of some enzymes in the Sertoli and germ cells and in levels of metal ions.J AppI Toxicol. 1990:10:447–53.
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Iona, S., Klinger, F., Sisti, R. et al. A Comparative Study of Cytotoxic Effects of N-Ethyl-N-Nitrosourea, Adriamycin, and Mono- 2-Ethylhexyl)phthalate on Mouse Primordial Germ Cells. Cell Biol Toxicol 18, 131–145 (2002). https://doi.org/10.1023/A:1015336318623
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DOI: https://doi.org/10.1023/A:1015336318623