Protective effects of lipoic acid on chrysene-induced toxicity on Müller cells in vitro.

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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Benzyl isothiocyanate: an effective inhibitor of polycyclic aromatic hydrocarbon tumorigenesis in A/J mouse lung.

Polycyclic aromatic hydrocarbons (PAH) are an important group of carcinogens that are likely to be involved as one of the causes of lung cancer in smokers and occupationally exposed individuals. Previous studies have shown that benzyl isothiocyanate (BITC), administered by gavage, is a good inhibitor of lung tumorigenesis in A/J mice induced by benzo[a]pyrene (BaP), a typical PAH carcinogen. In this study, we evaluated the effects of BITC on lung tumor induction in A/J mice by two other carcinogenic PAH in cigarette smoke - 5-methylchrysene (5-MeC) and dibenz[a,h]anthracene (DBahA). We also compared the effects of BITC with two other well known chemopreventive agents - butylated hydroxyanisole (BHA) and sulforaphane. In experiment 1, groups of A/J mice were treated by gavage once weekly for 8 weeks with BaP (3 micromol) or 5-MeC (2 micromol) or DBahA (1 micromol) in 0.1 ml cottonseed oil. Fifteen minutes before each treatment, the mice were gavaged with 0.1 ml cottonseed oil or 0.1 ml cottonseed oil containing 13.4 micromol or 6.7 micromol of BITC. The experiment was terminated 19 weeks after the final carcinogen treatment. BITC significantly reduced lung tumor multiplicity in all PAH-treated groups by 63.5-90.6%. In experiment 2, groups of A/J mice were treated with BaP or BITC and BaP as in experiment 1, or with BHA or sulforaphane at doses equimolar to those of BITC. BITC was significantly more effective as an inhibitor of lung tumor induction than either BHA or sulforaphane. These results firmly establish gavaged BITC as a strong inhibitor of lung tumorigenesis induced in A/J mice by PAH, and support its further development for chemoprevention of smoking-induced lung cancer.

Antagonistic interactions among nephrotoxic polycyclic aromatic hydrocarbons.

Although the liver and pulmonary toxicity of polycyclic aromatic hydrocarbons (PAHs) has been extensively characterized, limited data concerning the nephrotoxic potential of these chemicals are available. The present studies were conducted to define the kidney cell-specific toxic responses to anthracene (ANTH), benzo[a]pyrene (BaP), and chrysene (CHRY). Given that exposure to environmental chemicals from a specific source is rarely limited to a single compound, a second goal was to evaluate the nephrotoxic potential of binary and ternary mixtures of these chemicals. Cultured rat glomerular mesangial cells (rGMCs) and porcine cortico-tubular epithelial kidney cells (LLCPK-1) were challenged with hydrocarbon concentrations ranging from 0.03 to 30 microM for up to 24 h and were processed for measurements of mitochondrial membrane permeability, trypan blue dye exclusion, cytoplasmic enzyme leakage, and protein synthesis. BaP induced a threefold increase in mitochondrial fragility, a modest increase in cellular death, and 40% decrease in the rate of protein synthesis in rGMCs. Anthracene was also cytotoxic to rGMCs, inducing a twofold increase in mitochondrial fragility and a 40% decrease in the rate of protein synthesis, but no changes in cellular viability. Although CHRY was devoid of toxicity to rGMCs, a 40% decrease in the rate of protein synthesis was observed in LLCPK-1 cells treated with this hydrocarbon. BaP and ANTH were not overtly cytotoxic to LLCPK-1 cells at any of the concentrations tested. Binary and ternary mixtures of BaP with ANTH and CHRY in rGMCs, and mixtures of CHRY with ANTH and BaP in LLCPK-1 cells, yielded antagonistic interactions. Based on these data, it is concluded that PAHs exhibit chemical- and cell-specific nephrotoxicity, but that toxicological outcomes are influenced by the presence of multiple hydrocarbons in complex mixtures.