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2,5-Hexanedione induces human ovarian granulosa cell apoptosis through BCL-2, BAX, and CASPASE-3 signaling pathways

  • Molecular Toxicology
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Abstract

Studies have shown that 2,5-hexanedione (2,5-HD) is the main active metabolite of n-hexane in the human body. The toxicity of n-hexane and 2,5-hexanedione has been extensively researched, but toxicity to the reproductive system, especially the impact on female reproductive function, has been less frequently reported. In this study, we exposed human ovarian granulosa cells to 0, 16, 64, and 256 μM 2,5-HD in vitro for 24 h. Through hematoxylin–eosin (HE) staining, Hoechst 33342 staining, transmission electron microscopy, and flow cytometry using FITC-Annexin V/PI double staining, 2,5-HD was demonstrated to cause significant apoptosis of human ovarian granulosa cells in a dose-dependent manner. As part of our continuing studies, we investigated the underlying apoptosis mechanism of human ovarian granulosa cells exposed to 0, 16, 64, and 256 μM 2,5-HD in vitro for 24 h. Real-time quantitative PCR and Western blot analysis were used to detect changes in the expression of the apoptosis-related BCL-2 family (BCL-2, BAX) and CASPASE family (CASPASE-3) with increasing 2,5-HD concentration. The results showed that with increasing 2,5-HD doses, the expression of BCL-2 decreased. However, a marked dose-dependent increase in the expression of BAX and active CASPASE-3 (p17) was observed in human ovarian granulosa cells. These results suggest that the mechanisms of 2,5-HD causing increased apoptosis in human ovarian granulosa cells might be through BCL-2, BAX, and CASPASE-3 signaling pathways.

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References

  • Agnesi R, Valentini F, Mastrangelo G (1997) Risk of spontaneous abortion and maternal exposure to organic solvents in shoe industry. Int Arch Occup Environ Health 69:311–316

    Article  PubMed  CAS  Google Scholar 

  • Blanchard KT, Allard EK, Boekelheide K (1996) Fate of germ cells in 2, 5-hexanedione-induced testicular injury. I. Apoptosis is the mechanism of germ cell death. Toxicol Appl Pharmacol 137:141–148

    Article  PubMed  CAS  Google Scholar 

  • Boekelheide K, Fleming SL, Johnson KJ, Patel SR, Schoenfeld HA (2000) Role of Sertoli cells in injury-associated testicular germ cell apoptosis. Proc Soc Exp Biol Med 225:105–115

    Article  PubMed  CAS  Google Scholar 

  • Brauchi S, Cea C, Farias JG, Bacigalupo J, Reyes JG (2006) Apoptosis induced by prolonged exposure to odorants in cultured cells from rat olfactory epithelium. Brain Res 1103:114–122

    Article  PubMed  CAS  Google Scholar 

  • Cao JT, Huang ZX, Cheng X, Chen Y (2007) Experimental research in sex glands of SD rats after n-hexane inhalation. Chin J Ind Hygiene Occup Dis 25:275–278

    CAS  Google Scholar 

  • Carratù MR, Labate M, De Santis S, Giustino A, De Salvia MA, Cuomo V (1995) Lectin binding pattern of Schwann cells and macrophages in 2, 5-hexanedione- induced axonal degeneration in rats. Arch Toxicol 69:160–164

    Article  PubMed  Google Scholar 

  • Chen PC, Hsieh GY, Wang JD, Cheng TJ (2002) Prolonged time to pregnancy in female workers exposed to glycol ethers in semiconductor manufacturing. Epidemiology 13:191–196

    Article  PubMed  Google Scholar 

  • Chen Q, Yano T, Matsumi H, Osuga Y, Yano N, Xu J, Wada O, Koga K, Fujiwara T, Kugu K, Taketani Y (2005) Cross-Talk between Fas/Fas ligand system and nitric oxide in the pathway subserving granulosa cell apoptosis: a possible regulatory mechanism for ovarian follicle atresia. Endocrinology 146:808–815

    Article  PubMed  CAS  Google Scholar 

  • Cory S, Adams JM (2002) The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2:647–656

    Article  PubMed  CAS  Google Scholar 

  • Cui N, Li S, Zhao X, Zhang T, Zhang C, Yu L, Zhu Z, Xie K (2007) Expression of Bcl-2, Bax and Caspase-3 in nerve tissues of rats chronically exposed to 2, 5-hexanedione. Neurochem Res 32:1566–1572

    Article  PubMed  CAS  Google Scholar 

  • Depalo R, Nappi L, Loverro G, Bettocchi S, Caruso ML, Valentini AM, Selvaggi L (2003) Evidence of apoptosis in human primordial and primary follicles. Hum Reprod 18:2678–2682

    Article  PubMed  Google Scholar 

  • Doonan F, Cotter TG (2008) Morphological assessment of apoptosis. Methods 44:200–204

    Article  PubMed  CAS  Google Scholar 

  • Hernandez-Viadel M, Montoliu C, Monfort P, Canales JJ, Erceg S, Rowan M, Ceccatelli S, Felipo V (2003) Chronic exposure to 2, 5-hexanedione impairs the glutamate-nitric oxide-cyclic GMP pathway in cerebellar neurons in culture and in rat brain in vivo. Neurochem Int 42:525–533

    Article  PubMed  CAS  Google Scholar 

  • Host E, Gabrielsen A, Lindenberg S, Smidt-Jensen S (2002) Apoptosis in human cumulus cells in relation to zona pellucida thickness variation, maturation stage, and cleavage of the corresponding oocyte after intracytoplasmic sperm injection. Fertil Steril 77:511–515

    Article  PubMed  Google Scholar 

  • Hsu SY, Lai RJ, Finegold M, Hsueh AJ (1996) Targeted overexpression of Bcl-2 in ovaries of transgenic mice leads to decreased follicle apoptosis, enhanced folliculogenesis, and increased germ cell tumorigenesis. Endocrinology 137:4837–4843

    Article  PubMed  CAS  Google Scholar 

  • Iba MM, Fung J, Gonzalez FJ (2000) Functional Cyp2e1 is required for substantial in vivo formation of 2, 5-hexanedione from n-hexane in the mouse. Arch Toxicol 74:582–586

    Article  PubMed  CAS  Google Scholar 

  • Iwata T, Mori H, Dakeishi M, Onozaki I, Murata K (2005) Effects of mixed organic solvents on neuromotor functions among workers in Buddhist altar manufacturing factories. J Occup Health 47:143–148

    Article  PubMed  CAS  Google Scholar 

  • Kessler W, Heilmaier H, Kreuzer P, Shen JH, Filser M, Filser JG (1990) Spectrophotometric determination of pyrrole-like substances in urine of rat and man: an assay for the evaluation of 2, 5-hexanedione formed from n-hexane. Arch Toxicol 64:242–246

    Article  PubMed  CAS  Google Scholar 

  • Krajewski S, Krajewska M, Shabaik A, Miyashita T, Wang HG, Reed JC (1994) Immunohistochemical determination of in vivo distribution of Bax, a dominant inhibitor of Bcl-2. Am J Pathol 145:1323–1336

    PubMed  CAS  Google Scholar 

  • Kugu K, Ratts VS, Piquette GN, Tilly KI, Tao XJ, Martimbeau S, Aberdeen GW, Krajewski S, Reed JC, Pepe GJ, Albrecht ED, Tilly JL (1998) Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary. Cell Death Diff 5:67–76

    Article  CAS  Google Scholar 

  • Lakhani SA, Masud A, Kuida K, Porter GA Jr, Booth CJ, Mehal WZ, Inayat I, Flavell RA (2006) Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science 311:847–851

    Article  PubMed  CAS  Google Scholar 

  • Liu ZH, Yue KZ, Ma SF, Sun XS, Tan JH (2003) Effects of pregnant mare serum gonadotropin (eCG) on follicle development and granulosa-cell apoptosis in the pig. Theriogenology 59:775–785

    Article  PubMed  CAS  Google Scholar 

  • Lobb DK, Younglai EV (2006) A simplified method for preparing IVF granulosa cells for culture. J Assist Reprod Genet 23:93–95

    Article  PubMed  Google Scholar 

  • Mathai JP, Germain M, Shore GC (2005) BH3 only BIK regulates BAX, BAK-dependent release of Ca2+ from endoplasmic reticulum stores and mitochondrial apoptosis during stress-induced cell death. J Biol Chem 280:23829–23836

    Article  PubMed  CAS  Google Scholar 

  • Mattison DR, Plowchalk DR, Meadows MJ, al-Juburi AZ, Gandy J, Malek A (1990) Reproductive toxicity: male and female reproductive systems as targets for chemical injury. Med Clin North Am 74:391–411

    PubMed  CAS  Google Scholar 

  • McMartin KI, Chu M, Kopecky E, Einarson TR, Koren G (1998) Pregnancy outcome following maternal organic solvents exposure: a meta-analysis of epidemiologic studies. Am J Ind Med 34:288–292

    Article  PubMed  CAS  Google Scholar 

  • Métrailler-Ruchonnet I, Pagano A, Carnesecchi S, Ody C, Donati Y, Barazzone Argiroffo C (2007) Bcl-2 protects against hyperoxia-induced apoptosis through inhibition of the mitochondria-dependent pathway. Free Radic Biol Med 42:1062–1074

    Article  PubMed  Google Scholar 

  • Mishra DP, Pal R, Shaha C (2006) Changes in cytosolic Ca2+ levels regulate Bcl-xS and Bcl-xL expression in spermatogenic cells during apoptotic death. J Biol Chem 281:2133–2143

    Article  PubMed  CAS  Google Scholar 

  • Moffit JS, Bryant BH, Hall SJ, Boekelheide K (2007) Dose-dependent effects of Sertoli cell toxicants 2, 5-hexanedione, carbendazim, and mono-(2-ethylhexyl) phthalate in adult rat testis. Toxicol Pathol 35:719–727

    Article  PubMed  CAS  Google Scholar 

  • Morishima N, Nakanishi K, Takenouchi H, Shibata T, Yasuhiko Y (2002) An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12. J Biol Chem 277:34287–34294

    Article  PubMed  CAS  Google Scholar 

  • Ogawa Y, Shimizu H, Kim SU (1996) 2, 5-Hexanedione induced apoptosis in cultured mouse DRG neurons. Int Arch Occup Environ Health 68:495–497

    Article  PubMed  CAS  Google Scholar 

  • O’Shea JD, Hay MF, Cran DG (1978) Ultrastructural changes in the theca interna during follicular atresia in sheep. J Reprod Fertility 54:183–187

    Article  Google Scholar 

  • Ou YJ, Liu J, Pang F, Wang JL, Lin W, Zhang WC (2009) Experimental study of toxic effect on female gonad induced by n-hexane in rats. Strait J Prev Med 15:4–6

    Google Scholar 

  • Perez GI, Robles R, Knudson CM, Flaws JA, Korsmeyer SJ, Tilly JL (1999) Prolongation of ovarian lifespan into advanced chronological age by Bax-deficiency. Nat Genet 21:200–203

    Article  PubMed  CAS  Google Scholar 

  • Philchenkov AA (2003) Caspases as regulators of apoptosis and other cell functions. Biochemistry (Mosc) 68:365–376

    Article  CAS  Google Scholar 

  • Raman RS, Chan PJ, Corselli JU, Patton WC, Jacobson JD, Chan SR, King A (2001) Comet assay of cumulus cell DNA status and the relationship to oocyte fertilization via intracytoplasmic sperm injection. Hum Reprod 16:831–835

    Article  PubMed  CAS  Google Scholar 

  • Ranjan P, Boss JM (2006) C/EBPbeta regulates TNF induced MnSOD expression and protection against apoptosis. Apoptosis 11:1837–1849

    Article  PubMed  CAS  Google Scholar 

  • Ratts VS, Flaws JA, Kolp R, Sorenson CM, Tilly JL (1995) Ablation of bcl-2 gene expression decreases the numbers of oocytes and primordial follicles established in the post-natal female mouse gonad. Endocrinology 136:3665–3668

    Article  PubMed  CAS  Google Scholar 

  • Sallmén M, Neto M, Mayan ON (2008) Reduced fertility among shoe manufacturing workers. Occup Environ Med 5:518–524

    Google Scholar 

  • Sasson R, Amsterdam A (2002) Stimulation of apoptosis in human granulosa cells from in vitro fertilization patients and its prevention by dexamethasone: involvement of cell contact and bcl-2 expression. J Clin Endocrinol Metab 87:3441–3451

    Article  PubMed  CAS  Google Scholar 

  • Shiota M, Sugai N, Tamura M, Yamaguchi R, Fukushima N, Miyano T, Miyazaki H (2003) Correlation of mitogen-activated protein kinase activities with cell survival and apoptosis in porcine granulosa cells. Zoolog Sci 20:193–201

    Article  PubMed  CAS  Google Scholar 

  • Simoni M, Gromoll J, Nieschlag E (1997) The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology. Endocr Rev 18:739–773

    Article  PubMed  CAS  Google Scholar 

  • Vaskivuo TE, Anttonen M, Herva R, Billig H, Dorland M, te Velde ER, Stenbäck F, Heikinheimo M, Tapanainen JS (2001) Survival of human ovarian follicles from fetal to adult life: apoptosis, apoptosis-related proteins, and transcription factor GATA-4. J Clin Endocrinol Metab 86:3421–3429

    Article  PubMed  CAS  Google Scholar 

  • Verma Y, Rana SV (2009) Endocrinal toxicity of industrial solvents–a mini review. Indian J Exp Biol 47:537–549

    PubMed  CAS  Google Scholar 

  • Wennborg H, Bodin L, Vainio H, Axelsson G (2001) Solvent use and time to pregnancy among female personnel in biomedical laboratories in Sweden. Occup Environ Med 58:225–231

    Article  PubMed  CAS  Google Scholar 

  • Williams GT, Smith CA (1993) Molecular regulation of apoptosis: genetic controls on cell death. Cell 74:777–779

    Article  PubMed  CAS  Google Scholar 

  • Wyllie AH (1997) Apoptosis: an overview. Br Med Bull 53:451–465

    PubMed  CAS  Google Scholar 

  • Yu YS, Sui HS, Han ZB, Li W, Luo MJ, Tan JH (2004) Apoptosis in granulosa cells during follicular atresia: relationship with steroids and insulin-like growth factors. Cell Res 14:341–346

    Article  PubMed  CAS  Google Scholar 

  • Zhivotovsky B (2003) Caspases: the enzymes of death. Essays Biochem 39:25–40

    PubMed  CAS  Google Scholar 

  • Zhou DR, Yang LG, Jiang XP, Yang CF, Zhang SX (2001) Effect of bcl-2 gene and liposome on division and apoptosis of ovarian granular cells in laying hens. J Nanjing Agric Univ 24:75–78

    CAS  Google Scholar 

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Acknowledgments

This study was financially supported by the National Science Foundation of China (30972514) and the Foundation of Fujian Maternity and Child Health Hospital (2010-15).

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Authors and Affiliations

  1. Fujian Province Key Laboratory of Environment and Health, School of Public Health, Fujian Medical University, Fuzhou, 350108, People’s Republic of China

    Yan Sun, Jin Liu, Xiaohua Sheng & Wenchang Zhang

  2. Assisted Reproductive Technology Laboratory, Fujian Maternal and Child Health Hospital, Fuzhou, China

    Yan Sun & Yuan Lin

  3. Emergency Department, Fujian Center for Disease Control and Prevention, Fuzhou, China

    Hong Li

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  1. Yan Sun
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Correspondence to Wenchang Zhang.

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Sun, Y., Lin, Y., Li, H. et al. 2,5-Hexanedione induces human ovarian granulosa cell apoptosis through BCL-2, BAX, and CASPASE-3 signaling pathways. Arch Toxicol 86, 205–215 (2012). https://doi.org/10.1007/s00204-011-0745-7

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