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Abraham Spector, Ren–Rong Wang, Wanchao Ma, Norman J. Kleiman; Development and Characterization of an H2O2-Resistant Immortal Lens Epithelial Cell Line. Invest. Ophthalmol. Vis. Sci. 2000;41(3):832-843.
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purpose. To determine how nature would protect lens epithelial cells from
H2O2 stress, an immortal murine lens epithelial
cell line, αTN4-1, 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 μM 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-transferaseα
-1 and α-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
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
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