Development and characterization of an H2O2-resistant immortal lens epithelial cell line.
OBJECTIVE
To determine how nature would protect lens epithelial cells from H2O2 stress, an immortal murine lens epithelial cell line, alphaTN4-1, was subjected to gradually increasing H2O2 levels over a period of approximately 8 months. The resultant conditioned cells grew normally when exposed daily to 125 microM H2O2, whereas normal cells died within 9 hours. This communication describes changes in the cell biology of the conditioned cells that allowed them to remain viable. The manner in which critical biochemical parameters were affected in both conditioned and normal cells is also reported.


METHODS
Conditioned cells were obtained by gradually increasing the concentration of H2O2 over a period of approximately 8 months, introducing an aliquot of H2O2 every 24 hours. A wide spectra of biological parameters were evaluated, including catalase, GSH peroxidase and other antioxidative enzyme activities, cell number and cell viability, non-protein thiol, ATP, transport systems, thymidine incorporation, and DNA cleavage.


RESULTS
Surprisingly, the conditioned cells did not degrade the medium H2O2 more rapidly than normal cells. However, analyses of the antioxidative defenses indicated that catalase activity was increased 60-fold and glutathione peroxidase (GSH Px) approximately 2.7-fold. Glucose-6-phosphate dehydrogenase, GSH S-transferase, and GSSG reductase also had increased activity. Using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in situ trypsin digestion and matrix-assisted laser desorption/ionization mass spectrometry, a highly amplified doublet in the conditioned cell preparation was shown to be GSH S-transferase alpha-1 and alpha-2 isomers. Examination of key biochemical parameters indicated that while most such parameters in the conditioned cells showed marked decay in the first hour or so after stress, recovery was then observed and within a few hours, these parameters were back in the normal range. In contrast, damage in the normal cells was not repaired. The damage to DNA was shown to involve Fenton chemistry. In the presence of a metal ion chelator, normal cells survive H2O2 stress.


CONCLUSIONS
The overall conclusion from this investigation is that nature has chosen to respond to the H2O2 stress by not only increasing the activity of enzymes degrading H2O2 but also the systems involved in repair, generation of reducing potential, and detoxification. All but one system of those evaluated appears to be permanently modified.
Spector
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A
Department of Ophthalmology, Columbia University, New York, New York 10032, USA. as42@columbia.edu
Wang
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RR
Ma
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Kleiman
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NJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects; D004351:Drug Resistance; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D005978:Glutathione / Q000378:metabolism; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D018384:Oxidative Stress / Q000187:drug effects; D010088:Oxidoreductases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005986:Glyceraldehyde 3-Phosphate; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D005978:Glutathione