Molecular Biology Reports

, Volume 39, Issue 1, pp 493–500 | Cite as

The cyclophosphamide metabolite, acrolein, induces cytoskeletal changes and oxidative stress in Sertoli cells

  • Feng Liu
  • Xu-Liang Li
  • Tao Lin
  • Da-Wei He
  • Guang-Hui Wei
  • Jun-Hong Liu
  • Lu-Sheng Li


The aim of this study is to explore the mechanism by which acrolein (ACR), a metabolite of cyclophosphamide (CP), induces immature Sertoli cell cytoskeletal changes. Sertoli cells obtained from rats were cultivated and treated with 50 and 100 μM ACR. XTT assays were performed to detect cell viability. Activities of superoxide dismutase (SOD), glutathione peroxidases (GSH-Px), and catalase (CAT), as well as total anti-oxidation competence (T-AOC) were examined. Superoxide anion levels were detected by a fluorescent probe. Cell ultrastructure changes were observed by transmission fluorescent microscope. Actin filament (F-actin) distribution was detected by immunofluorescence, and ERK and p38MAPK expression were detected by western blot analysis. ACR significantly decreased the viability of Sertoli cells in a dose- and time-dependent manner. T-AOC and the antioxidant activity of SOD, CAT and GSH-Px, were decreased in ACR-treated groups compared with the control group. The levels of reactive oxygen species (ROS) in ACR-treated Sertoli cells were increased. In addition, characteristics of cell apoptosis such as mitochondrial swelling, aggregated chromatin, condensed cytoplasm, nuclei splitting, and nuclei vacuolization were observed in ACR-treated cells. Furthermore, ACR-treatment also induced microfilament aggregation, marginalization and regionalization. The expression levels of ERK and p38MAPK were also increased in ACR-treated cells in a dose- and time-dependent manner. ACR, a major CP metabolite, impairs the cytoskeleton which is likely caused by induction of the oxidative stress response through up-regulation of ERK and p38MAPK expression.


Sertoli cells Cyclophosphamide Acrolein Oxidative stress Cytoskeleton MAPK signal pathway 


  1. 1.
    Tripathi DN, Jena GB (2008) Astaxanthin inhibits cytotoxic and genotoxic effects of cyclophosphamide in mice germ cells. Toxicology 248:96–103PubMedCrossRefGoogle Scholar
  2. 2.
    Charak BS, Gupta R, Mandrekar P et al (1990) Testicular dysfunction after cyclophosphamide-vincristine-procarbazine-prednisolone chemotherapy for advanced Hodgkin’s disease: a long-term follow-up study. Cancer 65:1903–1906PubMedCrossRefGoogle Scholar
  3. 3.
    Kenney LB, Laufer MR, Grant FD et al (2001) High risk of infertility and long term gonadal damage in males treated with high dose cyclophosphamide for sarcoma during childhood. Cancer 91:613–621PubMedCrossRefGoogle Scholar
  4. 4.
    Trasler JM, Hales BF, Robaire B et al (1986) Chronic low dose cyclophosphamide treatment of adult male rats: effect on fertility, pregnancy outcome and progeny. Biol Reprod 34:275–283PubMedCrossRefGoogle Scholar
  5. 5.
    Hoorweg-Nijman JJ, Delemarrevande-Wall HA, De Wall FC et al (1992) Cyclophosphamide-induced disturbance of gonadotropin secretion manifesting testicular damage. Acta Endocrinol 126:143–148PubMedGoogle Scholar
  6. 6.
    Das UB, Mallick M, Debnath JM et al (2002) Protective effect of ascorbic acid on cyclophosphamide-induced testicular gametogenic and androgenic disorders in male rats. Asian J Androl 4:201–207PubMedGoogle Scholar
  7. 7.
    Ghosh D, Das UB, Ghosh S et al (2002) Testicular gametogenic and steroidogenic activities in cyclophosphamide treated rat: a correlative study with testicular oxidative stress. Drug Chem Toxicol 25:281–292PubMedCrossRefGoogle Scholar
  8. 8.
    Manda K, Bhatia AL (2003) Prophylactic action of melatonin against cyclophosphamide-induced oxidative stress in mice. Cell Biol Toxicol 19:367–372PubMedCrossRefGoogle Scholar
  9. 9.
    Agarwal A, Saleh RA, Bedaiwy MA (2003) Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 79:829–843PubMedCrossRefGoogle Scholar
  10. 10.
    Griveau JF, Le Lannou D (1997) Reactive oxygen species and human spermatozoa: physiology and pathology. Int J Androl 20:61–69PubMedCrossRefGoogle Scholar
  11. 11.
    Conte G, Milardi D, De Marinis L et al (1999) Reactive oxygen species in male infertility: review of literature and personal observations. Panminerva Med 41:45–53PubMedGoogle Scholar
  12. 12.
    Iuchi Y, Kaneko T, Matsuki S et al (2004) Carbonyl stress and detoxification ability in the male genital tract and testis of rats. Histochem Cell Biol 121:123–130PubMedCrossRefGoogle Scholar
  13. 13.
    Maiorino M, Ursini F (2002) Oxidative stress, spermatogenesis and fertility. Biol Chem 383:591–597PubMedCrossRefGoogle Scholar
  14. 14.
    Show MD, Anway MD, Folmer JS et al (2003) Reduced intratesticular testosterone concentration alters the polymerization state of the Sertoli cell intermediate filament cytoskeleton by degradation of Vimentin. Endocrinology 144:5530–5536PubMedCrossRefGoogle Scholar
  15. 15.
    Tindall DJ, Rowley DR, Murthy L et al (1985) Structure and biochemistry of the Sertoli cell. Int Rev Cytol 94:127–149PubMedCrossRefGoogle Scholar
  16. 16.
    Vogl AW, Vaid KS, Guttman JA (2009) The Sertoli cell cytoskeleton. Adv Exp Med Biol 636:186–211CrossRefGoogle Scholar
  17. 17.
    Monsees TK, Franz M, Gebhardt S et al (2000) Sertoli cells as a target for reproductive hazards. Andrologia 32:239–246PubMedCrossRefGoogle Scholar
  18. 18.
    de Jonge ME, Huitema AD, Rodenhuis S et al (2005) Clinical pharmacokinetics of cyclophosphamide. Clin Pharmacokinet 44:1135–1164PubMedCrossRefGoogle Scholar
  19. 19.
    Kehrer JP, Biswal SS (2000) The molecular effects of acrolein. Toxicol Sci 57:6–15PubMedCrossRefGoogle Scholar
  20. 20.
    Adams JD, Klaidman LK (1993) Acrolein-induced oxygen radical formation. Free Radic Biol Med 15:187–193PubMedCrossRefGoogle Scholar
  21. 21.
    Mythili Y, Sudharsan PT, Selvakumar E et al (2004) Protective effect of DL-alpha-lipoic acid on cyclophosphamide induced oxidative cardiac injury. Chem Biol Interact 30:13–19CrossRefGoogle Scholar
  22. 22.
    Luo J, Shi Ri (2004) Acrolein induces axolemmal disruption, oxidative stress, and mitochondrial impairment in spinal cord tissue. Neurochem Int 44:475–486PubMedCrossRefGoogle Scholar
  23. 23.
    Luo J, Shi R (2004) Acrolein induces oxidative stress in brain mitochondria. Neurochem Int 46:443–452Google Scholar
  24. 24.
    Arumugam N, Sivakumar V, Thanislass J et al (1997) Effects of acrolein on rat liver antioxidant defense system. Indian J Exp Biol 35:1373–1374PubMedGoogle Scholar
  25. 25.
    Gurtoo HL, Hipkens JH, Sharma SD (1981) Role of glutathione in the metabolism-dependent toxicity and chemotherapy of cyclophosphamide. Cancer Res 41:3584–3591PubMedGoogle Scholar
  26. 26.
    Westlind A, Malmebo S, Johansson I et al (2001) Cloning and tissue distribution of a novel human cytochrome p450 of the CYP3A subfamily, CYP3A43. Biochem Biophys Res Commun 281:1349–1355PubMedCrossRefGoogle Scholar
  27. 27.
    Clifton RJ, O’Donnell L, Robertson DM et al (2002) Pachytene spermatocytes in co-culture inhibit rat Sertoli cell synthesis of inhibin beta B-subunit and inhibin B but not the inhibin alpha-subunit. J Endocrinol 172:565–574PubMedCrossRefGoogle Scholar
  28. 28.
    Galdieri M, Zani B (1981) Hormonal induced changes in Sertoli cell glycoproteins. Cell Biol Int Rep 5:111PubMedCrossRefGoogle Scholar
  29. 29.
    Li LS, Li XL, Wei GH et al (2007) The oxidative stress impairment of immature Sertoli cells by acrolein. Chin J Pediatr Surg 28:318–321 (in Chinese)Google Scholar
  30. 30.
    Halliwell B, Gutteridge JMC (1998) Free radicals in biology and medicine, 3rd edn. Oxford Science, OxfordGoogle Scholar
  31. 31.
    Aitken J, Fisher H (1994) Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioessays 16:259–267PubMedCrossRefGoogle Scholar
  32. 32.
    Bauche F, Fouchard MH, Jegou B (1994) Antioxidant system in rat testicular cells. FEBS Lett 349:392–396PubMedCrossRefGoogle Scholar
  33. 33.
    Tramer F, Rocco F, Micali F et al (1998) Antioxidant systems in rat epididymal spermatozoa. Biol Reprod 59:753–758PubMedCrossRefGoogle Scholar
  34. 34.
    Esterbauer H, Schaur RJ, Zollner H (1991) Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med 11:81–128PubMedCrossRefGoogle Scholar
  35. 35.
    Roy J, Pallepati P, Bettaieb A et al (2009) Acrolein induces a cellular stress response and triggers mitochondrial apoptosis in A549 cells. Chem Biol Interact 181:154–167PubMedCrossRefGoogle Scholar
  36. 36.
    Angkeow P, Deshpande SS, Qi B et al (2002) Redox factor-1: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis. Cell Death Differ 9:717–725PubMedCrossRefGoogle Scholar
  37. 37.
    Deshpande SS, Angkeow P, Huang J et al (2000) Racl inhibits TNF-alpha-induced endothelial cell apoptosis: dual regulation by reactive oxygen species. FASB J 14:1705–1714CrossRefGoogle Scholar
  38. 38.
    Macho A, Hirsch T, Marzo I et al (1997) Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis. J Immunol 158:4612–4619PubMedGoogle Scholar
  39. 39.
    Guan X, Ruch RJ (1996) Gap junction endocytosis and lysosomal degradation of connexin43-P2 in WB-F344 rat liver epithelial cells treated with DDT and lindane. Carcinogenesis 17:1791–1798PubMedCrossRefGoogle Scholar
  40. 40.
    Defamie N, Mograbi B, Roger C et al (2001) Disruption of gap junctional intercellular communication by lindane is associated with aberrant localization of connexin43 and zonula occludens-1 in 42GPA9 Sertoli cells. Carcinogenesis 22:1537–1542PubMedCrossRefGoogle Scholar
  41. 41.
    Fiorini C, Tilloy-Ellul A, Chevalier S, Charuel C, Pointis G (2004) Sertoli cell junctional proteins as early targets for different classes of reproductive toxicants. Reprod Toxicol 18(3):413–421PubMedCrossRefGoogle Scholar
  42. 42.
    Calingasan NY, Uchida K, Gibson GE (1999) Protein-bound acrolein: a novel marker of oxidative stress in Alzheimer’s disease. J Neurochem 72:751–756PubMedCrossRefGoogle Scholar
  43. 43.
    Tirumalai R, Rajesh KT, Mai KH et al (2002) Acrolein causes transcriptional induction of phase II genes by activation of Nrf2 in human lung type II epithelial (A549) cells. Toxicol Lett 132:27–36PubMedCrossRefGoogle Scholar
  44. 44.
    Marshall CJ (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80:179–185PubMedCrossRefGoogle Scholar
  45. 45.
    Dalton TP, Shertzer HG, Puga A (1999) Regulation of gene expression by reactive oxygen. Annu Rev Pharmacol Toxicol 39:67–101PubMedCrossRefGoogle Scholar
  46. 46.
    Kyriakis JM, Avruch J (1996) Sounding the alarm: protein kinase cascades activated by stress and inflammation. J Biol Chem 271:24313–24316PubMedCrossRefGoogle Scholar
  47. 47.
    Li MW, Mruk DD, Lee WM et al (2009) Disruption of the blood-testis barrier integrity by bisphenol A in vitro: is this a suitable model for studying blood-testis barrier dynamics? Int J Biochem Cell Biol 41:2302–2314PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Feng Liu
    • 1
  • Xu-Liang Li
    • 1
  • Tao Lin
    • 1
  • Da-Wei He
    • 1
  • Guang-Hui Wei
    • 1
  • Jun-Hong Liu
    • 1
  • Lu-Sheng Li
    • 1
  1. 1.The Department of Pediatric UrologyMinistry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, CSTC2009CA5002, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Children’s Hospital of Chongqing Medical UniversityChongqingChina

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