Comprehensive characterization of serum clinical chemistry parameters and the identification of urinary superoxide dismutase in a carbon tetrachloride-induced model of hepatic fibrosis in the female Hanover Wistar rat

Abstract

Carbon tetrachloride (CCl(4)) was used to induce liver fibrosis in the rat. Using this model, we have identified changes in serum and urinary clinical chemistry parameters, and characterized histopathological lesions in the liver. Two experiments were conducted. In Experiment 1, rats were dosed at six levels of CCl(4) (0.06-0.36 ml/kg) twice weekly for 6 weeks, followed by a 6-week non-dosing recovery period (week 12). Livers were removed for histology at 6 and 12 weeks and serum parameters analysed. In Experiment 2, rats were given seven dose levels of CCl(4) (0.4-1.0 ml/kg) twice weekly for 6 weeks, followed by a 6-week recovery period (week 12); urine samples were analysed at 3, 6, 9 and 12 weeks using one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Liver fibrosis was evident at 6 weeks in Experiments 1 and 2, and the activity of serum enzymes (including alanine aminotransferase, aspartate aminotransferase and glutamate dehydrogenase) was increased. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis (Experiment 2) revealed a protein band at 18.4 kDa in urine from rats treated with CCl(4), not present in control urine, which was identified as copper/zinc superoxide dismutase (Cu/Zn SOD). Western blotting revealed that SOD was increased in urine from rats treated with CCl(4) at 3 and 6 weeks, but not at 9 and 12 weeks. We conclude that Cu/Zn SOD is a urinary marker of hepatic necrosis, but not hepatic fibrosis.

Figure 1

Histology of the liver from a rat treated with carbon tetrachloride (CCl₄) twice a week for 6 weeks and sampled at 6 weeks. (a) Liver from a control rat to show the normal arrangement and appearance of hepatocytes. (b) Centrilobular region in the liver of a rat treated with CCl₄ at 0.36 ml/kg twice weekly for 6 weeks showing diffuse, marked, hepatocellular vacuolation and degeneration, with necrotic cells. Original magnification of images; ×400 (haematoxylin and eosin).

Figure 2

Histopathology of the liver in rats treated with carbon tetrachloride (CCl₄) twice a week for 6 weeks and sampled at that time. (a) Liver from a rat treated with CCl₄ at 0.6 ml/kg twice weekly for 6 weeks. In the subcapsular region, there is hepatocellular vacuolation, degeneration and necrosis, with capsular pitting or contraction (black arrows). There is collagen formation in the capsular area which extends downwards into the liver parenchyma (open arrow). Original magnification of image; ×100 [haematoxylin and eosin (H&E)]. (b) Liver from a rat treated with CCl₄ twice weekly for 6 weeks at 1.0 ml/kg. There is significant fibrosis which is tending to link centrilobular and periportal areas. Hepatocytes are vacuolated with evidence of degeneration. Original magnification of image; ×100 (H&E). (c) Liver from a rat treated with CCl₄ twice weekly for 6 weeks at 0.7 ml/kg; there is perivascular fibrosis in the centrilobular and periportal areas, with some evidence of bridging along the sinusoidal tracts. Original magnification of image; ×100 (Sirius red). (d) Liver from a rat treated for 6 weeks, twice weekly at 1.0 ml/kg CCl₄. Perivascular fibrosis is evident with bridging along sinusoidal tracts within the liver parenchyma. Original magnification of image; ×100 (Sirius red).

Figure 3

Histopathology of the liver from rats treated with carbon tetrachloride (CCl₄) twice a week for 6 weeks, then left untreated for a 6-week period of recovery. (a) Liver from a rat treated with CCl₄ for 6 weeks at 0.4 ml/kg and left untreated for a 6-week recovery period. There is perivascular fibrosis with some evidence of delicate partial bridging within the liver parenchyma. (b) Liver from a rat treated with CCl₄ for 6 weeks at 1.0 ml/kg and left untreated for a 6-week recovery period. There is perivascular fibrosis in centrilobular and portal regions, with delicate bridging of fibrosis between these areas. Original magnification of both images; ×100 (Sirius red).

Figure 4

Western blots with a superoxide dismutase (SOD-1) antibody of rat urine samples collected during the induction of liver fibrosis with carbon tetrachloride (CCl₄) repeatedly dosed at 0.7 ml/kg. Samples for sodium dodecyl sulphate–polyacrylamide gel electrophoresis were prepared, with each sample containing 14 μl of urine. (a) Urine collected after 3 weeks of dosing twice a week with CCl₄. (b) Urine collected after 6 weeks of dosing twice a week with CCl₄. (c) Urine collected at the end of the 6-week non-dosing recovery period. In all blots: lane 1, 2, 3 = control urine from rats treated with vehicle, and lanes 4 and 5 = urine from rats treated with CCl₄ at 0.7 ml/kg.

Figure 5

Mean superoxide dismutase (SOD) (a) and glutamate dehydrogenase (GLDH) (b) levels in the urine of rats treated with carbon tetrachloride at 0.7 and 1.0 ml/kg twice a week for 6 weeks. Superoxide dismutase activity was determined by using a Ransod assay kit; GLDH activity was measured by using the kinetic Deutsche Gesellschaft für Klinische Chemie method. Values are means (error bars represent SD) of six animals. Values that differ from control by Student’s t-test are shown, *P < 0.05, ***P < 0.001.