Medicago glucosyltransferase UGT72L1: potential roles in proanthocyanidin biosynthesis

In the first reaction specific for proanthocyanidin (PA) biosynthesis in Arabidopsis thaliana and Medicago truncatula, anthocyanidin reductase (ANR) converts cyanidin to (?)-epicatechin. The glucosyltransferase UGT72L1 catalyzes formation of epicatechin 3′-O-glucoside (E3′OG), the preferred substrate for MATE transporters implicated in PA biosynthesis in both species. The mechanism of PA polymerization is still unclear, but may involve the laccase-like polyphenol oxidase TRANSPARENT TESTA 10 (TT10). We have employed a combination of cell biological, biochemical and genetic approaches to evaluate this PA pathway model. The promoter regions of UGT72L1 and MtANR share common cis-acting elements and direct overlapping, but partially distinct, expression patterns. UGT72L1 and MtANR are localized in the cytosol, whereas TT10 is localized to the vacuole. Over-expression of UGT72L1 in M. truncatula hairy roots results in increased accumulation of PA-like compounds, and loss of function of UGT72L1 partially reduces epicatechin, E3′OG and extractable PA levels in M. truncatula seeds. Expression of UGT72L1 in A. thaliana leads to a massive increase in E3′OG in immature seed, but reduced levels of extractable PAs. However, when UGT72L1 was expressed in the Arabidopsis tt10 mutant, extractable PA levels increased and seed coat browning was delayed. Our results suggest that glycosylation of epicatechin is important for both PA precursor transport and assembly, but that additional redundant pathways may exist.     

UDP-glucuronyltransferases in detoxification and activation metabolism of endogenous compounds and xenobiotics

Glucuronidation is a crucial pathway of metabolism and excretion of endogenous compounds and xenobiotics. UDP-glucuronyltransferases, UGT, catalyse transformations of bilirubine, steroids and thyroid hormones, bile acids as well as exogenous compounds, including drugs, carcinogens, environmental pollutants and nutrient components. From therapeutic point of view, the participation of UGTs in drug metabolism is of particular significance. Polymorphism of UGT1A and UGT2B genes resulted in various susceptibility of substrates to conjugation with glucuronic acid. Deactivation of xenobiotics and the following excretion of hydrophilic conjugates is a common task of glucuronidation, which should lead to detoxification. However, a lot of glucuronides were known, which expressed the comparable or even higher reactivity than that of the native compound. There are, among others, acyl glucuronides of carboxylic acids, morphine 6-O-glucuronide or retinoid glucuronides. They are able to bind cellular macromolecules with low or high strength and, as a consequence, their toxicity is saved or even increased, respectively.     

Uptake and metabolism of epicatechin and its access to the brain after oral ingestion

Epicatechin is a flavan-3-ol that is commonly present in green teas, red wine, cocoa products, and many fruits, such as apples. There is considerable interest in the bioavailability of epicatechin after oral ingestion. In vivo studies have shown that low levels of epicatechin are absorbed and found in the circulation as glucuronides, methylated and sulfated forms. Recent research has demonstrated protective effects of epicatechin and one of its in vivo metabolites, 3′-O-methyl epicatechin, against neuronal cell death induced by oxidative stress. Thus, we are interested in the ability of ingested epicatechin to cross the blood brain barrier and target the brain. Rats were administered 100 mg/kg body weight/d epicatechin orally for 1, 5, and 10 d. Plasma and brain extracts were analyzed by HPLC with photodiode array detection and LC-MS/MS. This study reports the presence of the epicatechin glucuronide and 3′-O-methyl epicatechin glucuronide formed after oral ingestion in the rat brain tissue.     

Flavonoid glucuronides are substrates for human liver beta-glucuronidase

Quercetin glucuronides are the main circulating metabolites of quercetin in humans. We hypothesise that the potential availability of the aglycone within tissues depends on the substrate specificity of the deconjugating enzyme beta-glucuronidase towards circulating flavonoid glucuronides. Human tissues (small intestine, liver and neutrophils) exhibited beta-glucuronidase against quercetin glucuronides. The various quercetin glucuronides were deconjugated at similar rates, but liver cell-free extracts were the most efficient and the activity was completely inhibited by saccharo-1,4-lactone (a beta-glucuronidase inhibitor). Furthermore, pure recombinant human beta-glucuronidase hydrolysed various flavonoid glucuronides, with a 20-fold variation in catalytic efficiency (k(cat)/K(m)=1.3x10(3) M(-1) s(-1) for equol-7-O-glucuronide and 26x10(3) M(-1) s(-1) for kaempferol-3-O-glucuronide). Similar catalytic efficiencies were obtained for quercetin O-glucuronides substituted at different positions. These results show that flavonoid glucuronides can be deconjugated by microsomal beta-glucuronidase from various human cells.     

Glucuronidation of oxidized fatty acids and prostaglandins B1 and E2 by human hepatic and recombinant UDP-glucuronosyltransferases

Arachidonic acid (AA) can be metabolized to various metabolites, which can act as mediators of cellular processes. The objective of this work was to identify whether AA, prostaglandin (PG) B1 and E2, and 15- and 20-hydroxyeicosatetraenoic acids (15- and 20-HETE) are metabolized via glucuronidation. Assays with human recombinant UDP-glucuronosyltransferase 1A (UGT1A) isoforms revealed that AA and 15-HETE were glucuronidated by UGT1A1, 1A3, 1A4, 1A9, and 1A10, whereas 20-HETE was glucuronidated by UGT1A1 and 1A4 and PGB1 was glucuronidated by UGT1A1, 1A9, and 1A10. All substrates were glucuronidated by recombinant UGT2B7, with AA and 20-HETE being the best substrates. Kinetic analysis of UGT1A1 and 1A9 with AA resulted in Km values of 37.9 and 45.8 microM, respectively. PGB1 was glucuronidated by UGT1A1 with a Km of 26.3 microM. The Km values for all substrates with UGT2B7 were significantly higher than with the UGT1A isoforms. Liquid chromatography-mass spectrometry of glucuronides biosynthesized from PGB1 and 15-HETE showed that hydroxyl groups were the major target of glucuronidation. This work demonstrates a novel metabolic pathway for HETEs and PGs and the role of UGT1A isoforms in this process. These results indicate that glucuronidation may play a significant role in modulation of the availability of these fatty acid derivatives for cellular processes.     

Inhibition of protein glycation by skins and seeds of the muscadine grape

The formation of advanced glycation end products (AGEs) leading to protein glycation and cross-linking is associated with the pathogenesis of diabetic complications. The inhibition of protein glycation by phenolic and flavonoid antioxidants demonstrates that the process is mediated, in part, by oxidative processes. In this study, the effects of seed and skin extracts of the muscadine grape on AGEs formation were examined. Seeds and skins were extracted (10% w/v) with 50% ethanol and incubated at 37 degrees C with a solution containing 250 mM fructose and 10 mg/ml albumin. After 72 h, fluorescence was measured at the wavelength pair of 370 and 440 nm as an index of the formation of AGEs. Both seed and skin extracts were found to be efficacious inhibitors of AGE formation. A 1:300 dilution of the seed extract decreased fluorescence by approximately 65%, whereas muscadine grape skin extract produced a 40% lowering. This difference correlates with the greater antioxidant activity found in muscadine seeds in comparison to skins, however, on a mass basis, the inhibitory activities of the seeds and skins were found to be nearly equivalent. Gallic acid, catechin and epicatechin, the three major polyphenols in the seeds, all significantly decreased the AGE product related fluorescence at a concentration of 50 microM. Neither muscadine seed extract nor skin extract inhibited the methylglyoxal-mediated glycation of albumin. These results suggest that consumption of the muscadine grape may have some benefit in altering the progression of diabetic complications.     

Use of the glucosyltransferase UGT71B6 to disturb abscisic acid homeostasis in Arabidopsis thaliana

A glucosyltransferase (GT) of Arabidopsis, UGT71B6, recognizing the naturally occurring enantiomer of abscisic acid (ABA) in vitro, has been used to disturb ABA homeostasis in planta. Transgenic plants constitutively overexpressing UGT71B6 (71B6-OE) have been analysed for changes in ABA and the related ABA metabolites abscisic acid glucose ester (ABA-GE), phaseic acid (PA), dihydrophaseic acid (DPA), 7'-hydroxyABA and neo-phaseic acid. Overexpression of the GT led to massive accumulation of ABA-GE and reduced levels of the oxidative metabolites PA and DPA, but had marginal effect on levels of free ABA. The control of ABA homeostasis, as reflected in levels of the different metabolites, differed in the 71B6-OEs whether the plants were grown under standard conditions or subjected to wilt stress. The impact of increased glucosylation of ABA on ABA-related phenotypes has also been assessed. Increased glucosylation of ABA led to phenotypic changes in post-germinative growth. The use of two structural analogues of ABA, known to have biological activity but to differ in their capacity to act as substrates for 71B6 in vitro, confirmed that the phenotypic changes arose specifically from the increased glucosylation caused by overexpression of 71B6. The phenotype and profile of ABA and related metabolites in a knockout line of 71B6, relative to wild type, has been assessed during Arabidopsis development and following stress treatments. The lack of major changes in these parameters is discussed in the context of functional redundancy of the multigene family of GTs in Arabidopsis.     

Expression of UDP-glucuronosyltransferases (UGTs) 2B7 and 1A6 in the human brain and identification of 5-hydroxytryptamine as a substrate

The extrahepatic expression of UDP-glucuronosyltransferases (UGTs) is important in the detoxification of a number of endogenous and exogenous compounds, including 5-hydroxytryptamine and morphine. Studies were designed to investigate the extrahepatic expression of human UGTs using RT-PCR techniques and to determine the UGTs involved in the glucuronidation of 5-hydroxytryptamine. Human UGT2B7 expression was found in the human liver, kidney, pancreas, and brain, while UGT1A6 expression is found in the liver, kidney, and brain. This is the first observation of UGTs present in the human central nervous system. Using glucuronidation assays, a significant amount of 5-hydroxytryptamine glucuronide was found to be catalyzed by UGT1A6. These studies suggest that UGT2B7 may play an important role in the overall contribution of morphine analgesia by serving to generate the potent morphine-6-O-glucuronide in situ. UGT1A6 could play an important role in the glucuronidation of 5-hydroxytryptamine in vivo, therefore terminating the actions of the neurotransmitter.     

Identification of human UDP-glucuronosyltransferase isoform(s) responsible for the C-glucuronidation of phenylbutazone

Glucuronidation is a major metabolic pathway in the biotransformation of many xenobiotics and endogeneous compounds. There have been many studies on the formation of O-, N- or S-glucuronides and identification of the UDP-glucuronosyltransferase (UGT) isoforms responsible for the formation of these glucuronides. However, there is no information available on which UGT isoform(s) catalyzes C-glucuronidation. In the present study, 16 human UGTs (UGTs 1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B11, 2B15, 2B17 and 2B28) were cloned and expressed in baculovirus-infected insect cells and investigated to determine their C-glucuronidating activity toward phenylbutazone (PB). Among the UGT isoforms investigated, only UGT1A9 catalyzed PB C-glucuronidation. Human liver and kidney microsomes, which are well known to express UGT1A9, had C-glucuronidating activity toward PB. However, the jejunum, which did not express UGT1A9, had no C-glucuronidating activity. These results demonstrate for the first time that PB C-glucuronidation is catalyzed by only UGT1A9.     

Defective biliary secretion of bile acid 3-O-glucuronides in rats with hereditary conjugated hyperbilirubinemia

Biliary secretion of bile acid glucuronides was studied in control rats and in rats with a congenital defect in hepatobiliary transport of organic anions (GY rats). In control animals, hepatobiliary transport of [3H]lithocholic acid 3-O-glucuronide and [3H]cholic acid 3-O-glucuronide was efficient (greater than 95% in 1 h) and comparable to that of [14C]taurocholic acid. Secretion of both glucuronides was impaired in GY rats (24% and 71% at 1 h), whereas that of taurocholate was similar to control values. However, recovery of the glucuronides in bile was nearly complete within 24 h; virtually no radioactivity was found in urine. In control rats, biliary secretion of lithocholic acid 3-O-glucuronide, but not that of cholic acid 3-O-glucuronide or taurocholate, could be delayed by simultaneous infusion of dibromosulphthalein. In mutant rats, dibromosulphthalein infusion was also able to inhibit secretion of cholic acid 3-O-glucuronide. [3H]Hydroxyetianic acid, a C20 short-chain bile acid, was secreted by control rats as a mixture of 20% carboxyl-linked and 80% hydroxyl-linked (3-O-)glucuronide; secretion was very efficient (99% in 1 h). In GY rats, secretion was drastically impaired (16% at 1 h and 74% over a 24-h period). Initially, the mutant secreted more carboxyl- than hydroxyl-linked glucuronide, but the ratio reached that of control animals after 24 h. The rates of formation of both types of hydroxyetianic acid glucuronide by hepatic microsomes from mutant rats were similar or even slightly higher than those of control microsomes. These findings indicate that bile acid 3-O-glucuronides, but probably not carboxyl-linked glucuronides, are secreted into bile by a transport system shared with organic anions such as conjugated bilirubin and dibromosulphthalein, but different from that for amino acid-conjugated bile acids.     

Extensive conjugation of dopamine (3,4-dihydroxyphenethylamine) metabolites in cultured human skin fibroblasts and rat hepatoma cells.

[G-3H]Dopamine (3,4-dihydroxyphenethylamine) metabolism in human skin fibroblasts and rat hepatoma cells in culture was determined by high-pressure liquid-chromatographic analysis of both cell extract and uptake medium. Conjugated metabolites were selectively hydrolysed by incubation with arylsulphatase or beta-glucuronidase before analysis. The principal metabolites of dopamine in fibroblast cells are 3-methoxytyramine 4-O-sulphate and 3-methoxytyramine. No significant differences, either in the amounts of these metabolites or in the amount of dopamine metabolism, were observed in fibroblasts from both normal and homocystinuric individuals. In rat hepatoma cells, the major metabolite of dopamine was 3-methoxytyramine 4- or 3-O-glucuronide; lower concentrations of dopamine 4- or 3-O-glucuronide, 4-hydroxy-3-methoxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid and two unidentified glucuronide conjugates were also observed. Significant differences in the relative concentrations of these metabolites in cell and uptake medium were observed in both cell systems.     

The reaction of flavonoid metabolites with peroxynitrite

There is much interest in the bioactivity of in vivo flavonoid metabolites. We report for the first time the hierarchy of reactivity of flavonoid metabolites with peroxynitrite and characterise novel reaction products. O-Methylation of the B-ring catechol containing flavonoids epicatechin and quercetin, and O-glucuronidation of all flavonoids reduced their reactivity with peroxynitrite. The reaction of the flavanones hesperetin and naringenin and their glucuronides resulted in the formation of multiple mono-nitrated and nitrosated products. In contrast, the catechol-containing flavonoids epicatechin and quercetin yielded oxidation products which when trapped with glutathione led to the production of glutathionyl-conjugates. However, the O-methylated metabolites of epicatechin yielded both mono- and di-nitrated products and nitrosated metabolites. The 3'-O-methyl metabolite of quercetin also yielded a nitrosated species, although its counterpart 4'-O-methyl quercetin yielded only oxidation products. Such products may represent novel metabolic products in vivo and may also express cellular activity.     

Glucuronidation of the soyabean isoflavones genistein and daidzein by human liver is related to levels of UGT1A1 and UGT1A9 activity and alters isoflavone response in the MCF-7 human breast cancer cell line

The soyabean isoflavones genistein and daidzein, which may protect against some cancers, cardiovascular disease and bone mineral loss, undergo substantial Phase 2 metabolism, predominantly glucuronidation. We observed a correlation between rates of metabolism of marker substrates of specific UGTs and rates of glucuronidation of genistein and daidzein in vitro by a panel of human liver microsomes, demonstrating that UGT1A1 and UGT1A9, but not UGT1A4, make a major contribution to the metabolism of these isoflavones by human liver. These findings were substantiated by observations that recombinant human UGT1A1 and UGT1A9, but not UGT1A4, catalysed the production of the major glucuronides of both genistein and daidzein in vitro. Recombinant human UGT1A8 also metabolised both genistein and daidzein, whereas UGT1A6 was specific to genistein and UGTs 2B7 and 2B15 were inactive, or only marginally active, with either isoflavone as substrate. The intestinal isoform UGT1A10 metabolised either both isoflavones or genistein only, depending on the commercial supplier of the recombinant enzyme, possibly as a result of a difference in amino acid sequence, which we were unable to confirm. Daidzein (16 microM) increased cell death in the MCF-7 human breast cancer cell line and this effect was reversed by glucuronidation. In view of a well-characterised functional polymorphism in UGT1A1, these observations may have implications for inter-individual variability in the potential health-beneficial effects of isoflavone consumption.     

Roles of glucuronidation and UDP-glucuronosyltransferases in xenobiotic bioactivation reactions

Glucuronide conjugates represent one of the major types of naturally occurring phase 2 metabolites of xenobiotics and endobiotics. The process underlying their formation, glucuronidation, is normally considered detoxifying, because glucuronides usually possess less intrinsic biological or chemical activity than their parent aglycones and they are rapid excreted. However, a number of glucuronide conjugates are known that are active and may contribute to pharmacological activities or toxicities associated with their parent compounds. These include two classes of glucuronides with electrophilic chemical reactivity (N-O-glucuronides of hydroxamic acids and acyl glucuronides of carboxylic acids) and several types of glucuronides that impart biological effects through non-covalent interactions (morphine 6-O-glucuronide, retinoid glucuronides, and D-ring glucuronides of estrogens). Glucuronides may thus contribute to clinically significant effects, including environmental arylamine-induced carcinogenesis, drug hypersensitivity and other toxicities associated with carboxylic acid drugs, morphine analgesia, and cholestasis from estrogens. This review summarizes the rat and human UDP-glucuronosyltransferases that may be involved in the formation of bioactive glucuronides, including their substrate- and tissue-specificity and genetic and environmental influences on their activity. This knowledge may be useful for enhancing the therapeutic efficacy and minimizing the risk of adverse effects associated with xenobiotics that undergo bioactivating glucuronidation reactions.     

Differential glucuronidation of bile acids, androgens and estrogens by human UGT1A3 and 2B7.

In this work, UDP-glucuronosyltransferases (UGTs), UGT1A3, 2B7(H268) and 2B7(Y268), stably expressed in human embryonic kidney cells (HK293) were used to assess glucuronidation activities with a variety of steroid hormone and bile acid substrates. The rate of synthesis of carboxyl- and hydroxyl-linked glucuronides was determined under optimal reaction conditions. Expressed UGT1A3 catalyzed bile acid glucuronidation at high rates exclusively at the carboxyl moiety for all compounds tested. In contrast, UGT1A4 catalyzed bile acid glucuronidation at very low rates exclusively at the 3alpha-hydroxyl function. Both UGT2B7 allelic variants glucuronidated the bile acid substrates at both carboxyl and hydroxyl moieties, however, the 3alpha-hydroxyl position was preferentially conjugated compared to the carboxyl function. Similarly, androsterone, a 3alpha-hydroxylated androgenic steroid, was glucuronidated at very high rates by expressed UGT2B7. Of the estrogenic compounds tested, UGT2B7 catalyzed the glucuronidation of estriol at rates comparable to those determined for androsterone. Other structural discrimination was found with UGT2B7 which had activity toward estriol and estradiol exclusively at the 17beta-OH position, yielding the cholestatic steroid D-ring glucuronides.     

High protection by grape seed proanthocyanidins (GSPC) of polyunsaturated fatty acids against UV-C induced peroxidation

The antioxidative effects of grape seed proanthocyanidins (GSPC) were studied in three in-vitro models in which polyunsaturated fatty acids (PUFAs) in aqueous solution and mice liver or brain microsomes were used as oxidative substrates, and UVC irradiation as the pro-oxidant system. Analysis of UV-C induced lipid peroxidation was carried out by two methods: gas liquid chromatography of residual PUFAs and release of thiobarbituric acid-reactive substances (TBARs) measured by TBA reaction. Results indicate that PUFAs are more radiosensitive when incorporated in single component micelles than in mixed component micelles or microsomes. In every case, PUFA peroxidation was inhibited by low concentrations of GSPC (2 rng/L) while epigallocatecin (EGC) and epigallocatechin gallate (EGCG) monomers, at an equivalent level of epicatechin, exhibited no efficacy in our experimental conditions. This latter effect might be explained by a synergistic action of flavan-3-ol monomers, dimers and oligomers contained in the grape seed extract.     

Rapid methods to determine procyanidins, anthocyanins, theobromine and caffeine in rat tissues by liquid chromatography-tandem mass spectrometry

Rapid, selective and sensitive methods were developed and validated to determine procyanidins, anthocyanins and alkaloids in different biological tissues, such as liver, brain, the aorta vein and adipose tissue. For this purpose, standards of procyanidins (catechin, epicatechin, and dimer B(2)), anthocyanins (cyanidin-3-glucoside and malvidin-3-glucoside) and alkaloids (theobromine, caffeine and theophylline) were used. The methods included the extraction of homogenized tissues by off-line liquid-solid extraction, and then solid-phase extraction to analyze alkaloids, or microelution solid-phase extraction plate for the analysis of procyanidins and anthocyanins. The eluted extracts were then analyzed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry, using a triple quadrupole as the analyzer. The optimum extraction solution was water/methanol/phosphoric acid 4% (94/4.5/1.5, v/v/v). The extraction recoveries were higher than 81% for all the studied compounds in all the tissues, except the anthocyanins, which were between 50 and 65% in the liver and brain. In order to show the applicability of the developed methods, different rat tissues were analyzed to determine the procyanidins, anthocyanins and alkaloids and their generated metabolites. The rats had previously consumed 1g of a grape pomace extract (to analyze procyanidins and anthocyanins) or a cocoa extract (to analyze alkaloids) per kilogram of body weight. Different tissues were extracted 4h after administration of the respective extracts. The analysis of the metabolites revealed a hepatic metabolism of procyanidins. The liver was the tissue which produced a greater accumulation of these metabolites.     

Roles of UDP-glucuronosyltransferases in chemical carcinogenesis

UDP-glucuronosyltransferases (UGT) play a major role in the elimination of nucleophilic metabolites of carcinogens, such as phenols and quinols of polycyclic aromatic hydrocarbons. In this way they prevent their further oxidation to electrophiles, which may react with DNA, RNA, and protein. They also inactivate carcinogenic, N-oxidized metabolites of aromatic amines. Furthermore, glucuronides may be stable transport forms of proximate carcinogens excreted via the biliary or urinary tract, thereby liberating the ultimate carcinogen at the target of carcinogenicity. Isozymes of the UGT enzyme superfamily that control the glucuronidation of metabolites of aromatic hydrocarbons and of N-oxidized aromatic amines have been identified in rats and humans. Phenol UGT appears to be coinduced with other drug-metabolizing enzymes via the Ah or dioxin receptor. This isozyme probably controls various proximate carcinogens and contributes to the persistently altered enzyme pattern, leading to the "toxin-resistance phenotype" at cancer prestages. Knowledge about UGTs in different species, their regulation, and their tissue distribution will improve the risk assessment of carcinogens.     

Roles of UDP-Glucuronosyltransferases in Chemical Carcinogenesi

Abstract

UDP-glucuronosyltransferases (UGT) play a major role in the elimination of nucleophilic metabolites of carcinogens, such as phenols and quinols of polycyclic aromatic hydrocarbons. In this way they prevent their further oxidation to electrophiles, which may react with DNA, RNA, and protein. They also inactivate carcinogenic, N-oxidized metabolites of aromatic amines. Furthermore, glucuronides may be stable transport forms of proximate carcinogens excreted via the CCor urinary tract, thereby liberating the ultimate carcinogen at the target of carcinogenicity. Isozymes of the UGT enzyme super family that control the glucuronidation of metabolites of aromatic hyharbons and of N-oxidized aromatic amines have been identified in rats and humans. Phenol UGT appears to be conduced with other drug-metabolizing enzymes via the Ah or dioxin receptor. This isozyme probably controls various proximate carcinogens and contributes to the persistently altered enzyme pattern, leading to the “toxin-resistance phenotype” at cancer prestages. Knowledge about UGTs in different species, their regulation, and their tissue distribution will improve the risk assessment of carcinogens.     

Acyl glucuronide reactivity in perspective: biological consequences

The metabolic conjugation of exogenous and endogenous carboxylic acid substrates with endogenous glucuronic acid, mediated by the uridine diphosphoglucuronosyl transferase (UGT) superfamily of enzymes, leads to the formation of acyl glucuronide metabolites. Since the late 1970s, acyl glucuronides have been increasingly identified as reactive electrophilic metabolites, capable of undergoing three reactions: intramolecular rearrangement, hydrolysis, and intermolecular reactions with proteins leading to covalent drug-protein adducts. This essential dogma has been accepted for over a decade. The key question proposed by researchers, and now the pharmaceutical industry, is: does or can the covalent modification of endogenous proteins, mediated by reactive acyl glucuronide metabolites, lead to adverse drug reactions, perhaps idiosyncratic in nature? This review evaluates the evidence for acyl glucuronide-derived perturbation of homeostasis, particularly that which might result from the covalent modification of endogenous proteins and other macromolecules. Because of the availability of acyl glucuronides for test tube/in vitro experiments, there is now a substantial literature documenting their rearrangement, hydrolysis and covalent modification of proteins in vitro. It is certain from in vitro experiments that serum albumin, dipeptidyl peptidase IV, tubulin and UGTs are covalently modified by acyl glucuronides. However, these in vitro experiments have been specifically designed to amplify any interference with a biological process in order to find biological effects. The in vivo situation is not at all clear. Certainly it must be concluded that all humans taking carboxylate drugs that form reactive acyl glucuronides will form covalent drug-protein adducts, and it must also be concluded that this in itself is normally benign. However, there is enough in vivo evidence implicating acyl glucuronides, which, when backed up by in vivo circumstantial and documented in vitro evidence, supports the view that reactive acyl glucuronides may initiate toxicity/immune responses. In summary, though acyl glucuronide-derived covalent modification of endogenous macromolecules is well-defined, the work ahead needs to provide detailed links between such modification and its possible biological consequences.