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. 2003 Jan;111(1):33–38. doi: 10.1289/ehp.5458

Polycyclic aromatic hydrocarbon-induced cytotoxicity in cultured rat Sertoli cells involves differential apoptotic response.

Samir S Raychoudhury 1, Dana Kubinski 1
PMCID: PMC1241303  PMID: 12515676

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and persistent environmental contaminants. Some PAHs are carcinogens and may affect the male reproductive system. Therefore, we exposed cultured rat Sertoli cells to a variety of PAHs to determine possible direct toxic effects on the cells of the seminiferous epithelium. Sertoli cells were chosen because they support germ cell development and maintain spermatogenesis. Sertoli cells were isolated from 19-21-day-old male rats and cultured in medium containing 0.08% dimethylsulfoxide as vehicle or in the presence of a variety of PAHs. In the first set of experiments, cultured Sertoli cells were incubated in the presence of 10(-4) M, 10(-6 )M, 10(-8) M, 10(-12) M, and 10(-16) M fluoranthene (FL) for 24 hr. After 24 hr, FL at 10(4), 10(-6), and 10(-8) M killed significant numbers of Sertoli cells as revealed by cell viability determinations. Sertoli cells cultured in the presence of 10(-6) M and 10(-8) M FL showed morphologic changes. Cell protein levels were decreased and lactate production in the medium increased in a concentration-dependent manner. In addition, Sertoli cells exposed to 10(6) M and 10(-8) M FL exhibited altered F-actin and alpha-tubulin distributions compared with untreated controls. Because FL killed about 62% of cells at 10(-4) M (100 micro g/mL) and 48% of cells at 10(-6) M (1 micro g/mL), increased lactate production about 3-fold at both concentrations, and decreased cell protein by half at 10(-4) M (100 micro g/mL), we decided to use a range of concentrations between 10 and 100 micro g/mL for the second set of experiments using benz[a]anthracene (BaA), benzo[a]pyrene (BaP), or benzo[b]fluoranthene (BbF). After 24 hr, BaA (100 micro g/mL), BaP (50 and 100 micro g/mL), and BbF (100 micro g/mL) significantly increased lactate level in the medium in a concentration-dependent manner. In a third set of experiments, cells were treated in culture uniformly with only 10 micro g/mL FL, BaA, BaP, or BbF for 24 hr. The cytotoxic effects exerted by these PAHs tested resulted in different apoptotic responses as characterized by in situ fluorescence staining. Microscopic analysis of apoptotic cells demonstrated nuclei of reduced size and labeled 3 -OH DNA ends when Sertoli cells had been incubated for 24 hr with 10 micro g BaP or BbF, but not with vehicle, media, FL, or BaA. Thus, our results demonstrate that the toxic effects of BaA and BbF on Sertoli cells are exerted through apoptosis, whereas FL and BaA do not elicit the apoptotic response.

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Selected References

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  1. Boekelheide K. 2,5-Hexanedione alters microtubule assembly. II. Enhanced polymerization of crosslinked tubulin. Toxicol Appl Pharmacol. 1987 May;88(3):383–396. doi: 10.1016/0041-008x(87)90213-4. [DOI] [PubMed] [Google Scholar]
  2. Boese BL, Lamberson JO, Swartz RC, Ozretich R, Cole F. Photoinduced toxicity of PAHs and alkylated PAHs to a marine infaunal amphipod . Arch Environ Contam Toxicol. 1998 Apr;34(3):235–240. doi: 10.1007/s002449900311. [DOI] [PubMed] [Google Scholar]
  3. Chapin R. E., Gray T. J., Phelps J. L., Dutton S. L. The effects of mono-(2-ethylhexyl)-phthalate on rat Sertoli cell-enriched primary cultures. Toxicol Appl Pharmacol. 1988 Mar 15;92(3):467–479. doi: 10.1016/0041-008x(88)90186-x. [DOI] [PubMed] [Google Scholar]
  4. Hall E. S., Hall S. J., Boekelheide K. Sertoli cells isolated from adult 2,5-hexanedione-exposed rats exhibit atypical morphology and actin distribution. Toxicol Appl Pharmacol. 1992 Nov;117(1):9–18. doi: 10.1016/0041-008x(92)90211-a. [DOI] [PubMed] [Google Scholar]
  5. Johnson K. J., Hall E. S., Boekelheide K. 2,5-Hexanedione exposure alters the rat Sertoli cell cytoskeleton. I. Microtubules and seminiferous tubule fluid secretion. Toxicol Appl Pharmacol. 1991 Dec;111(3):432–442. doi: 10.1016/0041-008x(91)90248-d. [DOI] [PubMed] [Google Scholar]
  6. Jégou B. The Sertoli-germ cell communication network in mammals. Int Rev Cytol. 1993;147:25–96. [PubMed] [Google Scholar]
  7. Levy J. I., Houseman E. A., Spengler J. D., Loh P., Ryan L. Fine particulate matter and polycyclic aromatic hydrocarbon concentration patterns in Roxbury, Massachusetts: a community-based GIS analysis. Environ Health Perspect. 2001 Apr;109(4):341–347. doi: 10.1289/ehp.01109341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. MacKenzie K. M., Angevine D. M. Infertility in mice exposed in utero to benzo(a)pyrene. Biol Reprod. 1981 Feb;24(1):183–191. doi: 10.1095/biolreprod24.1.183. [DOI] [PubMed] [Google Scholar]
  9. Mastrangelo G., Fadda E., Marzia V. Polycyclic aromatic hydrocarbons and cancer in man. Environ Health Perspect. 1996 Nov;104(11):1166–1170. doi: 10.1289/ehp.961041166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Means A. R., Tash J. S., Chafouleas J. G., Lagace L., Guerriero V. Regulation of the cytoskeleton by Ca2+-calmodulin and cAMP. Ann N Y Acad Sci. 1982;383:69–84. doi: 10.1111/j.1749-6632.1982.tb23162.x. [DOI] [PubMed] [Google Scholar]
  11. Miller K. P., Ramos K. S. Impact of cellular metabolism on the biological effects of benzo[a]pyrene and related hydrocarbons. Drug Metab Rev. 2001 Feb;33(1):1–35. doi: 10.1081/dmr-100000138. [DOI] [PubMed] [Google Scholar]
  12. Newton S. C., Millette C. F. Sertoli cell plasma membrane polypeptides involved in spermatogenic cell-Sertoli cell adhesion. J Androl. 1992 Mar-Apr;13(2):160–171. [PubMed] [Google Scholar]
  13. PAYNE S. The pathological effects of the intraperitoneal injection of 3:4-benzpyrene into rats and mice. Br J Cancer. 1958 Mar;12(1):65–74. doi: 10.1038/bjc.1958.10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Phillips D. H. Fifty years of benzo(a)pyrene. Nature. 1983 Jun 9;303(5917):468–472. doi: 10.1038/303468a0. [DOI] [PubMed] [Google Scholar]
  15. Raychoudhury S. S., Blackshaw A. W., Irving M. G. Hormonal modulation of the interactions of cultured rat testicular Sertoli and peritubular myoid cells. Effects on glycosaminoglycan synthesis. J Androl. 1993 Jan-Feb;14(1):9–16. [PubMed] [Google Scholar]
  16. Raychoudhury S. S., Blake C. A., Millette C. F. Toxic effects of octylphenol on cultured rat spermatogenic cells and Sertoli cells. Toxicol Appl Pharmacol. 1999 Jun 15;157(3):192–202. doi: 10.1006/taap.1999.8664. [DOI] [PubMed] [Google Scholar]
  17. Raychoudhury S. S., Flowers A. F., Millette C. F., Finlay M. F. Toxic effects of polychlorinated biphenyls on cultured rat Sertoli cells. J Androl. 2000 Nov-Dec;21(6):964–973. [PubMed] [Google Scholar]
  18. Raychoudhury S. S., Millette C. F. Multiple fucosyltransferases and their carbohydrate ligands are involved in spermatogenic cell-Sertoli cell adhesion in vitro in rats. Biol Reprod. 1997 May;56(5):1268–1273. doi: 10.1095/biolreprod56.5.1268. [DOI] [PubMed] [Google Scholar]
  19. Schwartzman R. A., Cidlowski J. A. Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocr Rev. 1993 Apr;14(2):133–151. doi: 10.1210/edrv-14-2-133. [DOI] [PubMed] [Google Scholar]
  20. Selevan S. G., Borkovec L., Slott V. L., Zudová Z., Rubes J., Evenson D. P., Perreault S. D. Semen quality and reproductive health of young Czech men exposed to seasonal air pollution. Environ Health Perspect. 2000 Sep;108(9):887–894. doi: 10.1289/ehp.00108887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Shaw G. R., Connell D. W. Prediction and monitoring of the carcinogenicity of polycyclic aromatic compounds (PACs). Rev Environ Contam Toxicol. 1994;135:1–62. doi: 10.1007/978-1-4612-2634-5_1. [DOI] [PubMed] [Google Scholar]
  22. Shum S., Jensen N. M., Nebert D. W. The murine Ah locus: in utero toxicity and teratogenesis associated with genetic differences in benzo[a]pyrene metabolism. Teratology. 1979 Dec;20(3):365–376. doi: 10.1002/tera.1420200307. [DOI] [PubMed] [Google Scholar]
  23. Skinner M. K., Norton J. N., Mullaney B. P., Rosselli M., Whaley P. D., Anthony C. T. Cell-cell interactions and the regulation of testis function. Ann N Y Acad Sci. 1991;637:354–363. doi: 10.1111/j.1749-6632.1991.tb27322.x. [DOI] [PubMed] [Google Scholar]
  24. Srám R. J., Binková B., Rössner P., Rubes J., Topinka J., Dejmek J. Adverse reproductive outcomes from exposure to environmental mutagens. Mutat Res. 1999 Jul 16;428(1-2):203–215. doi: 10.1016/s1383-5742(99)00048-4. [DOI] [PubMed] [Google Scholar]
  25. Tung P. S., Dorrington J. H., Fritz I. B. Structural changes inducted by follicle-stimulating hormone or dibutyryl cyclic AMP on presumptive Sertoli cells in culture. Proc Natl Acad Sci U S A. 1975 May;72(5):1838–1842. doi: 10.1073/pnas.72.5.1838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wang J. S., Busby W. F., Jr Induction of lung and liver tumors by fluoranthene in a preweanling CD-1 mouse bioassay. Carcinogenesis. 1993 Sep;14(9):1871–1874. doi: 10.1093/carcin/14.9.1871. [DOI] [PubMed] [Google Scholar]
  27. Wang J. S., Busby W. F., Jr, Wogan G. N. Formation and persistence of DNA adducts in organs of CD-1 mice treated with a tumorigenic dose of fluoranthene. Carcinogenesis. 1995 Nov;16(11):2609–2616. doi: 10.1093/carcin/16.11.2609. [DOI] [PubMed] [Google Scholar]
  28. Wells P. G., Kim P. M., Laposa R. R., Nicol C. J., Parman T., Winn L. M. Oxidative damage in chemical teratogenesis. Mutat Res. 1997 Dec 12;396(1-2):65–78. doi: 10.1016/s0027-5107(97)00175-9. [DOI] [PubMed] [Google Scholar]
  29. Welsh M. J., Wiebe J. P. Rat sertoli cells: a rapid method for obtaining viable cells. Endocrinology. 1975 Mar;96(3):618–624. doi: 10.1210/endo-96-3-618. [DOI] [PubMed] [Google Scholar]
  30. Willett K. L., Gardinali P. R., Sericano J. L., Wade T. L., Safe S. H. Characterization of the H4IIE rat hepatoma cell bioassay for evaluation of environmental samples containing polynuclear aromatic hydrocarbons (PAHs). Arch Environ Contam Toxicol. 1997 May;32(4):442–448. doi: 10.1007/s002449900211. [DOI] [PubMed] [Google Scholar]
  31. Willett K. L., Randerath K., Zhou G. D., Safe S. H. Inhibition of CYP1A1-dependent activity by the polynuclear aromatic hydrocarbon (PAH) fluoranthene. Biochem Pharmacol. 1998 Mar 15;55(6):831–839. doi: 10.1016/s0006-2952(97)00561-3. [DOI] [PubMed] [Google Scholar]
  32. Winn L. M., Wells P. G. Evidence for embryonic prostaglandin H synthase-catalyzed bioactivation and reactive oxygen species-mediated oxidation of cellular macromolecules in phenytoin and benzo[a]pyrene teratogenesis. Free Radic Biol Med. 1997;22(4):607–621. doi: 10.1016/s0891-5849(96)00340-1. [DOI] [PubMed] [Google Scholar]
  33. Wyllie A. H., Kerr J. F., Currie A. R. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251–306. doi: 10.1016/s0074-7696(08)62312-8. [DOI] [PubMed] [Google Scholar]

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