December 2002
Volume 43, Issue 13
Free
ARVO Annual Meeting Abstract  |   December 2002
4-Hydroxynonenal Induced Apoptosis in Human Lens Epithelial Cells and Rat Lens Epithelium
Author Affiliations & Notes
  • T Xiao
    Human Biol-Chem-Genetics Univ Texas Medical Branch Galveston TX
  • S Choudhary
    Human Biol-Chem-Genetics Univ Texas Medical Branch Galveston TX
  • NH Ansari
    Human Biol-Chem-Genetics Univ Texas Medical Branch Galveston TX
  • Footnotes
    Commercial Relationships   T. Xiao, None; S. Choudhary, None; N.H. Ansari, None. Grant Identification: NIH EY 13014-02
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 2361. doi:
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      T Xiao, S Choudhary, NH Ansari; 4-Hydroxynonenal Induced Apoptosis in Human Lens Epithelial Cells and Rat Lens Epithelium . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2361.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: Hydrogen peroxide (H2O2), an oxidant which causes lipid peroxidation leading to the formation of reactive end products, has been shown to induce apoptosis in various cell lines and due to the fact that it also imparts toxicity to the lens, we investigated if the end products of lipid peroxidation (lipid derived aldehydes, LDA) induce apoptosis in Human Lens Epithelial cells (HLEC) and the rat lens epithelium thereby contributing to oxidative stress induced- catractogenesis. Methods: HLEC were cultured in DMEM media containing 20% FBS and 20µg/ml gentamycin. The cells were seeded at the density of 0.2x106 cells/ml and were exposed to micromolar concentrations of H2O2 and 4-hydroxynonenal (HNE), a major LDA. The cells were harvested at the end of specified incubation period and used for determining apoptosis either by quantifying the cytosolic histone associated DNA fragments or morphologically assessing the apoptotic cells by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) using DeadenEndTM Colorimetric Apoptosis Detection System or Vybrant apoptosis assay kit (Hoechst staining). Since proteolysis is a requirement in apoptosis, we investigated H2O2 and HNE induced activation of various caspases such as caspase-1, -2, -3, -8 and –9 by using specific fluorogenic substrates. Rat lens were exposed to 200 µM of H2O2 or 40 µM of HNE at different time point The lens epithelia with the capsule were removed and attached to the slides. The sections were fixed and processed for assessing apoptosis by TUNEL and Hoechst staining. Protein-HNE was quantified by immunofluorescence Results: A time dependent increase in apoptosis was observed in HLEC treated with either 40µM HNE or 200 µM H2O2. Similarly TUNEL assay revealed higher apoptotic index in HNE exposed cells at all the intervals as compared to their respective controls. Caspase-9 was not activated. Rat lenses cultured in the presence of HNE or H2O2 resulted in apoptosis and formation of the protein –HNE adducts. Increase in the formation of HNE-protein adduct in the H2O2 treated cells suggests that HNE could be a mediator of H2O2 induced apoptosis. Conclusion: In the present study we have demonstrated that H2O2 and the LDAs, such as HNE, can induce a time-dependent loss of cell viability and a simultaneous increase in apoptosis involving activation of caspases in the cultured human lens epithelial cells. Furthermore, it also induces apoptosis in the rat lens epithelium, with a concomitant formation of protein HNE adducts. It thus appears that scavenging LDAs and preventing apoptosis could be a novel therapeutic strategy to prevent oxidation–induced cataract.

Keywords: 338 cataract • 341 cell death/apoptosis • 458 lipids 
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