Over 20 years have elapsed since aspartame was approved by regulatory agencies as a sweetener and flavor enhancer. The safety of aspartame and its metabolic constituents was established through extensive toxicology studies in laboratory animals, using much greater doses than people could possibly consume. Its safety was further confirmed through studies in several human subpopulations, including healthy infants, children, adolescents, and adults; obese individuals; diabetics; lactating women; and individuals heterozygous (PKUH) for the genetic disease phenylketonuria (PKU) who have a decreased ability to metabolize the essential amino acid, phenylalanine. Several scientific issues continued to be raised after approval, largely as a concern for theoretical toxicity from its metabolic components--the amino acids, aspartate and phenylalanine, and methanol--even though dietary exposure to these components is much greater than from aspartame. Nonetheless, additional research, including evaluations of possible associations between aspartame and headaches, seizures, behavior, cognition, and mood as well as allergic-type reactions and use by potentially sensitive subpopulations, has continued after approval. These findings are reviewed here. The safety testing of aspartame has gone well beyond that required to evaluate the safety of a food additive. When all the research on aspartame, including evaluations in both the premarketing and postmarketing periods, is examined as a whole, it is clear that aspartame is safe, and there are no unresolved questions regarding its safety under conditions of intended use.

Two human UDP-glucuronosyltransferases (UGTs), UGT2B7 and UGT1A1, catalyze the glucuronidation of many endo- and xenobiotics. Although UGT1A1 uniquely catalyzes the glucuronidation of the endobiotic, bilirubin, and UGT2B7 uniquely catalyzes the glucuronidation of morphine to both the 3-0 glucuronide and the 6-0 glucuronide, both catalyze the glucuronidation of the mixed opioid agonist/antagonist buprenorphine with high efficiency. Etonitazenyl, a mu opioid receptor antagonist, was found to inhibit competitively opioid, steroid, and other substrate glucuronidation reactions catalyzed by UGT2B7. Data showing several benzodiazepines and alternative substrates interacting competitively support previous work, which indicates a single binding domain within UGT2B7. Etonitazenyl also competitively inhibited the glucuronidation of buprenorphine catalyzed by UGT1A1. However, neither etonitazenyl nor buprenorphine inhibited bilirubin glucuronidation except at very high concentrations. Therefore, it is unlikely that buprenorphine therapy for opioid or other drug addiction would influence bilirubin glucuronidation and lead to hyperbilirubenmia. Anthraflavic acid and catechol estrogen glucuronidation, catalyzed by UGT1A1, was also not inhibited by etonitazenyl or buprenorphine. Reactions catalyzed by UGT1A6 were not affected by etonitazenyl. These studies indicate that UGT2B7 has one binding site and that UGT1A1 has two or more binding sites for xenobiotics and endobiotics.

The UDP-glucuronosyltransferase UGT2B7 is an important human UGT isoform that catalyzes the conjugation of many endogenous and exogenous compounds, among them opioids, resulting in the formation of D-glucuronides. The binding site of the aglycone is located in the N-terminal half of the protein. In this study, we demonstrate that the opioid binding site in UGT2B7 is within the first 119 amino-terminal amino acids. Two maltose binding protein fusion proteins, 2B7F1 and 2B7F2, incorporating the first 157 or 119 amino acids, respectively, of UGT2B7 were expressed in Escherichia coli and purified by affinity chromatography. NMR spectroscopy using one-dimensional spectra, the inversion recovery method, and the transferred nuclear Overhauser effect spectroscopy was used to study the binding properties of opioids to the fusion proteins. Morphine was found to bind at a single site within the first 119 amino acids and to undergo a conformational change upon binding, as demonstrated by transferred nuclear Overhauser effect spectroscopy. Dissociation constants were obtained for morphine, naloxone, buprenorphine, and zidovudine, and the results were confirmed by equilibrium dialysis determinations. Two possible opioid binding sites, based on the nearest neighbors from opioid binding to the micro-receptor and to cytochrome 2D6, are proposed.

Recent reports suggest that linoleic acid (LA) epoxides and diols are associated with important physiological, pharmacological, and pathological events in vivo. We have shown recently that LA-diols are excellent substrates for human liver microsomal UDP-glucuronosyltransferases (UGTs); however, it is not known if other human tissues glucuronidate LA-diols or which UGT isozyme(s) is involved. The present studies with human intestinal microsomes indicate that glucuronidation of LA-diols occurs throughout the gastrointestinal tract, with the highest activity in the small intestine. LA-diols yielded exclusively hydroxyl-linked glucuronides, whereas LA yielded the carboxyl-linked glucuronide. Studies with human recombinant UGTs demonstrated that only UGT2B7 glucuronidated LA and LA-diols. Kinetic analysis with UGT2B7 yielded apparent K(m) values in the range of 40-70 microM and V(max) values from 4.5 to 5.4 nmol/mg x min. These studies indicate that LA and LA-diols are excellent substrates for intestinal UGTs and provide the first evidence for UGT2B7 being the major isoform involved.

It is suggested that formation of more polar metabolites of all-trans-retinoic acid (atRA) via oxidative pathways limits its biological activity. In this report, we investigated the biotransformation of oxidized products of atRA via glucuronidation. For this purpose, we synthesized 4-hydroxy-RA (4-OH-RA) in radioactive and nonradioactive form, 4-hydroxy-retinyl acetate (4-OH-RAc), and 5,6-epoxy-RA, all of which are major products of atRA oxidation. Glucuronidation of these retinoids by human liver microsomes and human recombinant UDP-glucuronosyltransferases (UGTs) was characterized and compared with the glucuronidation of atRA. The human liver microsomes glucuronidated 4-OH-RA and 4-OH-RAc with 6- and 3-fold higher activity than atRA, respectively. Analysis of the glucuronidation products showed that the hydroxyl-linked glucuronides of 4-OH-RA and 4-OH-RAc were the major products, as opposed to the formation of the carboxyl-linked glucuronide with atRA, 4-oxo-RA, and 5,6-epoxy-RA. We have also determined that human recombinant UGT2B7 can glucuronidate atRA, 4-OH-RA, and 4-OH-RAc with activities similar to those found in human liver microsomes. We therefore postulate that this human isoenzyme, which is expressed in human liver, kidney, and intestine, plays a key role in the biological fate of atRA. We also propose that atRA induces its own oxidative metabolism via a cytochrome P450 (CYP26) and is further biotransformed into glucuronides via UGT-mediated pathways.

3'-Azido-3'-deoxythymidine (AZT) is frequently prescribed to patients infected with the human immunodeficiency virus. After absorption, AZT is rapidly metabolized into 3'-azido-3'-deoxy-5'-glucuronylthymidine by UDP-glucuronosyltransferase (UGT) enzymes. Using labeled [(14)C]UDP-glucuronic acid and microsomal preparations from human kidney 293 cells stably expressing the different human UGT2B isoenzymes, it was demonstrated that AZT glucuronidation is catalyzed specifically by human UGT2B7. The identity of the metabolite formed was confirmed as AZT-G by liquid chromatography coupled with mass spectrometry. UGT2B7 is encoded by a polymorphic gene and kinetic analysis of AZT glucuronidation by the two allelic variants UGT2B7(H(268)) and UGT2B7(Y(268)), yielded apparent K(m) values of 91.0 and 80.1 microM, respectively. Normalization to protein levels yielded glucuronidation efficiency ratios (V(max)/K(m)) of 21.3 and 11.0 microl. min(-1). mg protein(-1) for UGT2B7(H(268)) and UGT2B7(Y(268)), respectively. It remains possible that other UGT enzymes are also involved in AZT conjugation; however, the glucuronidation of AZT by UGT2B7, which is a UGT2B protein expressed in the liver, is consistent with previous findings and supports the physiological relevance of this enzyme in AZT conjugation.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a heterocyclic aromatic amine found in cooked meats and dietary exposure to PhIP has been implicated in the etiology of colon cancer in humans. PhIP, along with other heterocyclic aromatic amines, requires metabolic activation to exhibit genotoxic effects. PhIP is initially oxidized by the activity of cytochrome P4501A2 to produce 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), a reaction occurring primarily in the liver. Whereas subsequent biotransformation of N-OH-PhIP via acetylation or sulfation can produce reactive electrophiles that readily bind to DNA, N-glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), functions as a detoxification mechanism. Although hepatic glucuronidation of N-OH-PhIP has been well characterized, the extrahepatic metabolism of this compound is poorly understood. Studies in our laboratory now indicate that the intestinal tract, and particularly the colon, is a significant site of glucuronidation of N-OH-PhIP. When assays were performed with microsomes prepared from the mucosa of the intestinal tract, it was determined that glucuronidation of N-OH-PhIP occurs throughout the intestinal tract, with activity approximately three times higher in the colon as that found in the upper intestine. Glucuronidation rates from colon microsomes showed considerable interindividual variability and incubation with N-OH-PhIP yielded two glucuronides. HPLC analysis showed that the predominant product formed is the N-OH-PhIP-N2-glucuronide, while the N3-glucuronide accounts for <10% of the total glucuronidation product. These rates approach the rates found in human liver microsomes, demonstrating the significance of extrahepatic metabolism of this food-borne carcinogen. Subsequent assays with human recombinant UGTs demonstrated that at least four human UGT isoforms, all from the UGT1A subfamily, are capable of catalyzing the biotransformation of N-OH-PhIP. Members of the UGT2B family available for this study did not conjugate N-OH-PhIP, although immunoinhibition studies in human liver microsomes strongly suggest the involvement of a UGT2B isoform(s) in this organ.

A recent paper by Trocho et al. (1) describes experiments meant to show that formaldehyde adducts are formed when rats are administered the sweetener aspartame. These authors assume that the methanol carbon of aspartame generates formaldehyde which then forms adducts with protein, DNA, and RNA. Doses employed range widely. In this letter, studies which have been published previously and which were not cited by these authors are reviewed in order to put into perspective the disposition of methanol and formaldehyde in monkeys and humans, species relevant to the toxicity of methanol and its toxic metabolite, formic acid.

Although the liver has been considered the most important organ involved in glucuronidation, recent studies have focused on the role of the gastrointestinal tract in the glucuronidation of xenobiotics and endobiotics. Two UDP-glucuronosyltransferase (UGT) isoforms of human intestinal mucosa, which are absent in liver, have been identified by reverse transcriptase-polymerase chain reaction. mRNAs of UGT1A8 and UGT1A10 were detected in both the small intestine and the colon. The corresponding cDNAs for UGT1A8 and UGT1A10 were cloned from ileal RNA and inserted into the mammalian expression vector pcDNA3. Transfection of the cDNAs into human embryonic kidney 293 cells was carried out and stable expression was achieved. Membrane preparations from human embryonic kidney 293 cells expressing either UGT1A8 or UGT1A10 were isolated and the expression of each isoform was analyzed by Western blot. The catalytic activity of stably expressed UGT1A8 toward catechol estrogens, coumarins, flavonoids, anthraquinones, and phenolic compounds was much higher than that of UGT1A10. UGT1A8, but not UGT1A10, catalyzed the glucuronidation of opioids, bile acids, fatty acids, retinoids, and clinically useful drugs, such as ciprofibrate, furosemide, and diflunisal. These studies suggest that human intestinal UGTs may play an important role in the detoxification of xenobiotic compounds and, in some cases, limit the bioavailability of therapeutic agents.

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.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are potent lung carcinogens in animals. UDP-glucuronosyltransferase (UGT)-mediated glucuronidation of NNAL is a potentially important detoxification pathway for these carcinogens. To identify the UGT isozyme(s) involved in this pathway, we examined the glucuronidation of NNAL in rat liver microsomes and homogenates from cell lines overexpressing specific UGT isozymes. NNAL glucuronidation was induced in liver microsomes from rats treated with family 2 UGT inducers including phenobarbitol and 3, 5-di-tert-butyl-4-hydroxytoluene, which exhibited 1.7- and 2.6-fold higher rates of glucuronidation than microsomes from control rats. The rates of NNAL glucuronidation in liver microsomes from GUNN (deficient in family 1 UGTs) and RHA parental control rats were similar. All rat liver microsomes used in the present study catalyzed the glucuronidation of (S)-NNAL at a rate between 3.5 and 5.5 times that of the glucuronidation of (R)-NNAL. Liver microsomes from Wistar rats exhibiting the low-androsterone glucuronidation phenotype characteristic of the UGT2B2-deficient genotype glucuronidated NNAL at a rate similar to microsomes from Wistar rats exhibiting the high-androsterone glucuronidation phenotype/UGT2B2 [+] genotype. Homogenates from UGT2B1-overexpressing cells catalyzed the glucuronidation of NNAL at a K(m) of 745 microM. As with rat liver microsomes, NNAL-Gluc I was the major diastereomer formed by UGT2B1. Glucuronidation of NNAL was not detected with homogenates from UGT2B12-overexpressing cells. These results suggest that UGT2B1 plays an important role in the glucuronidation of NNAL in the rat.

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 3α-hydroxyl function. Both UGT2B7 allelic variants glucuronidated the bile acid substrates at both carboxyl and hydroxyl moieties, however, the 3α-hydroxyl position was preferentially conjugated compared to the carboxyl function. Similarly, androsterone, a 3α-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 17β-OH position, yielding the cholestatic steroid D-ring glucuronides.

Genetically modified cell lines can be very useful models for assessing the toxicologic effects of modulation of expression of individual gene products in comparison to their isogenic parental control cell lines. This symposium begins with an overview of general issues related to development and utilization of model systems created by transfection of cell lines to induce elevated expression of metabolic enzymes of toxicologic relevance. Selected studies that illustrate the heterologous expression rationale and various approaches to transgenic-cell model construction are represented. Results to date with cells engineered to express specific transfected genes are discussed, with emphasis on the effects of expression of selected phase I or phase II enzymes on cellular sensitivity to several toxic end-points. The individual sections highlight the utility of these model cell lines for examining the role of enzyme catalysis and function in metabolism of biologically active xenobiotic or endobiotic compounds of interest in toxicology. Both activating and detoxifying enzymes are discussed, with principal emphasis on the latter. This symposium includes talks on transfected cells that express aldehyde dehydrogenases, superoxide dismutase, UDP-glycosyltransferases, glutathione transferases, and cytochrome P450 isozymes. In addition to the general toxicologic utility and advantages of these genetically engineered cell lines, this overview emphasizes their particular contributions to the insights obtained to date with the specific model cell lines.

UGT2B7 has been cloned and expressed previously in COS cells and HK293 cells. Two forms have been identified: one with a tyrosine and one with a histidine at position 268. UGT2B7 has been shown to catalyze NSAIDs, catechol estrogens, and morphine-3- and -6-glucuronidation. cDNAs for UGT2B7Y268 and H268 were cloned and stably expressed in HK 293 cells. Studies were designed to test each form for reactivity toward a number of opioid compounds, xenobiotics such as menthol, oxazepam, and propranolol, and androgens such as androsterone and testosterone using membrane preparations derived from HK 293 cells. Both UGT2B7Y and UGT2B7H are highly reactive with many opioids, menthol, androsterone, and (R)- and (S)-propranolol, and similar kinetic values were observed. UGT2B7Y and UGT2B7H react poorly with oxazepam and no difference in (R)- or (S)-glucuronidation rate ratios was found. Thus, UGT2B7Y and H cannot account for the variability in the plasma or urine concentrations of these glucuronides in human populations. Our data suggest that UGT2B7 is a major isoform responsible for the glucuronidation of androsterone. Neither UGT2B7Y nor H catalyzes the glucuronidation of testosterone although each catalyzes the glucuronidation of epitestosterone. UGT2B7 seems to be a major human isoform responsible for the glucuronidation of opioids of the morphinan and oripavine class and is capable of catalyzing the glucuronidation of both the 3- and 6-hydroxyl moieties on these molecules. Thus, UGT2B7 plays a major role in the conversion of morphine to morphine-6-glucuronide, the potent analgesic metabolite of morphine.

Extrahepatic glucuronidation, such as that in the central nervous system (CNS), may play a very important role in xenobiotic disposition and may serve to protect the CNS from potentially toxic xenobiotics. UDP-glucuronosyltransferase (UGT) 1A6 is an important catalyst for phenol and polycyclic aromatic hydrocarbon glucuronidation. Studies were designed to determine the immunohistochemical localization of UGT1A6 and the steroid-reactive UGTs 2B2 and 2B3 in brain regions throughout the rat development. Neuronal cells, such as pyramidal cells, in sections from cerebral cortex and hippocampus displayed intensive UGT1A6-specific staining. UGT1A6-specific staining was also found in neuronal cells throughout the cerebral cortex, as well as in the cerebellar white matter. Glial cells revealed no apparent staining. In addition, staining for UGT1A6 was seen in choroid plexus at a later developmental stage. Although UGT1A6 staining was evident, brain sections analyzed for UGT2B2 and UGT2B3 immunoreactivity showed no significant staining. These results provide the first definitive evidence for the presence and cellular localization of UGT1A6, in neurons of developing rat brain, whereas UGT2B2 and UGT2B3 were not detected.

Catechol estrogens are major estrogen metabolites in mammals and are the most potent naturally occurring inhibitors of catecholamine metabolism. These estrogen compounds have been implicated in carcinogenic activity and the 4/2-hydroxyestradiol concentration has been shown to be elevated in neoplastic human mammary tissue compared to normal human breast tissue. Three human liver UDP-glucuronosyltransferases, UGT2B7, UGT1A1, and UGT1A3, have been shown to catalyze the glucuronidation of catechol estrogens and lead to their enhanced elimination via urine or bile. The present study was designed to study the kinetic interaction of expressed human UGT2B7(Y) or (H), UGT1A1, and UGT1A3 toward 2- and 4-hydroxycatechol estrogens. cDNAs encoding UGT2B7(Y) or (H), UGT1A1, and UGT1A3 were expressed in HK293 cells, and cell homogenates or membrane preparations were used to determine their glucuronidation ability. UGT2B7(Y) reacted with higher efficiency toward 4-hydroxyestrogenic catechols, whereas UGT1A1 and UGT1A3 showed higher activities toward 2-hydroxyestrogens. UGT2B7(H) catalyzed estrogen catechol glucuronidation with efficiencies similar to UGT2B7(Y). Flunitrazepam (FNZ), a competitive inhibitor of morphine glucuronidation in hepatic microsomes, competitively inhibited catechol estrogen glucuronidation catalyzed by UGT2B7(Y), UGT1A1, and UGT1A3. Buprenorphine, an opioid substrate that reacts at high efficiency with each of these UGTs, was also studied. FNZ competitively inhibited buprenorphine glucuronidation with UGT1A1 and UGT2B7 but had no inhibitory activity toward UGT1A3. This suggests that buprenorphine and 2-hydroxycatechol estrogens react with separate active sites of UGT1A3. A catecholamine, norepinephrine, did not inhibit UGT2B7(Y)-, UGT1A1-, and UGT1A3-catalyzed glucuronidation of catechol estrogens. These results also suggest that drug-endobiotic interactions are possible in humans and may have implication in carcinogenesis.

The mRNA expression of human UDP-glucuronosyltransferase 1A8 (UGT1A8) has been found in jejunum, ileum, and colon but not in liver. A cDNA with a complete UGT1A8 coding region was amplified from total human ileal RNA by reverse transcriptase–polymerase chain reaction and inserted into the mammalian expression vector, pcDNA3. Lysates of HK293 cells expressing UGT1A8 revealed the expression of a protein with a molecular mass of 56 kDa by Western blot analysis. Transiently expressed human UGT1A8 shows glucuronidation activities with coumarins, anthraquinones, flavonoids, phenolic compounds, catechol estrogens, 17-hydroxyandrogens, primary amines such as the carcinogen 4-aminobiphenyl, and certain opioids. This UGT may play an important role in the detoxification of xenobiotics in human intestine.

Glucuronide conjugation of xenobiotics containing a tertiary amine moiety represents a unique and important metabolic pathway for these compounds in humans. Previously, human UDP-glucuronosyltransferase (UGT) 1A4 was shown to be an important enzyme for the formation of quaternary ammonium-linked glucuronides. UGT1A3 is 93% identical to UGT1A4 in primary amino acid sequence. We show that human UGT1A3, transiently expressed in human embryonic kidney 293 cells, also catalyzes the N-glucuronidation of primary, secondary, and tertiary amine substrates, such as 4-aminobiphenyl, diphenylamine, and cyproheptadine. In contrast to expressed human UGT1A4, which catalyzes the glucuronidation of amines with high efficiency, glucuronidation of amines catalyzed by UGT1A3 exhibited low efficiency, suggesting that UGT1A3 makes only a limited contribution to the metabolic elimination of these compounds. The reactivity of expressed human UGT1A3 toward hydroxylated and carboxylic acid-containing compounds was also examined. In addition to amines, expressed human UGT1A3 catalyzed the glucuronidation of opioids (e.g. morphine and buprenorphine), coumarins, flavonoids (e.g. naringenin and quercetin), anthraquinones, and small phenolic compounds (e.g. 4-nitrophenol). Drugs containing a carboxylic acid moiety, such as nonsteroidal anti-inflammatory agents (e.g. naproxen and ibuprofen) and fibrates (e.g. ciprofibrate), were substrates for human UGT1A3. In contrast, compounds containing an aliphatic hydroxyl group, such as sapogenins, monoterpenoid alcohols (e.g. menthol and borneol), and androgens, were not conjugated by expressed human UGT1A3. Of the compounds tested, scopoletin, naringenin, and norbuprenorphine appeared to be the best xenobiotic substrates for human UGT1A3.