Effects of benzo(e)pyrene on the retinal neurosensory cells and human microvascular endothelial cells in vitro

Purpose: 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). Materials and Methods: R28 cells and HMVEC were treated for 24 hours with 1000, 400, 200, and 100 μ 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 μ M showed a decrease in cell viability but not at lower concentrations of 200 and 100 μ M. At 400, 200, and 100 μ M B(e)P, there was an increase in caspase-3/7 and at 200 and 100 μM B(e)P caspase-12 activities. Caspase-8 activity was increased only at 200 μ 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 μ 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 μ 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 μ M of B(e)P causes activation of caspase-3/7, -8 (200 μ 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.