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Acute exposure to thimerosal induces antiproliferative properties, apoptosis, and autophagy activation in human Chang conjunctival cells

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Abstract

Background

Previously we have shown that acute exposure to thimerosal (Thi) can induce oxidative stress and DNA damage in a human conjunctival cell line. However, the long-term effect of Thi on Chang conjunctival cells is not clear. Therefore, the aim of this study was to further investigate the fate of the cells after acute exposure to Thi.

Method

Cells were first exposed to various concentrations of Thi (0.00001 % ∼ 0.001 %) for 30 min, and then cells were assessed after a 24-h recovery period. Morphologic changes were observed under a light microscope and cell viability was evaluated. Cell apoptosis, cell cycle distribution and mitochondrial membrane potential (MMP) (rhodamine 123 assay) were detected by flow cytometry analysis. Poly (ADP-ribose) polymerase (PARP), activation of caspase-3 and microtubule-associated protein light chain 3 (LC-3) were examined by western blot analysis.

Results

DNA strand breaks were significantly increased in a dose-dependent manner with 30 min exposure to Thi, although no significant cell death was detected. However, after 24-h recovery, the ratio of apoptotic cells was significantly increased to 0.0005 % and 0.001 % in Thi treated groups (p < 0.001 compared to the control group). Apoptosis was confirmed by the cleavage of PARP and caspase-3 activation. In addition, G2/M cell cycle arrest and decrease of MMP were recorded. Finally, the LC-3 results indicated the occurrence of autophagy in Thi-treated cells.

Conclusion

Acute exposure to Thi can induce DNA damage, and eventually can lead to cell death, probably through the caspase-dependent apoptosis pathway, while autophagy might also be involved.

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References

  1. Tripathi BJ, Tripathi RC, Kolli SP (1992) Cytotoxicity of ophthalmic preservatives on human corneal epithelium. Lens Eye Toxic Res 9:361–375

    CAS  PubMed  Google Scholar 

  2. Burstein NL, Klyce SD (1977) Electrophysiologic and morphologic effects of ophthalmic preparations on rabbit cornea epithelium. Invest Ophthalmol Vis Sci 16:899–911

    CAS  PubMed  Google Scholar 

  3. Baines MG, Cai F, Backman HA (1991) Ocular hypersensitivity to thimerosal in rabbits. Invest Ophthalmol Vis Sci 32:2259–2265

    CAS  PubMed  Google Scholar 

  4. Wilson-Holt N, Dart JK (1989) Thiomersal keratoconjunctivitis, frequency, clinical spectrum and diagnosis. Eye (Lond) 3(Pt 5):581–587

    Article  Google Scholar 

  5. Buck SL, Rosenthal RA, Schlech BA (2000) Methods used to evaluate the effectiveness of contact lens care solutions and other compounds against Acanthamoeba: a review of the literature. CLAO J 26:72–84

    CAS  PubMed  Google Scholar 

  6. Wilson LA, McNatt J, Reitschel R (1981) Delayed hypersensitivity to thimerosal in soft contact lens wearers. Ophthalmology 88:804–809

    Article  CAS  PubMed  Google Scholar 

  7. Ferrat L, Romeo M, Gnassia-Barelli M, Pergent-Martini C (2002) Effects of mercury on antioxidant mechanisms in the marine phanerogam Posidonia oceanica. Dis Aquat Organ 50:157–160

    Article  CAS  PubMed  Google Scholar 

  8. Barzilai A, Yamamoto K (2004) DNA damage responses to oxidative stress. DNA Repair (Amst) 3:1109–1115

    Article  CAS  Google Scholar 

  9. Geier DA, Sykes LK, Geier MR (2007) A review of Thimerosal (Merthiolate) and its ethylmercury breakdown product: specific historical considerations regarding safety and effectiveness. J Toxicol Environ Health B Crit Rev 10:575–596

    Article  CAS  PubMed  Google Scholar 

  10. Ye J, Zhang H, Wu H, Wang C, Shi X, Xie J, He J, Yang J (2012) Cytoprotective effect of hyaluronic acid and hydroxypropyl methylcellulose against DNA damage induced by thimerosal in Chang conjunctival cells. Graefes Arch Clin Exp Ophthalmol 250:1459–1466

    Article  CAS  PubMed  Google Scholar 

  11. Perez MJ, Cederbaum AI (2002) Antioxidant and pro-oxidant effects of a manganese porphyrin complex against CYP2E1-dependent toxicity. Free Radic Biol Med 33:111–127

    Article  CAS  PubMed  Google Scholar 

  12. Baracca A, Sgarbi G, Solaini G, Lenaz G (2003) Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F(0) during ATP synthesis. Biochim Biophys Acta 1606:137–146

    Article  CAS  PubMed  Google Scholar 

  13. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728

    Article  CAS  PubMed  Google Scholar 

  14. Xu Y, Pang G, Zhao D, Gao C, Zhou L, Sun S, Wang B (2010) In vitro activity of thimerosal against ocular pathogenic fungi. Antimicrob Agents Chemother 54:536–539

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Van Horn DL, Edelhauser HF, Prodanovich G, Eiferman R, Pederson HF (1977) Effect of the ophthalmic preservative thimerosal on rabbit and human corneal endothelium. Invest Ophthalmol Vis Sci 16:273–280

    PubMed  Google Scholar 

  16. Mondino BJ, Groden LR (1980) Conjunctival hyperemia and corneal infiltrates with chemically disinfected soft contact lenses. Arch Ophthalmol 98:1767–1770

    Article  CAS  PubMed  Google Scholar 

  17. Morgan SE, Kastan MB (1997) p53 and ATM: cell cycle, cell death, and cancer. Adv Cancer Res 71:1–25

    Article  CAS  PubMed  Google Scholar 

  18. Moll UM, Slade N (2004) p63 and p73: roles in development and tumor formation. Mol Cancer Res 2:371–386

    CAS  PubMed  Google Scholar 

  19. Kaufmann WK, Paules RS (1996) DNA damage and cell cycle checkpoints. FASEB J 10:238–247

    CAS  PubMed  Google Scholar 

  20. Kastan MB, Onyekwere O, Sidransky D, Vogelstein B, Craig RW (1991) Participation of p53 protein in the cellular response to DNA damage. Cancer Res 51:6304–6311

    CAS  PubMed  Google Scholar 

  21. Cistulli CA, Kaufmann WK (1998) p53-dependent signaling sustains DNA replication and enhances clonogenic survival in 254 nm ultraviolet-irradiated human fibroblasts. Cancer Res 58:1993–2002

    CAS  PubMed  Google Scholar 

  22. Lock RB, Ross WE (1990) Possible role for p34cdc2 kinase in etoposide-induced cell death of Chinese hamster ovary cells. Cancer Res 50:3767–3771

    CAS  PubMed  Google Scholar 

  23. Cuddihy AR, O’Connell MJ (2003) Cell-cycle responses to DNA damage in G2. Int Rev Cytol 222:99–140

    Article  PubMed  Google Scholar 

  24. Shackelford RE, Kaufmann WK, Paules RS (2000) Oxidative stress and cell cycle checkpoint function. Free Radic Biol Med 28:1387–1404

    Article  CAS  PubMed  Google Scholar 

  25. Castedo M, Perfettini JL, Roumier T, Andreau K, Medema R, Kroemer G (2004) Cell death by mitotic catastrophe: a molecular definition. Oncogene 23:2825–2837

    Article  CAS  PubMed  Google Scholar 

  26. Shenker BJ, Guo TL OI, Shapiro IM (1999) Induction of apoptosis in human T-cells by methyl mercury: temporal relationship between mitochondrial dysfunction and loss of reductive reserve. Toxicol Appl Pharmacol 157:23–35

    Article  CAS  PubMed  Google Scholar 

  27. Shenker BJ, Guo TL, Shapiro IM (2000) Mercury-induced apoptosis in human lymphoid cells: evidence that the apoptotic pathway is mercurial species dependent. Environ Res 84:89–99

    Article  CAS  PubMed  Google Scholar 

  28. Kroemer G, Galluzzi L, Brenner C (2007) Mitochondrial membrane permeabilization in cell death. Physiol Rev 87:99–163

    Article  CAS  PubMed  Google Scholar 

  29. Scarlett JL, Sheard PW, Hughes G, Ledgerwood EC, Ku HH, Murphy MP (2000) Changes in mitochondrial membrane potential during staurosporine-induced apoptosis in Jurkat cells. FEBS Lett 475:267–272

    Article  CAS  PubMed  Google Scholar 

  30. Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T, Susin SA, Petit PX, Mignotte B, Kroemer G (1995) Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 182:367–377

    Article  CAS  PubMed  Google Scholar 

  31. Schulze-Osthoff K, Beyaert R, Vandevoorde V, Haegeman G, Fiers W (1993) Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF. EMBO J 12:3095–3104

    CAS  PubMed  Google Scholar 

  32. Putt KS, Beilman GJ, Hergenrother PJ (2005) Direct quantitation of poly(ADP-ribose) polymerase (PARP) activity as a means to distinguish necrotic and apoptotic death in cell and tissue samples. Chembiochem 6:53–55

    Article  CAS  PubMed  Google Scholar 

  33. Codogno P, Meijer AJ (2005) Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ 12(Suppl 2):1509–1518

    Article  CAS  PubMed  Google Scholar 

  34. Yla-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL (2009) Monitoring autophagy by electron microscopy in Mammalian cells. Methods Enzymol 452:143–164

    Article  CAS  PubMed  Google Scholar 

  35. Klionsky DJ, Elazar Z, Seglen PO, Rubinsztein DC (2008) Does bafilomycin A1 block the fusion of autophagosomes with lysosomes. Autophagy 4:849–950

    CAS  PubMed  Google Scholar 

  36. Priault M, Salin B, Schaeffer J, Vallette FM, di RJP, Martinou JC (2005) Impairing the bioenergetic status and the biogenesis of mitochondria triggers mitophagy in yeast. Cell Death Differ 12:1613–1621

    Article  CAS  PubMed  Google Scholar 

  37. Kissova I, Deffieu M, Manon S, Camougrand N (2004) Uth1p is involved in the autophagic degradation of mitochondria. J Biol Chem 279:39068–39074

    Article  CAS  PubMed  Google Scholar 

  38. Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y, Crowe RA, Cascio WE, Bradham CA, Brenner DA, Herman B (1998) The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta 1366:177–196

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The work was supported by project foundation : 1. Zhejiang province Key Innovation Team Project of China (No.2009R50039) 2. Zhejiang Key Laboratory Fund of China (No.2011E10006) 3. Natural Science Foundation of China (81070756) 4. National "Twelfth Five-Year" Plan for Science & Technology Support of China (2012BAI08B01) 5. The Specialized Key Science & Technology Foundation of Zhejiang Provincial S & T Department,China (No.2012C13023-2) 6. Zhejiang Provincial Key Project of Medical and Health (No.2011ZDA014)

The authors have no financial interest in any product or concept discussed in article

Grant: Supported by project foundation: 1. Zhejiang province Key Innovation Team Project of China (No.2009R50039) 2. Zhejiang Key Laboratory Fund of China (No.2011E10006) 3. Natural Science Foundation of China (81070756) 4. National "Twelfth Five-Year" Plan for Science & Technology Support of China (2012BAI08B01) 5. The Specialized Key Science & Technology Foundation of Zhejiang Provincial S & T Department,China (No.2012C13023-2) 6. Zhejiang Provincial Key Project of Medical and Health (No.2011ZDA014)

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Authors and Affiliations

  1. Department of Ophthalmology, The 2nd Affiliated Hospital of Zhejiang University, College of Medicine, Hangzhou, Zhejiang, China

    Huina Zhang, Han Wu, Changjun Wang, Jiajun Xie, Jinjing He & Juan Ye

  2. Department of Toxicology, Hangzhou Normal University School of Public Health, Hangzhou, Zhejiang, 310036, China

    Jun Yang

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  1. Huina Zhang
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Correspondence to Juan Ye.

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Zhang, H., Wu, H., Wang, C. et al. Acute exposure to thimerosal induces antiproliferative properties, apoptosis, and autophagy activation in human Chang conjunctival cells. Graefes Arch Clin Exp Ophthalmol 252, 275–284 (2014). https://doi.org/10.1007/s00417-013-2542-x

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  • DOI: https://doi.org/10.1007/s00417-013-2542-x

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