The effect of chronic and acute ethanol treatment on morphology, lipid peroxidation, enzyme activities and Na+ transport systems on WRL-68 cells

Abstract

<jats:p> In this study we measured some parameters that are asso ciated with ethanol damage to the liver. The method allowed us to determine the injury that chronic and acute ethanol treatments produce at the cellular level without interference from homeostatic or compensatory mecha nisms. The system used is a hepatic fetal human cell line, WRL-68, which retains, in culture, many of the liver-spe cific functions. WRL-68 cells do not metabolise ethanol, and consequently we could evaluate the effect of ethanol alone. We explored two different conditions: 30 days with 0.1 M ethanol (chronic treatment) and 24 h in the presence of 0.5 M ethanol (acute treatment). </jats:p><jats:p> 1 The transmission electron microscopy studies revealed, in both treatments, the presence of granules not usually present in the cytoplasm of control cells and morphologi cal mitochondrial alterations in chronically treated cells. </jats:p><jats:p> 2 Lipid peroxidation, measured as the rate of malondi aldehyde production, increased three and a half times in acutely treated cells and about twofold in chronically treated cells. </jats:p><jats:p> 3 The percentage of total activity (activity in the medi um/(activity in the medium + activity of the cells). 100) and the enzymatic activity in the culture medium of gamma glutamyl transpeptidase (GGT), alanine amino transferase (ALAT), aspartate amino transferase (ASAT) and alkaline phosphatase (AI-P), increased. </jats:p><jats:p> 4 We measured some parameters related to the transport of sodium across the membrane. Cells chronically treated with ethanol had higher rate constants and effluxes than control cells. There was no difference between the total and passive efflux. Ethanol treated cells apparently lacked the ouabain sensitive pathway. In acutely treated cells, the total sodium efflux and the rate constant were enhanced. Sodium pools in the acutely treated cells were diminished and active sodium pumping was seven times higher than in control cells. </jats:p><jats:p> 5 We determined the number of high affinity ouabain binding sites per cell. Ethanol did not alter the number of pumps, rather it seems to induce a functional alteration. Our results indicate that ethanol per se induces lipid per oxidation, alters enzymatic activities, sodium transport systems, sodium pools and cellular morphology, and that all these changes may be partly responsible for ethanol- induced hepatotoxicity. The data compare favourably with those reported in the literature for many different systems. Therefore our model for studying the mechanism of alco hol effects appears to be valid, with the advantage of being able to compare experiments that can be done in the same system and under the same conditions. </jats:p>

Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes

“…It is known that disturbances in lipid homeostasis induce cellular stress, particularly at mitochondria and endoplasmic reticulum level [ 10 , 12 ]. An analysis by TEM pointed out that ER was clearly compromised in HC cells, presenting dramatic loss in ER architecture ( Figure 3(d) ) in comparison with CW cells ( Figure 3(a) ), seeing that Et treatment induced megamitochondria formation (Figures 3(b) and 3(e) ), as we previously reported [ 13 ]. Interestingly we observed autophagosomes formation only in HC cells treated with Et (arrow, Figure 3(e) ), suggesting that autophagy could play a prominent role.…”

Cytotoxicity of millimolar concentrations of ethanol on HepG2 human tumor cell line compared to normal rat hepatocytes in vitro

“…Several studies address the eect of ethanol on HepG2 cells (Wu and Cederbaum 1996;Neuman et al 1993;Yang and Cederbaum 1997;Cameron et al 1998). There are many in vitro experimental models of ethanol-induced toxicity (Lieber 1994a;Neuman et al 1993;Gutierrez-Ruiz et al 1995). However, most of them have used high concentrations of ethanol (Kurose et al 1997;Neuman et al 1993;Cameron et al 1998;Neuman et al 1995).…”