Cytokines, growth factors, and oxidative stress in HepG2 cells treated with ethanol, acetaldehyde, and LPS.

Inflammatory mediators, including cytokines, growth factors, and reactive oxygen species, are associated with the pathology of chronic liver disease. In the liver, cytokine and growth factor secretion are usually associated with nonparenchymal cells, particularly Kupffer cells. In the present studies, the effect of 24 and 72 h administration of ethanol (50 mM). acetaldehyde (175 microM), and LPS (1 microg/ml) were studied on the expression and secretion of TNF-alpha, IL-1beta, IL-6, and TGF-beta3, lipid peroxidation damage and glutathion content in HepG2 cell cultures. A 24 h exposure to ethanol induced the expression of TNF-alpha and TGF-beta1, and the secretion of IL-1beta and TGF-beta1. With the same period of treatment, acetaldehyde markedly increased TNF-alpha expression, and stimulated IL-1beta secretion, while LPS exposure induced the expression of TNF-alpha, IL-6, and TGF-beta1, and the secretion of IL-1beta, IL-6, and TGF-beta1. A reduced in TNF-alpha response and TGF-beta1 expression were observed after 72 h exposure to ethanol. A 72 h acetaldehyde exposure decreased markedly TNF-alpha expression and stimulated a previously absent TGF-beta1 response. With the same time of exposure, LPS reduced slightly TGF-beta1 expression, and decreased its secretion. IL-1beta and IL-6 were not detected under 72 h exposure conditions. Lipid peroxidation damage was increased in all treatments, but higher values were found in 72 h treatments. Glutathion content diminished in all treatments. These findings suggest that HepG2 cells, independent of other cells such as Kupffer or macrophages, participate in a differential cytokine, growth factor and oxidative stress response, which differs according to the toxic agent and the time of exposure.

Comparative study of the damage produced by acute ethanol and acetaldehyde treatment in a human fetal hepatic cell line.

The effects of acute ethanol and acetaldehyde treatment on cell proliferation, cell adhesion capacity, neutral red incorporation into lysosomes, glutathione content, protein sulfhydryl compounds, lipid peroxidation, inner mitochondrial membrane integrity (MTT test), lactate dehydrogenase activity (LDH) and ultrastructural alterations were investigated in a human fetal hepatic cell line (WRL-68 cells). WRL-68 cells were used, due to the fact that, although this cell line expresses some hepatic characteristics, it does not express alcohol dehydrogenase or cytochrome P450 activity, so it could be a good model to study the effect of the toxic agents per se. Cells were exposed during 120 min with 200 mM ethanol or 10 mM acetaldehyde. Under these conditions, cells presented 100% viability and no morphological alteration was observed by light microscopy. Acetaldehyde-treated cells reduced their proliferative capacity drastically while the ethanol-treated ones presented no difference with control cells. Cell adhesion to substrate, measured as time required to adhere to the substrate and time required to detach from the substrate, was diminished in acetaldehyde WRL-68-treated cells. Cytotoxicity measures as neutral red and MTT test showed that acetaldehyde-treated cells presented more damage than ethanol-treated ones. Cellular respiratory capacity was compromised by acetaldehyde treatment due to 40% less oxygen consumption than control cells. Lipid peroxidation values, measured as malondialdehyde production, were higher in ethanol-treated WRL-68 cells (127%) than in acetaldehyde-treated ones (60%) to control cell values. Lactate dehydrogenase activity (LDH) in extracellular media of ethanol-treated cells presented the highest values. GSH content was reduced 95% and thiol protein content was diminished severely in acetaldehyde-treated cells. Transmission electron microscopy showed more ultrastructural alterations in cells treated with acetaldehyde. The results indicate that acetaldehyde, like ethanol, produced damage at cellular level, although more damage could be observed in acetaldehyde WRL-68-treated cells.