Effects of benzo(e)pyrene on the retinal neurosensory cells and human microvascular endothelial cells in vitro.
OBJECTIVE
To study the effects of benzo(e)pyrene (B(e)P), a toxic component of cigarette smoke, on retinal neurosensory (R28) cells and human microvascular endothelial cells (HMVEC).


METHODS
R28 cells and HMVEC were treated for 24 hours with 1000, 400, 200, and 100 micro M of B(e)P. Cell viability was measured by dye exclusion assay. Caspase-3/7, -8, -9, and -12 activities were measured by fluorochrome assays. DNA ladder was run on agarose gel, and lactate dehydrogenase (LDH) release rate was evaluated using a LDH cytotoxicity kit II.


RESULTS
R28 cells exposed to B(e)P 1000 and 400 micro M showed a decrease in cell viability but not at lower concentrations of 200 and 100 micro M. At 400, 200, and 100 micro M B(e)P, there was an increase in caspase-3/7 and at 200 and 100 microM B(e)P caspase-12 activities. Caspase-8 activity was increased only at 200 micro M B(e)P. Caspase-9 activity was not increased at any concentration. DNA ladder revealed bands at 200 bp intervals at lower concentrations and LDH activity at higher concentrations. HMVEC cultures exposed to B(e)P 1000, 400, and 200 micro M showed a decrease in cell viability. Caspase-3/7 activity was not increased at any concentration. DNA laddering revealed no bands at 200 bp intervals at any dose. LDH release rates increased at all three concentrations. However, 100 micro M B(e)P was found safe on HMVEC.


CONCLUSIONS
B(e)P has different mechanisms of action on R28 cells and HMVEC at different concentrations. In R28 cells, 200 and 100 micro M of B(e)P causes activation of caspase-3/7, -8 (200 micro M only) and -12 pathways, leading to apoptotic cell death, but, at higher concentrations, there is non-apoptotic cell death, which could be due to necrosis. In contrast, the HMVEC cell death is through non-caspase-dependent necrosis pathway. The molecular mechanisms of cell death vary with different cell types and concentrations of B(e)P.
Patil
A Jayaprakash
AJ
Department of Ophthalmology, Gavin S. Herbert Eye Institute, University of California, Irvine, California, USA.
Gramajo
Ana L
AL
Sharma
Ashish
A
Chwa
Marilyn
M
Seigel
Gail M
GM
Kuppermann
Baruch D
BD
Kenney
M Cristina
MC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001580:Benzopyrenes / Q000633:toxicity*; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D053938:DNA Fragmentation; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051381:Rats; D012160:Retina / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology
D001580:Benzopyrenes; C026487:benzo(e)pyrene; D007770:L-Lactate Dehydrogenase; D020169:Caspases