Liver Histology In Rats Research Articles

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 17α-Ethynylestradiol) and progesterone (2 μg/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-α production by Kupffer cells from either control or EP treatment group. The increased tumor necrosis factor-α 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.

A Study of Cyclic Nucleotide Metabolism and the Histology of Rat Liver During 3’-Methyl-4-Dimethylaminoazobenzene Carcinogenesis II. Cyclic AMP Metabolism

We have studied cAMP metabolism in rat livers undergoing carcinogenesis induced by dietary 3'-methyl-4-dimethylaminoazobenzene. A correlation between the biochemical and the histological changes described in the companion paper has been made. In this study, we saw 100% incidence of cholangiocarcinoma by 10 weeks. During weeks 1--10, the biochemistry of tumor-free areas of the livers only was studied; during weeks 11-13, the increased size of the tumors made possible a biochemical study of the tumor tissue as well as the non-tumor tissue, and a comparison between the two was made. Alterations in all parameters of cAMP metabolism were seen from the earliest stages of treatemnt. Most striking were those of adenylate cyclase activity which preceded and accompanied tumor formation, and were seen in both non-tumor and tumor tissue. In the first few weeks of treatment, small acidophilic glycogen-deficient hepatocytes appeared in the periportal areas of the liver lobules. During this time, there was an increase in maximal isoproterenol stimulation of adenylate cyclase and to a lesser extent in the basal activity of the enzyme; increases in phosphodiesterase activity were seen, and were greatest in weeks 1, 2; cAMP levels were diminished in weeks 1, 2 and slightly but not significantly elevated at week 3. From week 4 onwards an even smaller glycogen-deficient cell population appeared in perilobular areas amongst the acidophilic hepatocytes, and tumors began to appear elsewhere in the livers; at this time, there were further marked increases in the basal activity and isoproterenol responsiveness of adenylate cyclase, and the appearance of increased Gpp(NH)p responsiveness of the enzyme; the increase in phosphodiesterase activities seen at week 3 (smaller than that seen in weeks 1, 2) was sustained but did not further increase; cAMP levels were now significantly elevated also, but they did not rise steadily as did the activity of adenylate cyclase. There was a marked difference between the adenylate cyclase activities in non-tumor tissue from tumor-bearing and non-tumor-bearing livers in weeks 4--10, but there was no difference between the phosphodiesterase activities or cAMP levels in these two groups. Adenylate cyclase activity was extremely high in both non-tumor tissue of tumor-bearing livers from weeks 4--10 and tumors from weeks 11--13. Although phosphodiesterase activities were most elevated in the tumors, there were extremely high cyclic AMP levels in these tissues. The difference between the cAMP levels of tumor and non-tumor tissue was striking. Our findings are discussed with respect to the two-state model of carcinogenesis...

A Study of Cyclic Nucleotide Metabolism and the Histology of Rat Liver During 3’-Methyl-4-Dimethylaminoazobenzene Carcinogenesis I. Histology

ABSTRACT We have studied the histology of rat liver at weekly intervals for 13 weeks from the beginning of carcinogenesis induced by continuous feeding of 3’ -methyl- 4-dimethylaminoazobenzene (0⋅06%). We found 100% incidence of cholangiocarcinoma by 10 weeks. The first change seen was the appearance at 3 weeks in periportal regions of the liver lobule, of shrunken acidophilic hepatocytes. These smaller hepatocytes were not necrotic, were seen in mitosis and were glycogendeficient; they persisted throughout, even in tumors, but were replaced by smaller cells, also glycogen deficient, which latter formed the predominant cell type in early tumors. The small cells were increasingly seen to form dutcules in both apparently uninvolved tissue and tumors. In early tumors, some ducts resembled the bile ducts of portal tracts, but they had increasingly bizarre characteristics in later tumors, and were frankly malignant, composing tumors we have classified as cholangiocarcinoma. Others have called the small cells ‘bile duct’ cells or ‘oval’ cells. We have preferred not to use these terms because our data suggest a hepatocytic origin for these cells. This study of histogenesis is correlated with biochemical studies of cyclic nucleotide metabolism described in companion papers and is discussed in the context of the extensive literature on rat liver carcinogenesis.

A Study of Cyclic Nucleotide Metabolism and the Histology of Rat Liver During 3’-Methyl-4-Dimethylaminoazobenzene Carcinogenesis III. Cyclic GMP Metabolism

A Study of Cyclic Nucleotide Metabolism and the Histology of Rat Liver During 3’-Methyl-4-Dimethylaminoazobenzene Carcinogenesis III. Cyclic GMP Metabolism