UDP-glucuronosyltransferase Isoenzymes Research Articles

Drug-drug interaction potential of antitumor acridine agent C-1748: The substrate of UDP-glucuronosyltransferases 2B7, 2B17 and the inhibitor of 1A9 and 2B7

BackgroundThe compound 9-(2′-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748), the promising antitumor agent developed in our laboratory was determined to undergo phase I metabolic pathways. The present studies aimed to know its biotransformation with phase II enzymes — UDP-glucuronosyltransferases (UGTs) and its potential to be engaged in drug-drug interactions arising from the modulation of UGT activity.MethodsUGT-mediated transformations with rat liver (RLM), human liver (HLM), and human intestine (HIM) microsomes and with 10 recombinant human isoenzymes were investigated. Studies on the ability of C-1748 to inhibit UGT were performed with HLM, HT29 colorectal cancer cell homogenate and the selected recombinant UGT isoenzymes. The reactions were monitored using HPLC-UV/Vis method and the C-1748 metabolite structure was determined with ESI-TOF-MS/MS analysis.ResultsPseudo-molecular ion (m/z 474.1554) and the experiment with β-glucuronidase indicated that O-glucuronide of C-1748 was formed in the presence of microsomal fractions. This reaction was selectively catalyzed by UGT2B7 and 2B17. High inhibitory effect of C-1748 was shown towards isoenzyme UGT1A9 (IC50 = 39.7 μM) and significant but low inhibitory potential was expressed in HT29 cell homogenate (IC50 = 84.5 μM). The mixed-type inhibition mechanism (Ki = 17.0 μM; Ki ‘= 81.0 μM), induced by C-1748 was observed for recombinant UGT1A9 glucuronidation, whereas HT29 cell homogenate resulted in noncompetitive inhibition (Ki = 94.6 μM).ConclusionsThe observed UGT-mediated metabolism of C-1748 and its ability to inhibit UGT activity should be considered as the potency for drug resistance and drug-drug interactions in the prospective multidrug therapy.

In vitro stereoselective inhibition of ginsenosides toward UDP-glucuronosyltransferase (UGT) isoforms

We evaluated in vitro, the potential of the six pairs of ginsenoside isomers, stereoisomers at the chiral carbon on position 20, to inhibit the enzymatic activity of several UDP-glucuronosyltransferase (UGT) isoenzymes, major players in the human phase II drug metabolism. The results show that the tested six pairs of ginsenoside isomers exhibited stereoselective inhibitory effects of varying degrees on the ten UGT isoenzymes explored. Of the tested twelve stereoselective ginsenosides, 20(R)-Rg3 had the strongest inhibitory effect on the UGT1A8 isoform with the lowest IC50 value of 5.66±1.04μM. On the other hand, the (S)-isomers of Rg3 and Rh2 also exerted remarkable inhibition on UGT1A8, with IC50 values of 6.89±0.812μM and 5.85±0.821μM, respectively. Although the inhibitory effect was low, both 20(R)-PPT and 20(S)-PPT also inhibited UGT1A8 activity. Considering 1) that the relative contents of 20(R)-Rg3 in processed ginseng are high, 2) that higher exposure to (R)-isomers of ginsenosides occur in the intestine compared to that in the liver, and 3) the inhibitory effects of other ginsenosides on enzymatic activity [20(S)-Rg3, 20(S)-Rh2, 20(R)- and 20(S)-PPT], there may be a potential for herb-drug interactions between processed ginseng and UGT1A8 substrates when concomitantly administered.

In Vitro Glucuronidation of Fenofibric Acid by Human UDP-Glucuronosyltransferases and Liver Microsomes

Fenofibric acid (FA), the active moiety of fenofibrate, is an agonist of the peroxisome proliferator-activated nuclear receptor alpha that modulates triglyceride and cholesterol profiles. Lipid response to fenofibrate and FA serum concentrations is highly variable. Although FA is reported to be almost exclusively inactivated by UDP-glucuronosyltransferases (UGTs) into FA-glucuronide (FA-G), the contribution of UGT isoenzymes has never been systematically assessed. Heterologously expressed human UGT1A and UGT2B and their coding variants were tested for FA glucuronidation using liquid chromatography/mass spectrometry. Recombinant UGT2B7 presented the highest V(max)/K(m) value (2.10 microl/min/mg), 16-fold higher than the activity of other reactive UGTs, namely, UGT1A3, UGT1A6, and UGT1A9 (0.13, 0.09, and 0.02 microl/min/mg, respectively). UGT2B7.1 (His(268)) and UGT2B7.2 (Tyr(268)) enzyme activity was similar, whereas UGT1A3.2 (R(11)A(47)), UGT1A3.3 (Trp(11)), and UGT1A9.3 (Thr(33)) showed 61 to 96% reduced V(max)/K(m) values compared with the respective (1) reference proteins. FA-G formation by a human liver bank (n = 48) varied by 10-fold, but the rate of formation was not associated with common genetic variations in UGT1A3, UGT1A6, UGT1A9, and UGT2B7. Correlation with activities for the probe substrates zidovudine (UGT2B7; r(2) = 0.75), mycophenolic acid (UGT1A9; r(2) = 0.42), fulvestrant (UGT1A3; r(2) = 0.36), but not serotonin (UGT1A6; r(2) = 0.06) indicated a primary role for UGT2B7 and lesser roles of UGT1A9 and UGT1A3 in hepatic FA glucuronidation. This was confirmed by a strong correlation of FA-G formation with UGT2B7 protein content and inhibition by fluconazole, a known UGT2B7 selective inhibitor. Additional studies are required to identify genetic factors contributing to the observed FA glucuronidation variability.

The kinetic basis for age-associated changes in quercetin and genistein glucuronidation by rat liver microsomes

The dietary bioavailability of the isoflavone genistein is decreased in older rats compared to young adults. Since flavonoids are metabolized extensively by the UDP-glucuronosyltransferases (UGTs), we hypothesized that UGT flavonoid conjugating activity changes with age. The effect of age on flavonoid glucuronidation was determined using hepatic microsomes from male F344 rats. Kinetic models of UGT activity toward the flavonol quercetin and the isoflavone genistein were established using pooled hepatic microsomal fractions of rats at different ages, and glucuronidation rates were determined using individual samples. Intrinsic clearance ( V max/ K m) values in 4-, 18- and 28-month-old rats were 0.100, 0.078 and 0.087 ml/min/mg for quercetin-7- O-glucuronide; 0.138, 0.133 and 0.088 for quercetin-3′- O-glucuronide; and 0.075, 0.077 and 0.057 for quercetin-4′- O-glucuronide, respectively. While there were no differences in formation rates of total quercetin glucuronides in individual samples, the production of the primary metabolite, quercetin-7- O-glucuronide, at 30 μM quercetin concentration was increased from 3.4 and 3.1 nmol/min/mg at 4 and 18 months to 3.8 nmol/min/mg at 28 months, while quercetin-3′- O-glucuronide formation at 28 months declined by a similar degree ( P≤.05). At 30 and 300 μM quercetin concentration, the rate of quercetin-4′- O-glucuronide formation peaked at 18 months at 0.9 nmol/min/mg. Intrinsic clearance values of genistein 7- O-glucuronide increased with age, in contrast to quercetin glucuronidation. Thus, the capacity for flavonoid glucuronidation by rat liver microsomes is dependent on age, UGT isoenzymes and flavonoid structure.

Irinotecan Therapy in a 12-year-old Girl with Recurrent Brain Stem Glioma and without Functional Polymorphisms in UGT1A1 Activity: Case Report

A 10-year-old girl was diagnosed with astrocytoma grade 2. Immuno-chemo-radiotherapy (interferon, ranimustine, and radiation), second-line chemotherapy (carboplatin and etoposide, 7 cycles) and third-line chemotherapy (ifosfamide, carboplatin, and etoposide) was given to treat progressive disease. Finally, irinotecan therapy was initiated and led to dramatic clinical improvement. Irinotecan is metabolized by carboxylesterase to form an active SN-38, which is further conjugated and detoxified by UDP-glucuronosyltransferase (UGT) to yield its beta-glucuronide. The polymorphic UGT isoenzyme, UGT1A1 has genetic variants which decrease in SN-38 glucuronidating capacity and could help predict irinotecan-associated toxicity. The patient suffered excessive toxicity with low-dose irinotecan although no functional polymorphism in UGT1A1 was identified. We suggest that irinotecan offers an effective treatment option for children with recurrent brain stem glioma and other genetic variants except UGT1A1 may be a risk factor for irinotecan-induced toxicity.

Common human UGT1A polymorphisms and the altered metabolism of irinotecan active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38).

7-Ethyl-10-hydroxycamptothecin (SN-38) is the pharmacologically active metabolite of irinotecan, in addition to being responsible for severe toxicity. Glucuronidation is the main metabolic pathway of SN-38 and has been shown to protect against irinotecan-induced gastrointestinal toxicity. The purpose of this study was to determine whether common polymorphic UDP-glucuronosyltransferase (UGT) affects SN-38 glucuronidation. First, kinetic characterization of SN-38-glucuronide (SN-38-G) formation was assessed for all known human UGT1A and UGT2B overexpressed in human embryonic kidney 293 cells. To assess the relative activity of UGT isoenzymes for SN-38, rates of formation of SN-38-G were monitored by liquid chromatography/mass spectrometry analysis and normalized by level of UGT cellular expression. Determination of intrinsic clearances predicts that hepatic UGT1A1 and UGT1A9 and the extrahepatic UGT1A7 are major components in SN-38-G formation, whereas a minor role is suggested for UGT1A6, UGT1A8, and UGT1A10. In support of the involvement of UGT1A9, a strong coefficient of correlation was observed in the glucuronidation of SN-38 and a substrate, mainly glucuronidate, by UGT1A9 (flavopiridol) by human liver microsomes (coefficient of correlation, 0.905; p = 0.002). In vitro functional experiments revealed a negative impact of the UGT1A1 allelic variants. Residual activities of 49, 7, 8, and 11% were observed for UGT1A1*6 (G(71)R), UGT1A1*27 (P(229)Q), UGT1A1*35 (L(233)R), and UGT1A1*7 (Y(486)D), respectively. Common variants of UGT1A7, UGT1A7*3 (N(129)K;R(131)K;W(208)R), and UGT1A7*4 (W(208)R), displayed residual activities of 41 and 28% compared with the UGT1A7*1 allele. Taken together, these data provide the evidence that molecular determinants of irinotecan response may include the UGT1A polymorphisms studied herein and common genetic variants of the hepatic UGT1A9 isoenzyme yet to be described.

Regioselectivity of Phase II Metabolism of Luteolin and Quercetin by UDP-Glucuronosyl Transferases

The regioselectivity of phase II conjugation of flavonoids is expected to be of importance for their biological activity. In the present study, the regioselectivity of phase II biotransformation of the model flavonoids luteolin and quercetin by UDP-glucuronosyltransferases was investigated. Identification of the metabolites formed in microsomal incubations with luteolin or quercetin was done using HPLC, LC-MS, and (1)H NMR. The results obtained demonstrate the major sites for glucuronidation to be the 7-, 3-, 3'-, or 4'-hydroxyl moiety. Using these unequivocal identifications, the regioselectivity of the glucuronidation of luteolin and quercetin by microsomal samples from different origin, i.e., rat and human intestine and liver, as well as by various individual human UDP-glucuronosyltransferase isoenzymes was characterized. The results obtained reveal that regioselectivity is dependent on the model flavonoid of interest, glucuronidation of luteolin and quercetin not following the same pattern, depending on the isoenzyme of UDP-glucuronosyltransferases (UGT) involved. Human UGT1A1, UGT1A8, and UGT1A9 were shown to be especially active in conjugation of both flavonoids, whereas UGT1A4 and UGT1A10 and the isoenzymes from the UGTB family, UGT2B7 and UGT2B15, were less efficient. Due to the different regioselectivity and activity displayed by the various UDP-glucuronosyltransferases, regioselectivity and rate of flavonoid conjugation varies with species and organ. Qualitative comparison of the regioselectivities of glucuronidation obtained with human intestine and liver microsomes to those obtained with human UGT isoenzymes indicates that, in human liver, especially UGT1A9 and, in intestine, UGT1A1 and UGT1A8 are involved in glucuronidation of quercetin and luteolin. Taking into account the fact that the anti-oxidant action as well as the pro-oxidant toxicity of these catechol-type flavonoids is especially related to their 3',4'-dihydroxyl moiety, it is of interest to note that the human intestine UGT's appear to be especially effective in conjugating this 3',4' catechol unit. This would imply that upon glucuronidation along the transport across the intestinal border, the flavonoids loose a significant part of these biological activities.

N-Glucuronidation of some 4-arylalkyl-1H-imidazoles by rat, dog, and human liver microsomes.

N-Glucuronidation in vitro of six 4-arylalkyl-1H-imidazoles (both enantiomers of medetomidine, detomidine, atipamezole, and two other closely related compounds) by rat, dog, and human liver microsomes and by four expressed human UDP-glucuronosyltransferase isoenzymes was studied. Human liver microsomes formed N-glucuronides of 4-arylalkyl-1H-imidazoles with high activity, with apparent V(max) values ranging from 0.59 to 1.89 nmol/min/mg of protein. In comparison, apparent V(max) values for two model compounds forming the N-glucuronides 4-aminobiphenyl and amitriptyline were 5.07 and 0.56 nmol/min/mg of protein, respectively. Atipamezole showed an exceptionally low apparent K(m) value of 4.0 microM and a high specificity constant (V(max)/K(m)) of 256 compared with 4-aminobiphenyl (K(m), 265 microM; V(max)/K(m), 19) and amitriptyline (K(m), 728 microM; V(max)/K(m), 0.8). N-Glucuronidation of medetomidine was highly enantioselective in human liver microsomes; levomedetomidine exhibited a 60-fold V(max)/K(m) value compared with dexmedetomidine. Furthermore, two isomeric imidazole N-glucuronides were formed from dexmedetomidine, but only one was formed from levomedetomidine. Dog liver microsomes formed N-glucuronides of 4-arylalkyl-1H-imidazoles at a low rate and affinity, with apparent V(max) values ranging from 0.29 to 0.73 nmol/min/mg of protein and apparent K(m) values from 279 to 1640 microM. Rat liver microsomes glucuronidated these compounds at a barely detectable rate. Four expressed human UDP-glucuronosyltransferase isoenzymes (UGT1A3, UGT1A4, UGT1A6, and UGT1A9) were studied for 4-arylalkyl-1H-imidazole-conjugating activity. Only UGT1A4 glucuronidated these compounds at an activity of about 5% of that measured for 4-aminobiphenyl. The observed activity of UGT1A4 does not explain the high efficiency of glucuronidation of 4-arylalkyl-1H-imidazoles in human liver microsomes.

Characterization of the UDP-glucuronosyltransferase isoenzyme expressed in rat ovary and its regulation by gonadotropins.

Earlier studies have demonstrated that phenol UDP-glucuronosyltransferase (UGT) activity is up-regulated by pregnant mare's serum gonadotropin (PMSG) in rat ovary, but not liver. This phenomenon was investigated in more detail in the present study. Ovaries and livers of immature rats, rats synchronized with respect to their preovulatory and corpus luteal phases by treatment with PMSG, and mature rats hyperstimulated with PMSG were compared. Under all of these conditions, only one immunoreactive band of UGT, shown to be phenol UGT, was detected in the rat ovary. The effects of oestradiol, progesterone and/or human chorionic gonadotropin (hCG) on the level of phenol UGT in immature rat ovary were also examined. Partial up-regulation was caused by progesterone or oestradiol, together with hCG, whereas progesterone or oestradiol alone had no up-regulating effect. Follicle-stimulating hormone also seemed to be required for the up-regulation in ovaries enriched in corpus luteum. The present findings demonstrate that progesterone is involved in the regulation of phenol UGT in rat ovary by gonadotropins. Regulation by both progesterone and oestradiol was dependent on induction of ovulation and steroidogenesis by luteinizing hormone.

Evidence for an UDP-glucuronic acid/phenol glucuronide antiport in rat liver microsomal vesicles.

The transport of glucuronides synthesized in the luminal compartment of the endoplasmic reticulum by UDP-glucuronosyltransferase isoenzymes was studied in rat liver microsomal vesicles. Microsomal vesicles were loaded with p-nitrophenol glucuronide (5 mM), phenolphthalein glucuronide or UDP-glucuronic acid, by a freeze-thawing method. In was shown that: (i) the loading procedure resulted in millimolar intravesicular concentrations of the different loading compounds; (ii) addition of UDP-glucuronic acid (5 mM) to the vesicles released both intravesicular glucuronides within 1 min; (iii) glucuronides stimulated the release of UDP-glucuronic acid from UDP acid-loaded microsomal vesicles; (iv) trans-stimulation of UDP-glucuronic acid entry by loading of microsomal vesicles with p-nitrophenol glucuronide, phenolphthalein glucuronide, UDP-glucuronic acid and UDP-N-acetyl-glucosamine almost completely abolished the latency of UDP-glucuronosyltransferase, although mannose 6-phosphatase latency remained unaltered; (v) the loading compounds by themselves did not stimulate UDP-glucuronosyltransferase activity. This study indicates that glucuronides synthesized in the lumen of endoplasmic reticulum can leave by an antiport, which concurrently transports USP-glucuronic acid into the lumen of the endoplasmic reticulum.

Heterogeneity of Hepatic Microsomal UDP-Glucuronosyltransferase(s) Activities: A New Kinetic Approach for the Study of Induction and Specificity

UDP-glucuronosyltransferase (UGT) activities have been described as heterogeneous, i.e. supported by a family of isoenzymes, each of them being capable of conjugating a given chemical family of aglycons, including steroids, coumarines, and phenols. Some of these isoenzymes are specifically induced by xenobiotics. In order to discriminate between the different isoenzymes, we propose a new in situ approach that combines induction (gene regulation) and catalytic activities (specificity). The characterization of one isoenzyme is obtained by (i) increasing its amount by specific inductive stimulation and (ii) studying simultaneously the glucuronidation kinetics of a series of alternative substrates. Provided the substrates are of similar structure, a linear relationship can be established between their glucuronidation rates before versus after induction. We developed a simple mathematical model to analyze the kinetic behaviors observed. With this method, it is possible to know (i) the exact extent of induction of a given isoenzyme by a given inducer (induction factor, n) and (ii) its strict specificity. This in situ methodology is complementary to isolated protein or cDNAs, for the characterization of the real in situ substrate specificity of differentially regulated UGT isoenzymes.

Differential expression of hepatic UDP-Glucuronosyltransferase during dietary magnesium depletion

Male Sprague Dawley rats weighing 80±5 g were fed either a magnesium deficient (MgD) diet (30, 66 or 85 mg/kg) or a pair-fed control (PFC) diet (595 mg/kg) for 10 days. Serum Mg +2 was decreased 66% in the magnesium depleted animals (on diets of 30 and 66 mg of Mg +2/kg), no significant change was noted for total liver magnesium concentration. Hepatic microsmes were prepared and used fresh or stored at −70°C. The total UDP-glucuronosyl-transferase (UDP-GT) activities were evaluated using microsomes activated with Triton X-100. Three isoenzyme activities of UDP-GT were determined for specific substrates: p-nitrophenol (pNP), bilirubin (BR) and lithocholate (LiC). Addition of magnesium (10 mM) to the in vitro reaction system increased each isoenzyme activity, pNP UDP-GT (100%), BR UDP-GT (350%) and LiC UDP-GT (100%). The specific effect of dietary magnesium depletion was a decrease in pNP UDP-GT activity while the BR UDP-GT and LiC UDP-GT isoenzyme activites were not significantly changed. Below a dietary intake of about 2 mg of magnesium/day, the loss of pNP UDP-GT isoenzyme activity was proportional to the decrease in dietary Mg +2 intake. Thus, expression of the pNP UDP-GT was specifically diminished at early stages of magnesium depletion. The mechanism of this selective Mg +2 effect is under further investigation.

Differential expression and induction of UDP-glucuronosyltransferase isoforms in hepatic and extrahepatic tissues of a fish, Pleuronectes platessa: Immunochemical and functional characterization

Glucuronidation of three substrates prototypical for different UDP-glucuronosyltransferase (UDPGT) isoforms in hepatic, renal, intestinal, and branchial microsomes of corn oil, 3-methylcholanthrene, Aroclor 1254, and clofibrate-pretreated plaice was investigated. The differential expression of UDPGT in the four tissues clearly demonstrated for the first time that multiple isoforms with differing substrate specificities were present in fish. The liver was quantitatively the most important site for the glucuronidation of all three compounds studied. Phenol UDPGT activity was ubiquitous to all tissues and was induced by 3-methylcholanthrene and Aroclor 1254 in hepatic tissue and by Aroclor 1254 in renal tissue. The glucuronidation of testosterone was restricted to liver and intestinal tissue, while conjugation of bilirubin was expressed solely in hepatic tissue. The biotransformation of the endogenous compounds was not induced in the xenobiotic-treated animals. The presence of immunoreactive UDPGTs in the four tissues was demonstrated by immunoblot analysis using sheep anti-plaice UDPGT antibodies. Hepatic tissue displayed a range of immunoreactive polypeptides of 52 to 57 kDa, while a 55-kDa polypeptide was detected in extrahepatic tissues. An increased intensity of the latter polypeptide species was demonstrated in liver and kidney microsomes in which there was a concomitant induction of phenol UDPGT activity in xenobiotic-treated fish. The results indicate that the 55-kDa polypeptide was the major polyaromatic hydrocarbon-inducible UDPGT isoenzyme in hepatic and renal microsomes.

Delta 2-valproate biotransformation using human liver microsomal fractions.

The metabolism of 2-n-propyl-2-pentenoate (delta 2-VPA) was evaluated in human hepatic microsomal fractions. Two biotransformation pathways have been particularly investigated. In the presence of the cytochrome P-450 co-factor, NADPH, the main metabolites recovered were delta 3-VPA, delta 2,4-VPA and VPA. The glucuronidation of delta 2-VPA was also studied on various hepatic microsomal fractions using Brij 35 as activator and UDP-glucuronic acid as co-factor. A large interindividual variability occurred in this metabolic pathway. Km and Vmax were 0.85 mmol/l and 1.75 nmol.min-1.mg-1, respectively, for delta 2-VPA and 1.11 mmol/l and 5.71 nmol.min-1.mg-1 for VPA, respectively. The good correlation (r = 0.82; p less than 0.001) observed between the glucuronidation of VPA and delta 2-VPA as well as the mutual inhibition of each other's glucuronidation strongly suggests that (a) common single UDP-glucuronosyltransferase isoenzyme(s) was (were) involved in this glucuronidation step. The glucuronidation of specific substrates for various UDP-glucuronosyltransferase isoenzymes showed a good relationship between the glucuronidations of delta 2-VPA and morphine, a substrate for UDP-glucuronosyltransferase-2B. Moreover, morphine competitively inhibits delta 2-VPA glucuronidation. It seems the same isoenzyme or, at least, (a) very closely related isoenzyme(s) belonging to UDP-glucuronosyltransferase-2 isoenzyme, is involved in delta 2-VPA glucuronidation.

Multiplicity of UDP-glucuronosyltransferases in fish. Purification and characterization of a phenol UDP-glucuronosyltransferase from the liver of a marine teleost, Pleuronectes platessa.

The aim of this work was to determine if a non-mammalian species had multiple UDP-glucuronosyltransferase (UDPGT) isoforms. At least six highly purified UDPGT isoenzymes were partially resolved by anion-exchange chromatography and UDP-hexanolamine-Sepharose 4B affinity chromatography from liver microsomes of a fish, the plaice. Q-Sepharose FF, chromatofocusing and affinity-chromatographic procedures were employed to separate and purify the phenol UDPGT isoform to apparent homogeneity. The purified enzyme conjugated 1-naphthol, but not bilirubin or steroids, and displayed a pI of 7.0 and a subunit molecular mass of 55 kDa. Bilirubin and testosterone UDPGT activities were more labile and, although purified over 200-fold, these preparations also contained the phenol UDPGT and had multiple polypeptides with molecular masses of 52-57 kDa. Antisera to rat bilirubin/phenol UDPGT and testosterone/phenol UDPGT isoforms cross-reacted strongly with the partially purified plaice UDPGT isoforms of molecular masses 52, 53 and 57 kDa and less strongly with phenol UDPGT 54 kDa and 56 kDa isoforms. Fish and mammalian UDPGTs therefore apparently possess a high degree of evolutionary conservation.

Differential inhibition of hepatic morphine UDP-glucuronosyltransferases by metal ions

The major metabolites of morphine, morphine-3-glucuronide (M3G) and morphine-6-glu-curonide (M6G), possess significant pharmacological properties. Whilst both metabolites bind to μ-opioid binding sites, M6G is a potent agonist whereas M3G antagonizes some of the effects of morphine and M6G. An inter-species comparison of in vitro hepatic morphine processing was performed. The results showed that not all species were able to produce M6G whereas all those tested did produce M3G. Guinea-pig liver produced the greatest amounts of M6G and was therefore chosen as a model to study morphine glucuronidation in further detail. Inclusion of the detergent Brij 58 (0.33 mg/mg protein) and Mg 2+ (15 mM) in the standard assay incubation gave optimal production of both M3G and M6G by guinea-pig liver homogenates. A number of metal ions were investigated for their ability to inhibit glucuronidation of morphine in both the 3- and 6-positions. Some metal ions, namely Cu +, Cu 2+ and Cd 2+, were able to inhibit the production of M3G without affecting glucuronidation at the 6-position. Taken together, these data provide further evidence for the existence of UDP-glucuronosyltransferase isoenzymes responsible for the metabolism of morphine. In addition these isoenzymes can be differentially modulated and therefore it is possible to alter the ratio of M3G: M6G formed during in vitro metabolic studies.

Male rat hepatic udp-glucuronosyltransferase activity toward thyroxine: Activation and induction properties—Relation with thyroxine plasma disappearance rate

Detergent-activation of UDP-glucuronosyltransferase (UGT) isoenzyme(s) involved in thyroxine (T4) glucuronidation in control, phenobarbital (PB)- and 3-methylcholanthrene (3-MC)-treated rats showed that between the four tested detergents, i.e. Triton X-100, Brij 58, Lubrol Px and 3-(3-holamidopropyl) dimethylammonio-1-propanesulfonic acid (CHAPS), optimal activation of T4 UGT was displayed by the zwitterion CHAPS. “Native” versus optimal detergent-activated T4 UGT activity determination revealed that the latency of T4 UGT in microsomes from 3-MC-treated rats was decreased while the latency of T4 UGT in microsomes from PB-treated rats was increased compared to control, and suggest that the UGT isoenzyme(s) involved in the hepatic glucuronidation of T4 is (are) different in PB-treated rats than in 3-MC-treated rats. After a 7-day treatment with 20 mg/kg 3-MC, the activity of T4 UGT was increased 5-fold when determined in “native” and 4-fold when determined in optimal detergent-activated microsomes compared to controls. After a 7-day treatment with 75 mg/kg PB, T4 UGT was equivalent to the control when determined in “native”, and increased 1.3-fold when determined in optimal detergent-activated microsomes. The results thus extend evidence that both 3-MC and PB induce the synthesis of UGT protein(s) involved in the glucuronidation of T4, 3-MC being a strong and PB a weak inducer. Hyperthyroid and hypothyroid status, achieved respectively by a 7-day treatment with 100 μg kg T4 or a 7-day treatment with 10 mg/kg of one of the antithyroid drugs propylthiouracile or methymazole, did not modify T4 UGT activity, suggesting that the isoenzyme(s) conjugating T4 in microsomes from control rats is (are) unlikely to be either 4-nitrophenol or bilirubin UGT isoenzymes. After 14 days of treatment with 75 mg/kg PB, the hepatic glucuronidation rate of T4 was not different from the control when enzyme activity was expressed per mg microsomal protein but was significantly increased 1.4-fold when expressed per whole liver. A significant (1.5-fold) increase in the 125I-T4 plasma elimination rate was also observed in PB-treated rats compared to controls. A strong (3.6-fold) increase in the T4 glucuronidation rate was observed in rats treated with 5 mg/kg 3-MC for 14 days while the 125I-T4 plasma elimination rate was equivalent to the controls. These results demonstrate that there is no direct relation between T4 UGT activity (and subsequent biliary secretion of T4-glucuronides) and T4 plasma clearance and suggest an important contribution of the intestinal exchangeable thyroid hormone pool to the maintenance of blood thyroid hormone levels.

Recent Developments in the Research on Glucuronidation.

UDP-glucuronosyltransferase (UGT) catalyzes a transfer of glucuronic acid from UDP-glucuronic acid to various endogenous and exogenous compounds and is located in the endoplasmic reticulum of liver and other tissues. Glucuronidation is a major detoxication reaction and provides great effects on the metabolism, disposition and excretion of pharmacologically and toxicologically active compounds. There are several UGT isoenzymes with different and overlapping substrate specificities and under different regulation. During the last decade, there has been a dramatical increase in the understanding of the structure and function of UGT isoenzymes especially through the recombinant DNA technology. This review concentrates on the recent developments in the research on glucuronidation and deals with purification, cloning, gene structure, deficiency, chromosomal location and function of UGT isoenzymes and glucuronides as active metabolites.

Molecular basis of multiple UDP-glucuronosyltransferase isoenzyme deficiencies in the hyperbilirubinemic rat (Gunn rat).

The Gunn rat is a mutant strain of Wistar rat which has unconjugated hyperbilirubinemia as a result of the absence of hepatic UDP-glucuronosyltransferase (UDPGT) activity toward bilirubin. The Gunn rat is also deficient in UDPGT activities toward phenol substrates, and also toward digitoxigenin-monodigitoxiside. We have demonstrated that the defect of the isoenzyme for 4-nitrophenol (4NP) in Gunn rat liver arises from a -1 frameshift mutation that removes 115 amino acids from the COOH terminus (Iyanagi, T., Watanabe, T., and Uchiyama, Y. (1989) J. Biol. Chem. 264, 21302-21307). To investigate the molecular basis of defects in other UDPGT isoenzymes, we isolated and sequenced cDNAs from a Gunn rat liver library using mutant 4NP-UDPGT cDNA as a probe. Three novel cDNAs were identified that had identical 3'-regions of 1362 base pairs containing a single-base deletion in the same position as that of the mutant 4NP-UDPGT cDNA. However, their 5'-regions, encoding the substrate-binding domain, showed no more than 40% homology to that of 4NP-UDPGT. These data provide evidence that defects in some UDPGT isoenzymes in the Gunn rat are caused by a single mutation that results in the formation of a common truncated COOH terminus. Furthermore, the data also suggest that these mRNAs are transcribed from a single gene and that the 5'-exons are transcribed independently and differentially spliced to common 3'-exons encoding the conserved domain.

Selective induction of bilirbuin UDP-glucuronosyl-transferase by perfluorodecanoic acid

Differential effects of perfluorodecanoic acid (PFDA) on rat liver UDP-glucuronosyltransferase isoenzymes have been observed after a single i.p. administration of the compound to young male Sprague-Dawley rats. (1) Bilirubin glucuronidation was induced 2-fold. The induced state was stable for at least 3 weeks. (2) Glucuronidation of 1-naphthol, morphine and testosterone was decreased to half of the control values. These decreases were maximal after 12 days but all three activities returned to normal levels after 3 weeks. (3) Immunoblotting experiments indicated that the differential effects of PFDA on UDP-glucuronosyltransferase activities were due to modulation of enzyme protein concentrations rather than activation/inactivation mechanisms. With respect to its influence on UDP-glucuronosyltransferase isoenzymes, PFDA may be classified as a clofibrate-type inducer. The persistence of the induction after a single application however is unique among peroxisome proliferators and therefore PFDA may be a useful, elective inducer of bilirubin glucuronidation.