Studies detailed in this thesis were concerned with elucidating the function and molecular properties of UDP-glucuronosyltransferase(s) involved in detoxicative metabolism in the piscine species, Pleuronectes platessa. This study represents the first detailed investigation of such a system in a non-mammalian species. Comparative studies on UDPGT expression in the mammalian and piscine used in this investigation indicated that a number of marked species differences were apparent which were of potential toxicological significance. Analysis of piscine UDPGT activities in liver, kidney, intestine and gill demonstrated that the phenol UDPGT activity was ubiquitous to different degrees in all tissues whereas activities to endogenous substrates were more restricted. UDPGT expression in piscine microsomes was relatively non-latent compared to its mammalian counterpart, a possible reason for this being the marked species differences observed by microsomal lipid composition analysis. Assay of hepatic microsomal fractions indicated that glucuronidation of planar phenolic xenobiotics was actively catalysed in the piscine species, whilst there was low UDPGT activity towards several steroids and bilirubin, compared to the rat. Aglycone specificity for several bulky non-planar substrates was either very low or absent in the piscine species in contrast to rats. The metabolism of the proximate carcinogen benzo(a)pyrene was also investigated in isolated hepatocytes derived from plaice. The major metabolites formed were glucuronides of benzo(a)pyrene phenols, diols and quinols, indicating that glucuronidation plays an important role in the prevention of formation of cytotoxic and carcinogenic intermediates in these animals. Administration of various xenobiotic agents indicated that polychlorinated biphenyls specifically induced phenol UDPGT activity in hepatic and renal tissue, whilst the carcinogen, 3-methylcholanthrene induced this activity solely in hepatic tissue. A procedure for the purification of hepatic UDPGTs was reported that enabled the physical separation of different UDPGT isoforms from this species indicating that they are polymorphic. One isoform that catalysed the glucuronidation of the phenolic heterocyclic compound, L-naphthol was purified 380 fold over solubilised hepatic microsomes' to apparent homogeneity with a subunit molecular weight of 55kDa. A purified UDPGT preparation was used to raise polyclonal antibodies which were applied to investigating the aforementioned biological variations in UDPGT expression at the molecular level. Such immunoblot analysis indicated that differential expression was due to varied complements of UDPGT isoenzymes. Molecular biological analysis was also employed to give an insight into the evolution of the enzyme. This work indicated that UDPGT isoforms in this piscine species had epitopes in common with their mammalian counterparts, however preliminary studies indicated no such similarity between UDPGT genes in the respective species. The relevance of these data to interspecies toxicity and evolution of detoxication enzyme systems is discussed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:358793 |
| Date | January 1990 |
| Creators | Clarke, Douglas James |
| Publisher | University of Dundee |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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