Piscine UDP-glucuronosyltransferase 1B

Abstract

Glucuronidation is an important detoxification pathway for organic pollutants in fish. We report here the isolation and characterisation of UDP-glucuronosyltransferases (UGT) genes from the closely related marine flatfish, plaice (Pleuronectes platessa) and flounder (Platichthys flesus). The deduced amino acid sequences share greater similarity with mammalian UGT1 family genes than UGT2 genes (44-47% and 39-40% amino acid identity respectively) and have been designated UGT1B. Both plaice and flounder UGT1B mRNAs are most highly expressed in liver, but are also expressed in intestine, gill, kidney and adipose tissue to a greater extent than muscle, heart or brain. Plaice UGT1B mRNA was undetectable in gametes or fertilised eggs and there was a large increase in expression between gastrulation and myotome formation after which levels declined some 5-10 fold. Flounder UGT1B mRNA was increased in liver after intraperitoneal injection of Arochlor 1254 or lindane, but not after perflourooctanoic acid or 3-methylcholanthrene. In isolated flounder hepatocytes UGT1B mRNA was increased by benzo(a)pyrene but not by ethynylestradiol. Expression of a cDNA for plaice UGT1B in cos7 cells resulted in higher 1-naphthol conjugation in cell homogenates compared to steroid conjugation, whilst bilirubin and bile acid conjugation undetectable. This indicates that the plaice gene codes for the phenol-conjugating UGT previously purified in our laboratory from this species and that it is likely to play a major role in the detoxification of polyaromatic hydrocarbons in flatfish. Its role in development is unknown. UGT1B genes are also present in pufferfish (Tetraodon nigroviridis) and zebrafish (Danio rerio) genomes, but that they differ in their genic organisation. Pufferfish possess multiple (repeated) complete UGT1 genes and Southern blots indicate that the homologous plaice UGT1B gene may also be organised in this way. In contrast, zebrafish appear to have two UGT1 loci whose sequences and intron/exon structures are closely related to that of plaice, however, the organisation of these genes is similar to the mammalian UGT1 family since each has multiple repeated exon 1's which are alternatively spliced to a common set of exons encoding the aglycone binding domain. Taken together with evidence from phylogenetic comparison of fish sequences with UGT1 and UGT2 families in mammals, we suggest these homologous fish UGTs should all be included within the vertebrate UGT1 family and designated as UGT1B.

Hepatic gene expression in flounder chronically exposed to multiply polluted estuarine sediment: Absence of classical exposure ‘biomarker’ signals and induction of inflammatory, innate immune and apoptotic pathways

“…These effects were confirmed by QPCR (Fig 4), along with measurements on other genes which were associated with sediment type (Diablo, transthyretin, hepcidin) and genes which are regularly reported in biomarker studies (UGT1B1, CuZnSOD, glutathone reductase, GSTA) Together these measurements demonstrate that 'clean' and 'polluted' hepatocytes respond similarly, although not identically when treated with the BAP or E2 or Cu. Thus, although BAP treatment caused large increases in both CYP1A and UGT1B, both previously shown to be responsive to BAP in flounder (Leaver et al, 2007), it is apparent that BAP and Cu also increased vitellogenin mRNA levels in 'clean' hepatocyes, but not in 'polluted' hepatocytes. Co-administration of BAP, E2 and Cu caused a similar level of increase in CYP1A and UGT1B as BAP alone, but levels of vitellogenin mRNA were much reduced in comparison to levels seen with E2 alone.…”

“…A sub-sample of each liver was frozen in liquid N 2 for individual analyses. The remainder of each liver was finely chopped and hepatocytes prepared as described previously (Leaver et al, 2007). Following hepatocyte preparation washed cell pellets were resuspended in 10ml of DMEM, 10%FBS, PSK and cell concentration determined in each preparation using a hemocytometer.…”

Characterization of phase-II conjugation reaction of polycyclic aromatic hydrocarbons in fish species: Unique pyrene metabolism and species specificity observed in fish species

“…The conjugated metabolite of PAHs in fish is primarily glucuronic acid (Bayer et al, 2010;Hornung et al, 2007), and it is known that glucuronidation of xenobiotics is performed by the action of UGT (Leaver et al, 2007). For example, in Pleuronectes platessa hepatocytes, the extent of glucuronide formation was higher than sulfate formation of 1-naphthol and phenolphthalein (Morrison et al, 1985).…”

The effects of 4-methylumbelliferone on hyaluronan synthesis, MMP2 activity, proliferation, and motility of human aortic smooth muscle cells

“…As UGTs have typical detoxification properties, it is noteworthy to find these enzymes in cells not belonging to the gastroenteric tract, where UGTs are abundantly expressed. However, UGTs have been found in the circulatory system of birds and fishes although their physiological role is still debated (Bond et al 1980;Yang et al 1986;Leaver et al 2007).…”