The influence of zinc on caspase-3 and DNA breakdown in cultured human retinal pigment epithelial cells.
OBJECTIVE
To investigate the role of extracellular zinc on the death process of cultured human retinal pigment epithelial (RPE) cells.


METHODS
Confluent cells on borosilicate glass coverslips were treated with substances in serum-free growth medium for various times and were analyzed for death by means of changes in morphologic features, numbers of attached cells, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) procedure. Some cultures were also exposed to experimental ischemia (defined as a lack of oxygen, glucose, and serum). Electrophoresis and Western blotting and enzyme assays were used to investigate changes in expression of the protease enzyme, caspase-3.


RESULTS
Experimental ischemia caused death of RPE cells. Zinc sulfate had no effect on these cells at low concentrations (100 pmol/L to 10 nmol/L), but protected them at higher concentrations (< or = 10 micromol/L) and appeared to exacerbate cell death at still greater concentrations. Moreover, zinc compounds (>10 micromol/L) also induced death of cells in control cultures that could be blocked by zinc chelators and partially by the caspase-3 inhibitor, DEVD-FMK. Zinc also increased the amount of the active form of caspase-3 in RPE cells.


CONCLUSIONS
Zinc salts protect RPE cells from experimental ischemia-induced death at low concentrations (100 pmol/L-10 nmol/L). However, at higher concentrations, zinc causes cell death and alters the cellular level of caspase-3. These observations are consistent with the death process being apoptosis.


CONCLUSIONS
Zinc supplements are taken by many individuals. Low doses of zinc can protect RPE cells against ischemic-type insults as may occur in certain ocular complaints. Furthermore, high concentrations of zinc can damage RPE cells. Because zinc ions are known to be taken up by RPE cells from the choroidal circulation, the actual therapeutic dose taken by patients is critical.
Wood
J P
JP
The Nuffield Laboratory of Ophthalmology, University of Oxford, Walton Street Oxford, OX2 6AW, England. john.wood@eye.ox.ac.uk
Osborne
N N
NN
D000328:Adult; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D015687:Cell Hypoxia / Q000187:drug effects; D002478:Cells, Cultured; D002648:Child; D053938:DNA Fragmentation / Q000187:drug effects*; D006801:Humans; D020287:In Situ Nick-End Labeling; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D019287:Zinc Sulfate / Q000494:pharmacology*
D019287:Zinc Sulfate; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases