Brian Burchell

Genetic variation in bilirubin UDP-glucuronosyltransferase gene promoter and Gilbert's syndrome

BACKGROUNDnThe genetic basis of Gilberts syndrome is ill-defined. This common mild hyperbilirubinaemia sometimes presents as an intermittent jaundice. A reduced hepatic bilirubin UPD- glucuronosyltransferase (UGT) is associated with this syndrome. We have examined variation in the gene encoding the UGT1*1 enzyme and serum bilirubin levels in a Scottish population.nnnMETHODSnBlood was collected from 12 patients with confirmed or suspected Gilberts syndrome, from 6 members of a family with 4 Gilbert members, and from 77 non-smoking, alcohol-free, drug-free volunteers recruited from the staff of a teaching hospital in Dundee. Polymerase chain reaction amplification was used to examine sequence variation of the promoter upstream of the UGT1*1 exon I. Genotypes were assigned as follows: 6/6 (homozygous for a common allele bearing the sequence [TA](6)TAA), 7/7 (homozygous for a rarer allele with the sequence [TA](7)TAA), and 6/7 (heterozygous with one of each allele).nnnFINDINGSnIndividuals in the population with the 7/7 genotype had significantly higher bilirubin concentrations than those who had the 6/7 or 6/6 genotype. 14 volunteers underwent a 24 h fasting test to see if they had Gilberts syndrome, and all four positives had the 7/7 genotype. One confirmed Gilberts patient, two recurrent jaundice patients (with suspected Gilberts syndrome), and nine clinically diagnosed cases had the 7/7 genotype. Segregation of the 7/7 genotype with the Gilbert phenotype was also demonstrated in the family with four affected members. The frequency of the 7/7 genotype in this eastern Scottish population was 10-13%.nnnINTERPRETATIONnIn a healthy population there was an association between variation in bilirubin concentration and a mutation within the gene encoding the enzyme bilirubin UGT. This and other findings suggest the existence of a mild and a more severe form of Gilberts syndrome, depending on whether the gene defect lies in the promoter sequence upstream of UGT1*I exon I, as here (mild), or in the coding sequence (severe) of the gene.

Cloning and fnctional expression of an apparent splice variant of the murine 5-HT3 receptor A subunit

The polymerase chain reaction has been employed to isolate a cDNA encoding a functional 5-HT3 receptor subunit from the murine neuroblastoma cell line N1E-115. Overall, the amino acid sequence predicted from this clone demonstrates a 98% homology with the 5-HT3 receptor A subunit cloned from NCB-20 hybridoma cells. A deletion of 6 amino acid residues located within the putative large intracellular loop, which may result from alternative splicing, represents the principal difference between the two clones. Upon expression in Xenopus oocytes, the homo-oligomeric receptor displayed pharmacological properties which define it as a functional 5-HT3 receptor.

Paracetamol glucuronidation by recombinant rat and human phenol UDP-glucuronosyltransferases

Stably expressed human and rat phenol UDP-glucuronosyltransferases (UGTs) of the UGT1 complex (HlugP1, HlugP4 and 3-methylcholanthrene-inducible rat UGT1A1, the latter considered to be an orthologous enzyme to HlugP1) have been used to investigate the role of UGTs in paracetamol glucuronidation. Kinetic analysis of recombinant UGTs was compared to that of total UGT activities in liver microsomes. Paracetamol was found to be an overlapping substrate of several UGTs. It shows higher affinity for HlugP1 and rat UGT1A1 (apparent Km values of 2 and 3 mM, respectively) than for HlugP4 (Km = 50 mM) and other UGTs present in liver microsomes (Km values of > 12 mM). Glucuronidation of paracetamol with HlugP1 contrasts with that of 6-hydroxychrysene and of 4-methylumbelliferone, which are conjugated with higher affinity by HlugP4 than by HlugP1. Due to the wide tissue distribution of rat UGT1A1, paracetamol glucuronidation was also investigated in extrahepatic rat and human tissues. Paracetamol UGT activity was present and inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat kidney, lung and spleen. It was also detected in human kidney. A selective cDNA probe for exon 1 of HlugP1 cross-reacted with mRNA from both human liver and kidney. The results demonstrate that paracetamol is conjugated by HlugP1 and its rat orthologue UGT1A1 with higher affinity than by HlugP4 and other UGTs.

Coexpression of a human P450 (CYP3A4) and P450 reductase generates a highly functional monooxygenase system in Escherichia coli.

The catalytic activities of recombinant cytochrome P450s expressed in E. coli have been impeded by the absence of endogenous P450 reductase. To solve this problem, we coexpressed P450 reductase with CYP3A4. Membranes from this strain contained 215 pmol P450/mg protein and a reductase activity of 1315 nmol cytochrome c reduced/min per mg. We detected 6β‐hydroxylation of testosterone and oxidation of nifedipine in vivo with turnover numbers of 15.2 and 17.3 min−1, respectively. These values compare favourably with those obtained using an optimally reconstituted system. Our data demonstrate that a catalytically efficient human P450 system can be generated in E. coli.

The structure and function of the UDP-glucuronosyltransferase gene family.

Publisher Summary The UDP-glucuronosyltransferases (UGTs) have evolved to catalyze the glucuronidation of hazardous endogenous compounds such as catecholamines and bilirubin and environmental chemicals. The UGT gene family is a major participant in the chemical defense required by plant and animal environmental warfare. The role of glucuronidation may not be confined to chemical defense, because rat and bovine olfactory epithelia have been shown to contain specific UGT isoforms responsible for the glucuronidation of odorants catalyzing signal termination and excretion of stimulants. An array of chemicals is encountered in the modern environment, where the chemical revolution has outpaced biological evolution of humans leading to serious unpredictable toxicities. Even some conjugates remain biologically active. Nonetheless, the majority of chemicals are effectively glucuronidated by a large family of enzymes. Individual UGTs are differentially regulated in tissues and during development by hormones and xenobiotics, providing a different panel of enzymes for organ-specific function. The chapter discusses UGT1 and UGT2 subfamilies. 20 different human UGT cDNAs have been identified and classified into two subfamilies based on sequence identities. The UGT1 subfamily proteins share an identical C-terminal coding sequence, whereas the N-terminal 246 amino acids were as little as 38% similar. Genetic defects have been observed in the UGT1 gene associated with hyperbilirubinemia. Defects in the coding regions of the gene are responsible for loss of single or many isoforms. Defects in regulatory regions are implicated in polymorphic variation in drug and endobiotic glucuronidation. The UGT2 subfamily has been subdivided into the UGT2A (olfactory-specific isoforms) and the UGT2B (steroid/bile and specific isoforms).10 distinct UGT2B cDNAs have been isolated. UGT2B isoenzymes are encoded by independent genes. There is discussion on substrate specificity of cloned-expressed human liver UGTs. Glucuronidation of drug molecules containing a wide range of acceptor groups has been reported, including phenols, alcohols, acidic carbon atoms, and carboxylic acids.

Genetic analysis of microsomal epoxide hydrolase in patients with carbamazepine hypersensitivity

Carbamazepine therapy is occasionally complicated by hypersensitivity reactions, the mechanism of which is poorly understood. It has been suggested that affected individuals may have a genetically-determined defect of microsomal epoxide hydrolase. The aim of this study was to determine whether a single genetic mutation or pattern of mutations could be used to predict individual susceptibility to carbamazepine-hypersensitivity. DNA was isolated from 10 carbamazepine-hypersensitive patients and 10 healthy volunteers. The patients had developed various forms of toxicity with carbamazepine, including toxic epidermal necrolysis, Stevens-Johnson syndrome, hepatitis and pneumonitis. The technique of polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP) was used to screen for mutations in all nine exons of the microsomal epoxide hydrolase gene. Any new mutations detected by this method were characterised by direct sequencing of the DNA. In addition, in the most severely affected patient, we sequenced all nine exons of the gene. There was a higher frequency of mutations in the hypersensitive group when compared with the controls, but there was no consistent mutation (or pattern of mutations) in the microsomal epoxide hydrolase gene which was common to the hypersensitive group. DNA sequencing of all nine exons of the microsomal epoxide hydrolase gene from the most severely affected patient showed the sequence to be wild-type, when compared to the previously published sequences. The results of this study suggest that a single mutation within the coding region of the microsomal epoxide hydrolase gene cannot be the sole determinant of the predisposition to carbamazepine hypersensitivity.

Formation of mono- and diglucuronides and other glycosides of benzo(a)pyrene-3,6-quinol by V79 cell-expressed human phenol UDP-glucuronosyltransferases of the UGT1 gene complex

Glucuronidation of quinols of polycyclic aromatic hydrocarbons (PAHs) represents an important detoxication pathway preventing toxic quinone/quinol redox cycles. Therefore, mono- and diglucuronide formation of benzo(a)pyrene-3,6-quinol was investigated and compared to that of structurally related 3,6-dihydroxychrysene and simple phenols (1-naphthol and 4-methylumbelliferone) using V79 cell-expressed human UGT1.6 (= P1) and human UGT1.7 (= P4). Properties of human UGT1.6 were compared to those of the rat ortholog. Cofactors related to UDP-glucuronic acid such as UDP-galacturonic acid and UDP-glucose were also studied. It was found that rat and human UGT1.6 and human UGT1.7 catalyse monoglucuronide formation of planar PAH quinols. Diglucuronide formation was only detectable with human UGT1.7. The UGT isozymes studied also formed galacturonides and, although only to a minor extent, glucosides. Rat UGT1.6 (but not the human ortholog) catalysed digalacturonide formation of benzo(a)pyrene-3,6-quinol; the in vivo significance of galacturonide formation remains to be established. The results suggest that planar PAH phenols and quinols are conjugated more efficiently by human UGT1.7 than by UGT1.6, which preferentially conjugates simple planar phenols.

Enhanced clearance of topoisomerase I inhibitors from human colon cancer cells by glucuronidation

As part of a program to identify novel mechanisms of resistance to topoisomerase I (topo I) inhibitors, the cellular pharmacology of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of clinically used irinotecan (CPT-11) and NU/ICRF 505, an anthraquinone-tyrosine conjugate, has been investigated in two human colorectal cancer (CRC) cell lines. Two novel metabolites of NU/ICRF 505 (M1 and M2) and a single metabolite of SN-38 (M1) were detected by high performance liquid chromatography in the culture medium of HT29 cells but were absent in HCT116 cells. Identities of all three metabolites were established by a combination of biochemical and physicochemical techniques. M1 of SN-38 was the C10-(beta)-glucuronide of the parent lactone while M1 of NU/ICRF 505 was the C4-O-glucuronide and M2 the tyrosine-O-glucuronide, both of the parent compound. Drug transport studies revealed that by 24hr HT29 cells had effectively cleared 82.5% of NU/ICRF 505 (10 microM) into the culture medium as the two glucuronides. In contrast, intracellular concentrations of NU/ICRF 505 were maintained in HCT116 cells in the absence of glucuronidation at a level 550 times greater than in HT29 cells. HT29 cells cleared 40.9% of SN-38 (1 microM) as the glucuronide to the culture medium, while the parent drug was maintained at a level 2-fold greater in HCT116 cells. Enhanced drug clearance due to glucuronidation may contribute to intrinsic drug resistance of human CRC.

Chromosomal assignment of human phenol and bilirubin UDP‐glucuronosyltransferase genes (UGT1A‐subfamily)

DNA probes were prepared from the 5′ ‐terminal portion of four cDNA clones encoding human phenol and bilirubin UDP‐glucuronosyltransferases (UGTs). An additional sequence common to all four clones was isolated from the 3′ ‐terminal portion of one of the clones (UGT1A1).

Evaluation of the marmoset as a model species for drug glucuronidation

1. The in vitro glucuronidation of a wide range of compounds has been studied in microsomes prepared from marmoset liver and kidney. These studies have been undertaken to evaluate the marmoset as a model species for drug glucuronidation and for comparison with conjugation by other species. 2. The compounds studied were glucuronidated by marmoset liver microsomes to varying extents (e.g. naproxen CL int 0.4 µlu2009min -1u2009mg -1, 1-naphthol CLint 43 µlu2009min -1u2009mg -1). Both marmoset and rat liver microsomes glucuronidated morphine at the 3-position (marmoset CL int 19 µlu2009min -1u2009mg -1, rat CL int 6.3 µlu2009min -1u2009mg -1) but glucuronidation at the 6-position was below the level of radiodetection in both species. 3. Interestingly, marmoset liver microsomes were able to catalyse the glucuronidation of the tertiary amine imipramine to a significant extent (0.05 nmolu2009min -1u2009mg -1). However, no glucuronidation was detected by rat liver microsomes. 4. Conjugation of a range of substrates was detectable by marmoset kidney microsomes in contrast to rat kidney microsomes, which only catalysed the glucurondation of bilirubin and 1-naphthol (CLint 17 µlu2009min -1u2009mg -1 and 18 µlu2009min -1u2009mg -1, respectively). 5. This report and previous work in dog and human tissue microsomes suggest that the marmoset may be an alternative animal model for human drug glucuronidation, especially when the pathway of drug glucuronidation is known to differ between lower laboratory species and man.