Functional and molecular characterization of piscine UDP-glucuronosyltransferases.
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
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
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
The relevance of these data to interspecies toxicity and evolution of
detoxication enzyme systems is discussed.