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    STUDIA BIOLOGIA - Issue no. 2 / 2002  
         
  Article:   DOSE-DEPENDENT EFFECTS OF BACTERIAL ENDOTOXIN TREATMENT ON MEMBRANE PERMEABILITY OF LIVER MITOCHONDRIA IN ETHANOL-FED RATS.

Authors:  CORNELIU TARBA, ION V. DEACIUC.
 
       
         
  Abstract:  Male Sprague-Dawley rats were maintained for 14-15 weeks on a Lieber-DeCarli liquid diet, supplemented isocalorically with 6% ethanol. One day before the decisive experiment, a part of the animals from both the control (pair-fed) and the ethanol-fed group were injected with different concentrations of lipopolysaccharide (LPS; E. coli O26:B2), also known as bacterial endotoxin (0.2, 0.8, 1.5 and 3 mg/kg body weight), while their counterpart groups received only saline injections. 24 hours after the injection of LPS or saline solution the rats were anesthetised, their livers perfused with collagenase and the hepatocytes isolated by centrifugation. Mitochondria were obtained from the homogenised hepatocytes by differential centrifugations. Appropriate aliquots were suspended in 4 flasks containing a basal swelling medium and kept at 30OC until thermal equilibration. At this moment, 5 mM succinate and different concentrations of calcium chloride (0, 10, 50 and 250 μM) were added to the 4 flasks, initiating a swelling process of mitochondrial matrix. At different times of incubation (0, 5, 10, 20 and 30 min), aliquots of 0.5 ml were extracted, placed in appropriate cuvettes and swelling monitored by the absorbance decrease recorded at 540 nm. Statistically significant differences between ethanol-fed and pair-fed rats were observed at higher Ca2+ concentrations (50 and 250 μM) after 30 min of incubation, the swelling being more pronounced in mitochondria of ethanol-fed rats. Differences were also seen in LPS-injected rats, both in pair-fed and in ethanol-fed animals. The extent of swelling, expressed as percent of absorbance decrease was significantly smaller, especially for the rats injected with 0.8 mg LPS/kg body weight (b.w.), suggesting a decreased membrane permeability. Differences were observed for incubation times varying from 5 to 30 min, mainly for low concentrations of calcium (0 and 10 μM). Similar but somewhat smaller differences were observed for rats injected with 0.2 mg LPS/kg b.w., whereas for rats injected with 3 mg LPS/kg b.w. there were no significant differences; however, in this last case the percent absorbance decrease (i.e., swelling) tended to be larger than for the saline-injected rats. Although at least one literature account reports on the increased capacity of liver mitochondria isolated from rats injected with 0.5 mg LPS/kg body weight to maintain a high membrane potential, it is not clear yet whether this type of behaviour reflects a general decrease of membrane permeability, with physiological significance.  
         
     
         
         
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