Cytotoxic effects of oxysterols produced during ozonolysis of cholesterol in murine GT1-7 hypothalamic neurons

Ozone present in the photochemical smog or generated at the inflammatory sites is known to oxidize cholesterol and its 3-acyl esters. The oxidation results in the formation of multiple "ozone-specific" oxysterols, some of which are known to cause abnormalities in the metabolism of cholesterol and exert cytotoxicity. The ozone-specific oxysterols have been shown to favor the formation of atherosclerotic plaques and amyloid fibrils involving pro-oxidant processes. In the present communication, cultured murine GT1-7 hypothalamic neurons were studied in the context of cholesterol metabolism, formation of reactive oxygen species, intracellular Ca²⁺ levels and cytotoxicity using two most commonly occurring cholesterol ozonolysis products, 3β- hydroxy-5-oxo-5,6-secocholestan-6-al (ChSeco) and 5β, 6β-epoxy-cholesterol (ChEpo). It was found that ChSeco elicited cytotoxicity at lower concentration (IC₅₀ = 21 ± 2.4 μM) than did ChEpo (IC₅₀ = 43 ± 3.7 μM). When tested at their IC₅₀ concentrations in GT1-7 cells, both ChSeco and ChEpo resulted in the generation of ROS, the magnitude of which was comparable. N-acetyl-l-cysteine and Trolox attenuated the cytotoxic effects of ChSeco and ChEpo. The intracellular Ca²⁺ levels were not altered by either ChSeco or ChEpo. Methyl-β-cyclodextrins, which cause depletion of cellular cholesterol, prevented ChSeco- but not ChEpo-induced cytotoxicity. The cell death caused by ChEpo, but not ChSeco, was prevented by exogenous cholesterol. Although oxidative stress plays a significant role, the results of the present study indicate differences in the pathways of cell death induced by ChSeco and ChEpo in murine GT1-7 hypothalamic neurons.