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.

Proteomic investigation of urinary markers of carbon-tetrachloride-induced hepatic fibrosis in the Hanover Wistar rat

“…There was no evidence of CCl 4 -induced histological changes in the kidneys of any animals. This confirms findings in previous studies where it had been shown that CCl 4 only induced kidney lesions at dose levels of 1.2 ml/kg CCl 4 and above (Smyth et al 2007(Smyth et al , 2008. Accordingly, in the present study, in the absence of the histological evidence of kidney injury, it was assumed that any proteins in the urine of rats with fibrosis, and not present in the urine of control animals, were due to the induction of hepatocellular degeneration and necrosis, the resulting inflammatory response, and the development of hepatic fibrosis.…”

“…CCl 4 -induced oxidative stress plays a role in Kupffer cell activation and fibrogenesis (Rivera et al 2001). We have previously reported histopathological changes and markers of inflammation in CCl 4 -induced hepatic injury in the female Hanover Wistar rat (Giffen et al 2003), and more recently described CCl 4 -induced models of hepatic fibrosis and acute liver injury, also in the female Hanover Wistar rat (Smyth et al 2007(Smyth et al , 2008.…”

“…Urine samples were collected for 24 h at weeks 3 and 6 during the dosing period and at 9 and 12 weeks during a 6 week recovery period during which the animals received no treatment. One dimensional gel electrophoresis of the urine samples showed that SOD was present in urine from fibrotic rats at weeks 3 and 6 of the dosing period but not during the recovery period (at weeks 9 and 12) (Smyth et al 2007). However, as SOD is not a liver-specific protein the present studies describe further preliminary work, using two-dimensional gel electrophoresis (2-DGE) in an attempt to identify a more specific urinary biomarker of hepatic injury and fibrosis.…”

“…In a separate study, (Smyth et al 2007) fibrosis was induced in female Hanover Wistar rats following the administration of CCl 4 twice weekly for 6 weeks. The presence of fibrosis at 6 weeks was confirmed by histopathological assessment (haematoxylin and eosin routine staining (H&E) and Sirius red special staining), and serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH) were significantly increased in the fibrotic animals.…”

Emodin protects rat liver from CCl4-induced fibrogenesis via inhibition of hepatic stellate cells activation

“…Since then many methods to establish a model of liver fibrosis have been tried [30] . Among them, hepatic fibrosis caused by CCl4 has been extensively used in experimental models in rats because hepatic responses in rats to chronic CCl4 stimulation are shown to be superficially similar to human cirrhosis [31] . Hepatocyte damage is the initial factor of hepatic fibrogenesis and activities of ALT and AST in serum are the most commonly used biochemical markers of liver injuries [32] .…”