Development of a new, simple rat model of early alcohol-induced liver injury based on sensitization of Kupffer cells.
The continuous intragastric in vivo enteral feeding model in the rat developed by Tsukamoto and French has been very useful; however, it requires surgical expertise. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hours later. Accordingly, these experiments were designed to determine if a new, simple animal model of ethanol hepatotoxicity could be developed based on Kupffer cell sensitization. Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hours intragastrically. Livers were stained with hematoxylin-eosin to assess steatosis, inflammation, and necrosis, and tissue triglycerides, serum transaminases, and plasma endotoxin were measured. Kupffer cells were isolated 0 to 24 hours after one intragastric dose of ethanol daily, and intracellular Ca2+ ([Ca2+]i) was measured using fura-2, while tumor necrosis factor alpha (TNF-alpha) was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western and Northern analysis. Ethanol caused steatosis, necrosis, and inflammation in only a few weeks, and after 8 weeks, serum aspartate transaminase (AST) levels were doubled. Values were similar to levels achieved in the enteral feeding model. Triglycerides were also increased significantly by ethanol as expected, and endotoxin levels were increased to 70 to 80 pg/mL. This latter increase was prevented (<20 pg/mL) by antibiotics implicating endotoxin. In isolated Kupffer cells from untreated control rats, [Ca2+]i increased to 82 +/- 7 nmol/L after addition of lipopolysaccharide (LPS) (100 ng/mL), and levels were elevated about twofold by ethanol given 24 hours earlier (174 +/- 15 nmol/L). In addition, TNF-alpha production by Kupffer cells was increased fourfold in cells isolated from rats treated with ethanol 24 hours earlier. Sterilization of the gut with antibiotics blocked all effects of ethanol on [Ca2+]i and TNF-alpha release completely. Moreover, 4 weeks after ethanol, CD14 in Kupffer cells was elevated about twofold. A new, simple chronic model of ethanol hepatotoxicity has been developed here based on sensitization of Kupffer cells to endotoxin.
Enomoto
N
N
Laboratory of Hepatobiology and Toxicology, Chapel Hill, NC, USA.
Yamashina
S
S
Kono
H
H
Schemmer
P
P
Rivera
C A
CA
Enomoto
A
A
Nishiura
T
T
Nishimura
T
T
Brenner
D A
DA
Thurman
R G
RG
D000284:Administration, Oral; D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D001219:Aspartate Aminotransferases / Q000097:blood; D001835:Body Weight; D002118:Calcium / Q000378:metabolism; D004041:Dietary Fats; D004195:Disease Models, Animal; D004359:Drug Therapy, Combination / Q000494:pharmacology; D000431:Ethanol / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000633:toxicity; D005260:Female; D007249:Inflammation; D007728:Kupffer Cells / Q000187:drug effects / Q000473:pathology*; D008070:Lipopolysaccharides / Q000633:toxicity; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D008108:Liver Diseases, Alcoholic / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology*; D008657:Metabolic Clearance Rate; D009336:Necrosis; D009355:Neomycin / Q000494:pharmacology; D011112:Polymyxin B / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014280:Triglycerides / Q000097:blood; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D004041:Dietary Fats; D008070:Lipopolysaccharides; D014280:Triglycerides; D014409:Tumor Necrosis Factor-alpha; D000431:Ethanol; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase; D009355:Neomycin; D011112:Polymyxin B; D002118:Calcium