Page 204 - The Toxicology of Fishes

184 The Toxicology of Fishes

Glucuronidation involves the transfer of an activated sugar group to the substrate. This process
increases the water solubility and the molecular weight of the substrates, thus facilitating transport and
excretion. The preferred cofactor for most UGT isoforms is uridine diphosphoglucuronic acid (UDPGA).
However, a number of other nucleotide–sugar cofactors have been reported for these enzymes; therefore,
the name UDP-glycosyltransferase has been proposed as a better name for this superfamily (Mackenzie
et al., 1997). UDPGA is synthesized in the cytosol from uridine-triphosphate and glucose-1-phosphate.
Contrary to many phase I reactions, glucuronidation does not require additional energy supply in the
form of ATP or NADPH. The synthesis of UDPGA in the cytosol introduces an extra threshold for the
glucuronidation reaction rate, as the water-soluble cofactor has to be actively transported through the
ER membrane to reach the active site on the UGT enzyme. Mechanisms surrounding transport are still
unknown.

UGT Gene Structure
Multiple UGT genes have been characterized in mammals (17 in humans) and are subdivided into two
families. All UGT1 family members are encoded by a unique single locus gene, which in humans has
13 different first exons spanning some 300 kb. They encode the aglycone binding site, which is alter-
natively spliced onto identical exons 2 to 5, which encode the C-terminal half of the protein responsible
for UDP-glucuronic acid binding. Each exon 1 appears to have its own promoter in the 5′ upstream
region between its transcription start site and the preceding exon 1. Elaboration of the gene appears to
have arisen from duplication into two clusters: (1) a bilirubin cluster (1A1 to 1A5 isoforms), primarily
involved in the conjugation of amines, bilirubin, carboxylic acids, and thyroxine, and (2) a phenol cluster,
which conjugates planar polyaromatic hydrocarbons (1A6) and bulky phenols (1A7 to 1A13). A similar
although slightly less complex elaboration of the UGT1A family has been found in rodents and the
rabbit. In humans and rats, at least one gene of the bilirubin cluster is a pseudogene resulting in reduced
conjugation of planar phenols. In contrast, the genes of UGT2 family members, each comprised of six
exons, are located at distinct loci on another chromosome. The UGT2A subfamily has postulated roles
in olfaction, and the products of the UGT2B family genes appear to be particularly involved in bile acid
and steroid hormone conjugation. Although many of the enzymes were successfully purified from rodents,
the majority of the genes have been heterologously expressed for activity studies, and indeed most of
the human enzymes are now commercially available.
The gene encoding the major phenol-conjugating isoform of plaice has been cloned (UGT1B1) and
found to share an almost identical five-exon structure with mammalian UGT1 family members (George
and Leaver, 2002; George, unpublished data). When heterologously expressed, it displays specificity for
planar phenols such as 1-naphthol and 4-nitrophenol as aglycones but no demonstrable activity with
bilirubin or steroids (Leaver et al., 2007).
Expressed sequence tags (ESTs) for UGTs have subsequently been cloned from flounder, plaice, and
zebrafish. Analysis of the currently published zebrafish EST sequences and the latest assembly of the
genome reveals that at least 14 distinct UGTs are expressed (George and Taylor, 2002; George, unpub-
lished data, Leaver et al.., 2007). More putative genes can be identified in the genome; however, they
are found on numerous chromosomes (in both mouse and humans, the UGT1 family occurs on one
chromosome and the UGT2 family on another). The phylogenetic relationships between these expressed
zebrafish UGT homologs and the human genes is shown in Figure 4.8. The zebrafish genes are divisible
into three groups; the group with closest similarity to the characterized UGT1B1 gene comprises two
alternatively spliced six-exon genes. Gene 6220 is comprised of six exon 1’s, three of which are known
to be expressed (Figure 4.9). The products arise by alternative splicing of the primary transcripts.
Inspection of the nucleotide sequences of the intervening introns between the multiple exon 1’s reveals
the presence of peroxisomal proliferator response elements (PPREs) and xenobiotic response elements
(XREs), which would support the plaice induction data. A further three zebrafish genes are comprised
of six exons and divide with the mammalian UGT2 family, thus inferring the closest homology. Inter-
estingly, gene 4649 also appears to exhibit alternative splicing. Intronless UGT genes can also be
identified in zebrafish (four putative genes, two reported as ESTs) and in the pufferfish (two genes).
These genes (family 3) have an unknown function and appear to be unique to fish.