Development of phenobarbital-sensitive control mechanisms for uridine diphosphate glucuronyltransferase activity in chick embryo liver.

Uridine diphosphate (UDP) glucuronyltransferase activity in chick liver rises at hatching from near zero to adult levels. This rise will occur prematurely in embryo liver during organ culture. Increase in enzyme activity during organ culture differs with embryo age: in liver from 11-day old embryos it ceases at adult values; in liver from 5-day old embryos it continues to much higher-than-adult levels. Phenobarbital added to culture medium accelerates these rises in enzyme activity and elevates the plateau reached in 11-day embryo liver to that observed in 5-day embryo liver. Kinetic analysis of the changes in enzyme activity induced by phenobarbital during culture suggests that the regulatory mechanisms for enzyme activity are different in 5- and 11-day embryo liver and that these differences reflect developmental changes occurring in ovo.

Preclinical prediction of factors influencing the elimination of 5,6-dimethylxanthenone-4-acetic acid, a new anticancer drug.

The glucuronidation of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a newly developed anticancer drug, was investigated in vitro to determine factors likely to affect the elimination of this compound in patients. Human liver microsomal DMXAA glucuronidation followed Michaelis-Menten kinetics, with a mean apparent Km of approximately 100 microM. Two cDNA-expressed UGT isoforms, UGT1*02 and UGT2B7, had the capacity to glucuronidate DMXAA, although comparative kinetic and inhibitor studies were more consistent with a greater contribution of UGT2B7 to the human hepatic reaction. Microsomal DMXAA glucuronide formation was screened for inhibition by drugs known to be eliminated by glucuronidation. Of the drugs screened, significant inhibition was observed with diclofenac, epirubicin, indomethacin, R,S-ketoprofen, lorazepam, S-naproxen, oxazepam, and temazepam; apparent Ki values ranged from 9.5-318 microM. These values are substantially above unbound concentrations of the individual drugs achieved in vivo. DMXAA glucuronide was found to be unstable at physiological pH values, and the rate of degradation was marginally increased in the presence of albumin. Taken together, these data indicate that the kinetics of DMXAA glucuronidation in vivo are likely to be linear and unaffected by the coadministration of most glucuronidated drugs, but plasma DMXAA clearance may be decreased in patients with renal dysfunction. This study illustrates the utility of in vitro techniques for the prediction of potential drug interactions and other dispositional characteristics of newly developed anticancer drugs before their administration to patients.

Latency of epoxide hydratase and its relationship to that of UDPglucuronyltransferase.

Epoxide hydratase activity in liver microsomal preparations from adult made rats is latent to a slight extent. Maximal activations with neutral or anionic detergents were 30-60% whilst UDPglucuronyltransferase was maximally activated by 160-830% by the same detergents. Activation of microsomal epoxide hydratase requires much higher amounts of neutral or anionic detergents than activation of microsomal UDPglucuronyltransferase. High concentrations of inorganic salt, sonication or freeze-thawing which activate microsomal UDPglucuronyltransferase have no influence on microsomal epoxide hydratase activity. From this it appears that the activation which may involve either removal of a permeability barrier or release from conformational restraint occurs more easily for UDPglucuronyltransferase than for epoxide hydratase and that the activation of microsomal epoxide hydratase requires breakage of some hydrophobic bonds between the enzyme and membrrane component(s).

Evidence indicating that UDP-N-acetylglucosamine does not appear to stimulate hepatic microsomal UDP-glucuronosyltransferase by interaction with the catalytic unit of the enzyme.

The binding studies in this paper indicate that the catalytic unit(s) of microsomal UDP glucuronosyltransferase(s) is not accessible to N-ethylmaleimide or UDP-N-acetylglucosamine, when the enzyme is in its membrane environment. Thus a separate regulatory factor may exist within the endoplasmic reticulum membrane that mediates the stimulation of UDPglucuronosyltransferase(s) by UDP-N-acetylglucosamine. The possible role and the mode of interaction of the putative regulatory factor with the multiple forms of UDPglucuronosyltransferase are discussed.

Membrane topology of epoxide hydrolase.

The amino acid sequences of epoxide hydrolase from rat, rabbit and human have been subjected to hydropathy analysis and a novel model for the membrane topology of this enzyme is presented. The enzyme would appear to be retained in microsomal membranes by a single transmembrane segment located at the N-terminus and the majority (96%) of the protein is exposed at the cytosolic membrane surface. This model is significantly different from a scheme suggested by analysis of the rat enzyme alone which proposed six transmembrane domains (Porter et al. (1988) Arch. Biochem. Biophys. 248, 121-129). Experiments with rat microsomal membranes were conducted to distinguish between the two models and used proteolytic enzymes and non-permeant chemical probes. Epoxide hydrolase of intact and permeabilised membranes was resistant to digestion by a number of proteinases. However, this is likely to be related to a compact fold of the protein rather than membrane association since purified, delipidated enzyme preparations were also resistant to proteolysis. While the use of proteinases did not provide useful membrane topological information, experiments with the fluorescent probe, 3-azido-2,7-naphthalenedisulphonate strongly support the view that the majority of the protein is indeed exposed at the cytosolic surface of the membranes. The analysis illustrates the caution which must be employed in the formulation of topological models based on hydropathy plots alone and the value of considering homologous proteins.

UDP-glucuronosyltransferases: a family of detoxifying enzymes.

Glucuronidation is an important process in the metabolism of xenobiotic and endogenous substances leading to enhancement of excretion of these compounds from the body. A multigene family encodes a number of UDP-glucuronosyltransferase enzymes which catalyse this route of metabolism. Recent advances in biochemical and molecular biological approaches, reviewed here by Thomas Tephly and Brian Burchell, have given new insight into the function and structure of UDP-glucuronosyltransferases. These proteins have surprising similarities and yet appear to be capable of conjugating a remarkable number of different chemicals.

Characterization of the uridine diphosphate-glucuronosyltransferase-catalyzing thyroid hormone glucuronidation in man.

Increased thyroid hormone glucuronidation in rats caused by exposure to xenobiotics has stimulated a search for the individual uridine diphosphate-glucuronosyltransferases (UGTs) catalyzing this reaction in rats and man. Microsomal preparations from Crigler-Najjar liver, normal human liver, and kidney have been used to try to identify the UGT isoforms responsible for glucuronidation of the thyroid hormones. The predominant thyroid hormone released from the thyroid gland, T4, and the inactive rT3 are glucuronidated by cloned expressed bilirubin UGT1A1 and also phenol UGT1A9. Results from Crigler-Najjar microsomal samples indicate that UGT1A1 is the main contributor to thyroid hormone glucuronidation in the liver, with rT3 being the preferential substrate. In kidney microsomes thyroid hormone glucuronidation is more complex, suggesting that more than just the UGT1A9 isoform may be involved. Bioactive T3 is not significantly glucuronidated by these isoforms and other UGTs, and sulfotransferases may be involved.

DD angiotensin-converting enzyme gene polymorphism is associated with endothelial dysfunction in normal humans.

A polymorphism within the angiotensin-converting enzyme (ACE) gene may increase the risk of myocardial infarction in individuals previously thought to be at low cardiovascular risk. The mechanism through which it exerts this effect is unknown but may be due to increased angiotensin II-induced nitric oxide (NO) breakdown and/or reduced bradykinin-mediated NO release. We investigated whether endothelial function was different between different ACE genotypes. We performed a cross-sectional study comparing the endothelial function of the 3 genotypes (II: n=25; ID: n=31; DD: n=12). Mean+/-SD ages of the subjects were 24+/-4 (II), 25+/-6 (ID), and 25+/-6 (DD) years. We assessed the impact of the genotypes on endothelial function and found that the DD genotype was associated with a significant blunting in endothelial-dependent vasodilatation (forearm blood flow data are presented as mean+/-SD ratio of blood flow in response to 3 incrementally increasing doses of each vasoactive agent in the test arm to blood flow in the control arm; the comparison is between DD versus ID versus II; the P value is an expression of an overall difference by ANOVA, and the 95% CIs are of a pairwise comparison between genotypes): acetylcholine, 2.88+/-1.45 versus 3.81+/-1.93 versus 4.23+/-2.37 (P=0.002; 95% CI [II versus ID], -0.19 to 0.91; 95% CI [II versus DD], 0.36 to 1.80; 95% CI [ID versus DD], 0.02 to 1.42). There was also a significant difference with the endothelial-independent vasodilator sodium nitroprusside, with values of 2.11+/-1.00 versus 2.55+/-1.36 versus 2.75+/-1.18 (P<0.05; 95% CI [II versus ID], -0.15 to 0.51; 95% CI [II versus DD], 0.03 to 0.89; 95% CI [ID versus DD], -0.13 to 0.71), but not with verapamil. There was no effect of the ACE genotype on endothelial-dependent or -independent vasoconstrictors NG-monomethyl-L-arginine or norepinephrine. Investigating the effects of cigarette smoking on each genotype demonstrated that for II and DD genotypes, acetylcholine responses were further blunted if subjects smoked. These data demonstrate that the DD ACE genotype in a young population is associated with a blunting of stimulated endothelial NO and donated NO responses but not to non-NO vasodilators or vasoconstrictors.

Tissue distribution and interindividual variation in human UDP-glucuronosyltransferase activity: relationship between UGT1A1 promoter genotype and variability in a liver bank.

The variability in a liver bank and tissue distribution of three probe UDP-glucuronosyltransferase (UGT) activities were determined as a means to predict interindividual differences in expression and the contribution of extrahepatic metabolism to presystemic and systemic clearance. Formation rates of acetaminophen-O-glucuronide (APAPG), morphine-3-glucuronide (M3G), and oestradiol-3-glucuronide (E3G) as probes for UGT1A6, 2B7, and 1A1, respectively, were determined in human kidney, liver, and lung microsomes, and in microsomes from intestinal mucosa corresponding to duodenum, jejunum and ileum. While formation of E3G and APAPG were detectable in human kidney microsomes, M3G formation rates from kidney microsomes approached the levels seen in liver, indicating significant expression of UGT2B7. Interestingly, rates of E3G formation in human intestine exceeded the hepatic rates by several fold, while APAPG and M3G formation rates were low. The intestinal apparent Km value for E3G formation was essentially identical to that seen in liver, consistent with intestinal UGT1A1 expression. No UGT activities were observed in lung. Variability in APAPG and M3G activity across a bank of 20 human livers was modest (< or = 7-fold), compared to E3G formation, which varied approximately 30-fold. The E3G formation rates were found to segregate by UGT1A1 promoter genotype, with wild-type (TA)6 rates significantly greater than homozygous mutant (TA)7 individuals. Kinetic analyses were performed to demonstrate that the promoter mutation altered apparent Vmax without significantly affecting apparent Km. These results suggest that glucuronidation, and specifically UGT1A1 activity, can profoundly contribute to intestinal first pass metabolism and interindividual variability due to the expression of common allelic variants.

Functional co-expression of CYP2D6 and human NADPH-cytochrome P450 reductase in Escherichia coli.

The polymorphic human CYP2D6 has been co-expressed with human NADPH-cytochrome P450 oxidoreductase in Escherichia coli in order to generate a functional recombinant monooxygenase system for the study of xenobiotic metabolism. The two cDNAs were co-expressed from separate, compatible plasmids with different antibiotic selection markers. The CYP2D6 could be detected in bacterial cells at levels up to 700 nmol I-1 culture by Fe(2+)-CO versus Fe2+ difference spectroscopy, exhibiting the characteristic absorbance peak at 450 nm. Immunoblotting demonstrated the presence of both proteins in bacterial membranes, where they were expressed at levels significantly higher than those found in human liver microsomes. Membrane content was 150-200 pmol CYP2D6 (determined spectrally) and 100-230 pmol CYP-reductase (determined enzymatically) per mg protein. Critically, the two co-expressed proteins were able to couple to form a NADPH-dependent monooxygenase which metabolized the CYP2D6 substrate bufuralol (Vmax 3.30 nmol min-1 mg-1 protein; K(m) 11.1 microM) in isolated membrane fractions. This K(m) value was similar to the K(m) determined in human liver microsomes. Activity could be inhibited by the specific inhibitor quinidine. Of greater significance however, was the finding that intact E. coli cells, even in the absence of exogenous NADPH, were able to metabolize bufuralol at rates almost as high as those measured in membranes (4.6 +/- 0.4 min-1 versus 5.7 +/- 0.2 min-1 at 50 microM substrate). Such recombinant strains will greatly facilitate the molecular characterization of allelic variants of cytochrome P450 isoenzymes.

The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence.

This review represents an update of the nomenclature system for the UDP glucuronosyltransferase gene superfamily, which is based on divergent evolution. Since the previous review in 1991, sequences of many related UDP glycosyltransferases from lower organisms have appeared in the database, which expand our database considerably. At latest count, in animals, yeast, plants and bacteria there are 110 distinct cDNAs/genes whose protein products all contain a characteristic 'signature sequence' and, thus, are regarded as members of the same superfamily. Comparison of a relatedness tree of proteins leads to the definition of 33 families. It should be emphasized that at least six cloned UDP-GlcNAc N-acetylglucosaminyltransferases are not sufficiently homologous to be included as members of this superfamily and may represent an example of convergent evolution. For naming each gene, it is recommended that the root symbol UGT for human (Ugt for mouse and Drosophila), denoting 'UDP glycosyltransferase,' be followed by an Arabic number representing the family, a letter designating the subfamily, and an Arabic numeral denoting the individual gene within the family or subfamily, e.g. 'human UGT2B4' and 'mouse Ugt2b5'. We recommend the name 'UDP glycosyltransferase' because many of the proteins do not preferentially use UDP glucuronic acid, or their nucleotide sugar preference is unknown. Whereas the gene is italicized, the corresponding cDNA, transcript, protein and enzyme activity should be written with upper-case letters and without italics, e.g. 'human or mouse UGT1A1.' The UGT1 gene (spanning > 500 kb) contains at least 12 promoters/first exons, which can be spliced and joined with common exons 2 through 5, leading to different N-terminal halves but identical C-terminal halves of the gene products; in this scheme each first exon is regarded as a distinct gene (e.g. UGT1A1, UGT1A2, ... UGT1A12). When an orthologous gene between species cannot be identified with certainty, as occurs in the UGT2B subfamily, sequential naming of the genes is being carried out chronologically as they become characterized. We suggest that the Human Gene Nomenclature Guidelines (http://www.gene.acl.ac.uk/nomenclature/guidelines.html++ +) be used for all species other than the mouse and Drosophila. Thirty published human UGT1A1 mutant alleles responsible for clinical hyperbilirubinemias are listed herein, and given numbers following an asterisk (e.g. UGT1A1*30) consistent with the Human Gene Nomenclature Guidelines. It is anticipated that this UGT gene nomenclature system will require updating on a regular basis.

Cloning of cDNAs coding for rat hepatic microsomal UDP-glucuronyltransferases.

Radioiodinated, affinity-purified, anti-UDP-glucuronyltransferase (UDPGT) antibodies have been used to isolate cDNAs coding for UDPGT(s) from a rat liver cDNA library cloned in the expression vector bacteriophage lambda gt11. The sizes of ten cloned cDNAs range from 0.3-2.1 kb. The identity of the cDNAs was confirmed by the hybrid-select translation and immunochemical analyses. Restriction mapping indicates that two classes of cDNA coding for different UDPGT mRNAs have been cloned.

Congenital jaundice in rats due to the absence of hepatic bilirubin UDP-glucuronyltransferase enzyme protein.

Three major UDP-glucuronyltransferase isoenzymes (50-54 kDa) have been identified by immunoblot analysis. Bilirubin UDP-glucuronyltransferase (54 kDa) was specially induced by treatment of the rats with clofibrate. This isoenzyme was not detectable in liver microsomal extracts from congenitally jaundiced Gunn rats and was not induced by treatment of these animals with clofibrate. Phenol UDP-glucuronyltransferase, the only isoenzyme determined to be present in foetal Wistar rat liver microsomes was not detected by enzyme assay or immunoblot analysis of foetal Gunn rat liver microsomal extracts. These results provide the first indication that bilirubin UDP-glucuronyltransferase and possible phenol UDP-glucuronyltransferase proteins are not present in the congenitally jaundiced Gunn rat.

Isolation and characterisation of a new hepatic bilirubin UDP-glucuronosyltransferase. Absence from Gunn rat liver.

A novel UDP-glucuronosyltransferase that conjugates bilirubin IX alpha, bilirubin monoglucuronide and an arylalkanoic acid was purified to homogeneity from clofibrate treated Wistar rats. The enzyme displayed a subunit molecular mass of 54 kDa, a pI of 7.6 and was demonstrated to be N-glycosylated. Sequence analysis of peptides derived by endoproteinase Glu-C cleavage of the purified enzyme indicated that it was a new member of the recently identified UGT1 subfamily. Immunoblot analysis demonstrated that this enzyme was absent from Gunn rat liver. The molecular derivation of this enzyme and the lack of it in Gunn rats is discussed.

The molecular basis of the inherited deficiency of androsterone UDP-glucuronyltransferase in Wistar rats.

A major UDP-glucuronyltransferase isoenzyme in rat liver (51 kDa), corresponding to androsterone glucuronidating activity, has been identified by immunoblot analysis. This isoenzyme is absent from Wistar rats exhibiting the low androsterone (LA) UDP-glucuronyltransferase activity exhibiting the low androsterone (LA) UDP-glucuronyltransferase activity phenotype. Northern blot analysis of total RNA from normal and androsterone glucuronidation deficient Wistar rats demonstrated that the mRNA encoding this protein was not synthesised. Differences in restriction fragment length observed on Southern blotting of genomic DNA from LA Wistar rats indicate that this inherited deficiency is the result of a deletion in the androsterone UDP-glucuronyltransferase gene.

Expression of a human liver cDNA encoding a UDP-glucuronosyltransferase catalysing the glucuronidation of hyodeoxycholic acid in cell culture.

A cDNA encoding a human liver UDPGT (HLUG 25) transcribed and translated in vitro showed that the encoded protein was synthesized as a precursor and was cleaved and glycosylated when dog pancreatic microsomes were present during translation. The UDPGT cDNA was transiently expressed in mammalian cell culture (COS-7 cells) resulting in the biosynthesis of a polypeptide of 52 kDa. This expressed UDPGT glycoprotein catalysed the glucuronidation of hyodeoxycholic acid forming an ether glucuronide. These results suggest that this UDPGT isoenzyme may be responsible for the glucuronidation of 6 alpha-hydroxy bile acids in human liver.

Expression of a functionally active human hepatic UDP-glucuronosyltransferase (UGT1A6) lacking the N-terminal signal sequence in the endoplasmic reticulum.

UDP-glucuronosyltransferase 1A6 (UGT1A6) is a membrane glycoprotein of the endoplasmic reticulum playing a key role in drug metabolism. It is synthesized as a precursor with an N-terminal cleavable signal peptide. We demonstrate that deletion of the signal peptide sequence does not prevent membrane targeting and integration of this human isoform when expressed in an in vitro transcription-translation system, as shown by N-glycosylation, resistance to alkaline treatment and protease protection. Furthermore, UGT1A6 lacking the signal peptide (UGT1A6delta sp) was targeted to the endoplasmic reticulum in mammalian cells as shown by immunofluorescence microscopy and was catalytically active with kinetic constants for 4-methylumbelliferone glucuronidation similar to that of the wild-type. These results provide evidence that the signal peptide is not essential for the membrane assembly and activity of UGT1A6 suggesting that additional topogenic element(s) mediate(s) this process.

Characterisation of a human bilirubin UDP-glucuronosyltransferase stably expressed in hamster lung fibroblast cell cultures.

A cDNA encoding a human bilirubin UDP-glucuronosyltransferase has been isolated and stably expressed in Chinese hamster V79 lung fibroblast cell line. Western blotting of cell homogenates with anti-UGT antibody revealed a highly expressed protein of approx. 55.5 kDa in size. The expressed enzyme specifically catalysed the formation of bilirubin mono- and diglucuronides, and also catalysed the glucuronidation of two phenolic compounds, which are good substrates for other human UGT isoenzymes, at low rates.

Expression of the human UDP-glucuronosyltransferase UGT1*6 in Escherichia coli. Influence of bacterial signal peptides on the production and localization of the recombinant protein.

The membrane-bound human liver UDP-glucuronosyltransferase UGT1*6 was expressed in Escherichia coli. Exchange of the natural signal peptide by the bacterial signal peptides of pclB or OmpT proteins considerably increased the level of expression and, as the natural signal peptide, targeted the protein to the membranes. The extent of maturation of SpelB-UGT1*6 precursor was about 30%. No processing of sOmpT-UGT1*6 occurred but the processing rate of this precursor could be significantly increased by mutagenesis of the first two amino acid residues of the mature sequence. These expression vectors allowed us to produce high levels of recombinant mature UGT1*6 required for further structural studies.