Endotoxin potentiates hepatocyte apoptosis in cholestasis.
BACKGROUND
Cholestasis is a component of liver disease of almost any etiology, including septic liver injury. The cellular mechanisms of liver injury in cholestasis and sepsis remain unresolved. We evaluated apoptosis, a well-orchestrated and potentially reversible mechanism of cell death, in bile duct-ligated and endotoxin-injected rats.


METHODS
Male Sprague-Dawley rats were randomly assigned to six groups (n = 6-9): bile duct-ligated+endotoxin (B+E), sham+endotoxin (S+E), bile duct-ligated (B), sham (S), endotoxin (E), and normal (N). On day 1, the bile ducts of B+E and B rats were ligated and severed. S+E and S animals underwent biliary manipulation only. On day 3, B+E, S+E, and E groups received 3 mg/kg endotoxin i.v.. On day 4, livers from all rats were excised, fixed, and stained (hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick-end labeling [TUNEL]). Portions were frozen for DNA fragmentation analysis. Caspase 3 activity was determined using isolated hepatocytes.


RESULTS
Livers from all groups (B+E, S+E, E, and B) except normal and sham displayed apoptosis by hematoxylin and eosin staining, TUNEL staining, and DNA fragmentation. Histologic evaluation revealed 10% to 20% necrosis in endotoxin-treated animals (B+E, S+E, and E). Caspase 3 activity was significantly higher in endotoxin-treated animals versus animals without endotoxin (treated 0.450 +/- 0.08 versus nontreated 0.135 +/- 0.05, p < 0.0001) (mean +/- SD).


CONCLUSIONS
Cholestatic livers had apoptosis without progression to necrosis. When exposed to the second insult of endotoxin, cholestatic livers received an acute on chronic apoptotic trigger, and proceeded to necrosis. Endotoxin was a potent hepatotoxic insult because all treated rat livers displayed both apoptosis and necrosis.
Moazzam
Farnaz N
FN
Department of Surgery, Burn & Shock Trauma Institute, Loyola University Medical Center, Maywood, IL 60153, USA.
Brems
John J
JJ
Yong
Sherri L
SL
Filkins
James P
JP
Fisher
Susan G
SG
Holt
David R
DR
Gamelli
Richard L
RL
Ding
Jin Wen
JW
D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001663:Bilirubin / Q000097:blood; D053148:Caspase 3; D020169:Caspases / Q000032:analysis; D001651:Cholestasis, Extrahepatic / Q000150:complications / Q000473:pathology*; D003135:Common Bile Duct / Q000293:injuries; D053938:DNA Fragmentation; D004731:Endotoxins / Q000494:pharmacology*; D022781:Hepatocytes / Q000382:microbiology / Q000473:pathology*; D020287:In Situ Nick-End Labeling; D008026:Ligation; D008297:Male; D023421:Models, Animal; D009336:Necrosis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012477:Salmonella enteritidis; D018805:Sepsis / Q000150:complications
D004731:Endotoxins; D000410:Alanine Transaminase; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D001663:Bilirubin