Cytoprotective effect of hyaluronic acid and hydroxypropyl methylcellulose against DNA damage induced by thimerosal in Chang conjunctival cells.
BACKGROUND
To investigate genotoxicity of the preservative thimerosal (Thi), and the cytoprotective and antioxidant effects of hyaluronic Acid (HA) and hydroxypropyl methylcellulose (HPMC) on Chang conjunctival cells.


METHODS
Cells were divided into three groups. One group was exposed to Thi at various concentrations (0.00001 %∼0.001 %) for 30 min; the other two groups were pretreated with 0.3 % HA or 0.3 % HPMC for 30 min before the Thi exposure. After cell viability was evaluated, alkaline comet assay and detection of the phosphorylated form of the histone variant H2AX (γH2AX) foci were used to determine DNA damage. Reactive oxygen species (ROS) production was assessed by the fluorescent probe, 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA).


RESULTS
A significant change of cell viability was observed after exposure to 0.001 % Thi for 30 min. DNA single- and double-strand breaks were significantly increased in a dose-dependent manner with Thi exposure. In addition, intracellular ROS induced by Thi was dose-dependent, except at 0.001 % less ROS was induced than at 0.0005 %. However, cells pretreated with 0.3 % HA or 0.3 % HPMC showed significantly increased cell survival, decreased DNA damage, and decreased ROS production compared to cells exposed to Thi alone. Pretreatment with 0.3 % HA was found to be even more protective than 0.3 % HPMC.


CONCLUSIONS
Thi can induce DNA damage in human conjunctival epithelial cells, probably due to oxidative stress. HA and HPMC are protective agents that have antioxidant properties and can decrease DNA damage induced by Thi. Pretreatment of 0.3 % HA may be more protective of the ocular surface than 0.3 % HPMC.
Department of Ophthalmology, The 2nd Affiliated Hospital of Zhejiang University, College of Medicine, Hangzhou, Zhejiang, China. yejuan@zju.edu.cn
Juan
J
Ye
Huina
H
Zhang
Han
H
Wu
Changjun
C
Wang
Xin
X
Shi
Jiajun
J
Xie
Jinjing
J
He
Jun
J
Yang
D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D020552:Comet Assay; D003228:Conjunctiva / Q000166:cytology*; D019610:Cytoprotection; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D006820:Hyaluronic Acid / Q000494:pharmacology*; D065347:Hypromellose Derivatives; D008747:Methylcellulose / Q000031:analogs & derivatives* / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009883:Ophthalmic Solutions / Q000494:pharmacology; D011310:Preservatives, Pharmaceutical / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013849:Thimerosal / Q000633:toxicity*; D055675:Viscosupplements / Q000494:pharmacology
D009883:Ophthalmic Solutions; D011310:Preservatives, Pharmaceutical; D017382:Reactive Oxygen Species; D055675:Viscosupplements; D013849:Thimerosal; D065347:Hypromellose Derivatives; D006820:Hyaluronic Acid; D008747:Methylcellulose