UC Irvine

Purpose

This study evaluates the toxic effects of chrysene (a component from cigarette smoke) on Müller cells (MIO-M1) in vitro and investigates whether the inhibitor lipoic acid can reverse the chrysene-induced toxic effects.

MIO-M1 cells were exposed to varying concentrations of chrysene with or without lipoic acid. Cell viability was measured by a trypan blue dye exclusion assay. Caspase-3/7 activity was measured by a fluorochrome assay. Lactate dehydrogenase (LDH) release was quantified by an LDH assay. The production of reactive oxygen/nitrogen species (ROS/RNS) was measured with a 2’,7’-dichlorodihydrofluorescein diacetate dye assay. Mitochondrial membrane potential (ΔΨm) was measured using the JC-1 assay. Intracellular ATP content was determined by the ATPLite kit.

MIO-M1 cells showed significantly decreased cell viability, increased caspase-3/7 activity, LDH release at the highest chrysene concentration, elevated ROS/RNS levels, decreased ΔΨm value, and decreased intracellular ATP content after exposure to 300, 500, and 1,000 µM chrysene compared with the control. Pretreatment with 80 µM lipoic acid reversed loss of cell viability in 500-µM-chrysene-treated cultures (24.7%, p<0.001). Similarly, pretreatment with 80 µM lipoic acid before chrysene resulted in decreased caspase-3/7 activities (75.7%, p<0.001), decreased ROS/RNS levels (80.02%, p<0.001), increased ΔΨm values (86%, p<0.001), and increased ATP levels (40.5%, p<0.001) compared to 500-µM-chrysene-treated cultures.

Chrysene, a component of cigarette smoke, can diminish cell viability in MIO-M1 cells in vitro by apoptosis at the lower concentrations of Chrysene (300 and 500 µM) and necrosis at the highest concentration. Moreover, mitochondrial function was particularly altered. However, lipoic acid can partially reverse the cytotoxic effect of chrysene. Lipoic acid administration may reduce or prevent Müller cell degeneration in retinal degenerative disorders.