Oral contraceptives worsen endotoxin-induced liver injury in rats.
BACKGROUND
Oral contraceptives are widely used; however, these drugs occasionally cause liver injury. Recently, it was reported that estriol worsens alcoholic liver injury by the mechanism involving activation of Kupffer cells as a result of gut-derived endotoxin. However, the relationship between oral contraceptives and endotoxin-induced liver injury has not been elucidated. Here we show that oral contraceptives sensitize Kupffer cells via a mechanism dependent on increased gut permeability to endotoxin.


METHODS
Female Wistar rats (200-250 g) were given intraperitoneally a combination of estradiol (35 ng/kg of 17 alpha-Ethynylestradiol) and progesterone (2 microg/kg of Norethindrone), each dose being similar to that contained in oral contraceptives (EP treatment). After 24 hr, a sublethal dose of lipopolysaccharide (LPS; 5 mg/kg) was injected via the tail vein. In some experiments, antibiotics (150 mg/kg/day of polymyxin B and 450 mg/kg/day of neomycin) were administered orally for 4 days before EP treatment. Gut permeability was measured in isolated segments of ileum by translocation of horseradish peroxidase. Kupffer cells were isolated and cultured in RPMI 1640 + 10% fetal bovine serum for 24 hr. After addition of LPS (100 ng/ml) to the culture medium, intracellular calcium concentration ([Ca2+](i) ) was measured with fura-2.


RESULTS
Liver histology in rats given EP treatment intraperitoneally followed by an injection of LPS (5 mg/kg) 24 hr later revealed pronounced liver damage with massive necrosis. Whereas mean values of alanine aminotransferase (ALT) in the control, nontreated rats were 30 +/- 6 IU/liter, ALT increased to 75 +/- 21 IU/liter 24 hr after LPS injection. This increase was aggravated 6-fold (483 +/- 118 IU/liter; p< 0.05) by EP treatment. The EP treatment-induced increase in ALT was completely blocked by antibiotics (82 +/- 26 IU/liter; p< 0.05). Gut permeability was increased approximately 10-fold with EP treatment. This increase in gut permeability was not altered by treatment with nonabsorbable antibiotics. In isolated Kupffer cells, LPS increased [Ca2+](i) of Kupffer cells in control rats from basal levels (36 +/- 8 nmol/liter) to 100 +/- 8 nmol/liter. In contrast, the LPS-induced [Ca2+](i) elevation was approximately 3-fold greater in the group given EP treatment before 24 hr (305 +/- 17 nmol/liter; p< 0.05). This increase was also blocked completely by treatment with antibiotics (128 +/- 13 nmol/liter). Similar results were obtained for LPS-induced tumor necrosis factor-alpha production by Kupffer cells from either control or EP treatment group. The increased tumor necrosis factor-alpha production as a result of EP treatment was blocked completely by antibiotics.


CONCLUSIONS
These results indicate that EP treatment in vivo sensitizes Kupffer cells to LPS via a mechanism dependent on the portal increase of gut-derived endotoxin. This event suggests that oral contraceptives exacerbate alcoholic liver injury.
Department of Gastroenterology, Juntendo University School of Medicine, Tokyo, Japan.
Akira
A
Konno
Nobuyuki
N
Enomoto
Yoshiyuki
Y
Takei
Miyoko
M
Hirose
Kenichi
K
Ikejima
Nobuhiro
N
Sato
D000818:Animals; D003276:Contraceptives, Oral / Q000633:toxicity*; D004357:Drug Synergism; D004926:Escherichia coli; D004997:Ethinyl Estradiol / Q000633:toxicity*; D005260:Female; D007728:Kupffer Cells / Q000187:drug effects* / Q000473:pathology; D008070:Lipopolysaccharides / Q000633:toxicity*; D008104:Liver Cirrhosis, Alcoholic / Q000473:pathology; D008106:Liver Cirrhosis, Experimental / Q000473:pathology*; D008111:Liver Function Tests; D009640:Norethindrone / Q000633:toxicity*; D051381:Rats; D017208:Rats, Wistar
D003276:Contraceptives, Oral; D008070:Lipopolysaccharides; D004997:Ethinyl Estradiol; D009640:Norethindrone