Smyth, Rosemary;
(2004)
Investigation of novel urinary markers of hepatotoxicity.
Doctoral thesis (Ph.D.), University College London.
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Abstract
The aim of this study was to identify novel, sensitive, and specific protein markers of hepatotoxicity in rat urine. Collection of urine is non-invasive compared to biopsy or serum analysis and therefore preferable when screening for toxicity. Carbon tetrachloride (CCI4) was used both acutely to produce hepatotoxicity and chronically to produce a rat model of liver fibrosis. An optimal dose of CCl4, and the time post-dosing for maximum acute liver injury, were established by histopathological examination and by assaying serum enzyme markers of liver injury. Urine was analysed using Surface Enhanced Laser Desorption/Ionisation (SELDI) ProteinChip® technology. SELDI revealed the appearance of a protein peak at 15.7 KDa in response to CCU-treatment, while one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) identified an 18.4 KDa protein in urine from CCl4-treated rats. This protein was identified by in-gel digestion and tandem mass spectrometry as Cu/Zn superoxide dismutase (SOD-1). SOD-1 catalyses the destruction of the superoxide anion and acts as a defence against oxidative damage. SOD-1 exists as a 32.5 KDa homodimer and it was concluded that the 15.7 KDa SELDI and 18.4 KDa SDS PAGE proteins are the SOD-1 subunit that runs at an anomalous MW on SDS PAGE. SOD-1 in the rat urine was confirmed by Western blotting with a commercial antibody and by measuring SOD activity. Further studies revealed that SOD-1 was increased in urine from CCU-treated rats between 12 hours and 60 hours post-dosing, at dose levels as low as 0.4 ml/kg CCI4. Western blots of homogenates showed that SOD-1 was being lost from rat liver presumably by necrosis. Although the enzyme SOD-1 is not specifically located in the liver, its appearance in the rat urine following hepatotoxicity is a novel finding. Since changes in SOD-1 levels were detected following low dose levels of CCI4 and the response was measured using non-invasive methods suggests that SOD-1 is thus a potential marker of hepatotoxicity. Liver fibrosis was induced by repeat dosing with CCI4 and confirmed by histopathological examination. Analysis of the urine samples by SDS-PAGE revealed an increase in SOD-1 in fibrotic rats but no other differences were evident. Examination of urine samples from rats with fibrosis, or acute hepatotoxicity, by two-dimensional gel electrophoresis revealed a number of proteins that were increased in these models. Two-dimensional gel electrophoresis of liver homogenates from the acute model showed a number of proteins that decreased. These results suggest that although the urinary proteins have not yet been identified it is highly probable that one or more will be a specific marker for the non-invasive identification of hepatotoxicity.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D. |
| Title: | Investigation of novel urinary markers of hepatotoxicity. |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Thesis Digitised by Proquest. |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10122243 |
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