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Estrogen plays a pivotal role in the pathological development of breast cancer. Resveratrol has chemo-preventive effects against breast cancer, whereas, the mechanism of antitumor activities of resveratrol remains unanswered. In this study, we showed that estrogen homeostasis profile was disturbed in both breast cancer patients and in experimental breast cancer model rats, with carcinogenic catechol estrogens significantly accumulated in the mammary tissues. UDP-glucuronosyltransferase 1A8 (UGT1A8) is an important phase II drug-metabolizing enzymes which involved in the metabolism of catechol estrogens. Here we found that the mammary nuclear factor erythroid 2-related factor 2 (NRF2) - UGT1A8 signaling was down-regulated in breast cancer rats, whereas treatment with resveratrol could upregulate the expression of NRF2 and UGT1A8, accelerate metabolic elimination of catechol estrogens, inhibit estrogen-induced DNA damage and suppress the pathological development of breast cancer. In addition, luciferase reporter assay suggested that resveratrol activated the expression of UGT1A8 by up-regulating the transcriptional activity of NRF2. Small-interfering RNA-mediated silencing of NRF2 abolished resveratrol-mediated preventive effects indicated that the antitumor effect of resveratrol is based on NRF2-UGT1A8-estrogen metabolism axis. Taken together, we established the resveratrol regulating potential on estrogen homeostasis based on NRF2-UGT1A8 signaling pathway, and also provided a novel link between estrogen glucuronidation metabolism and breast cancer pathological development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The initial identification of traditionally hepatic enzymes expressed in the gut has led to the hypothesis that the gut may function as a metabolic organ. The UDP glucuronosyltransferases (UDP-GTs) play an important role as phase II metabolizing enzymes. Previously members of this family have been identified in the gut by non-isoform specific immunoreactivity, and a small amount of bilirubin glucuronosyltransferase activity was detected in the colon. Recent reports of gut transplantation to reverse the metabolic defect in Gunn rats raised further interest in the expression and distribution of human bilirubin (UDP-GTs (HUG Br 1 and HUG Br 2) in the human alimentary tract. The availability of molecular genetic probes for HUG Br 1 and HUG Br 2 permits the screening of the alimentary tract for the presence of isoform specific message. RNA samples extracted from pinch biopsy specimens of buccal mucosa, esophagus, stomach body, antrum, duodenum, and colon were analyzed for expression of HUG Br 1 and HUG Br 2. HUG Br 1 hybridization was detected in duodenum > colon, whereas HUG Br2 hybridization was detected in duodenum > esophagus > colon. Immunoreactivity data confirmed the presence of HUG Br 1 protein at low levels in the duodenum, whereas the less abundant HUG Br 2 protein was below the limits of detection of isoform specific anti-peptide antibodies. Bilirubin specific reactivity was demonstrated in duodenal samples but not antrum samples, consistent with the molecular genetic data. The presence of functional bilirubin UDP-GT isoforms in human alimentary tract supports the notion that the gut may function as a metabolic organ and may have diagnostic and therapeutic implications for disorders of bilirubin metabolism.

Interleukin-1 beta (IL-1) is one of the major inflammation mediators, commonly reported to be an inhibitor of hepatic drug metabolism. We studied the effect of IL-1 treatment on various drug-metabolizing enzymes in male and female rats. IL-1 induced both cytochrome P450 (P450) 3A1 activity and protein in females, but in males, IL-1 repressed P450 3A2 activity, without decreasing the protein. P450 1A1 was impaired in males, but was retained after dexamethasone pretreatment. IL-1 did not change P450 2B1/2 activity and protein, but counteracted their induction by dexamethasone. Uridine diphospho-glucuronosyltransferase (UGT) 1A2 (bilirubin) activity and its induction by dexamethasone were not affected by IL-1 treatment. Both P450 2C11 and epoxide hydrolase activities were repressed by IL-1 treatment, and both activities were impaired after dexamethasone treatment. These results clearly demonstrate that IL-1 acts at different steps of protein synthesis and gene expression. The effect of IL-1 on P450 was isoform-dependent, indicating that IL-1 can act on pretranscriptional events. The discrepancy between the variations of the activities and the protein of P450 3A2 suggests a post-translational regulation. For P450 2C11, 3A1, and for microsomal epoxide hydrolase, but not for P450 1A1 and 2B1/2, IL-1 mimics the glucocorticoid effects. These differential effects can affect the kinetics and the bioavailability of drugs used in pathologies in which IL-1 is increased.

Others have shown recently that dietary fish oil protects against acetaminophen-induced liver injury in vivo. Fish oil was protective because it increased the clearance of acetaminophen via glucuronidation. This work left unresolved the basis for increased rates of glucuronidation in animals fed fish oil. We therefore have determined how the amount and type of lipid in the diet affect the activity of liver microsomal UDP-glucuronosyltransferase activity. Male Wistar rats were fed a fat-free diet or isocaloric diets containing 5% corn oil, olive oil or fish oil for 4 weeks. The activity of UDP-glucuronosyltransferase was highest in rats fed fish oil and lowest in rats fed the fat-free diet. Treatment with corn oil and olive oil resulted in intermediate levels of activity. Diet-induced differences in amounts of UDP-glucuronosyltransferase were shown by immunoblotting and kinetic measurements. Treatment with fish oil resulted in a 3-fold increase in the amount of UDP-glucuronosyltransferase versus the fat-free diet. Corn oil and olive oil diets caused 2-fold increases in the amount of UDP-glucuronosyltransferase versus the fat-free diet. Fatty acid analysis of microsomal lipids showed that the fat-free diet was associated with decreased levels of arachidonic acid versus the corn oil or olive oil diets. The fish oil diet resulted in increased levels of omega-3 fatty acids versus the other diets.

Studies were performed to determine the effects of chronic hypoxia on enzymes that catalyze various detoxication reactions. Rats were exposed to room air or 10.5% O2 for 10 days, and microsomes and postmicrosomal supernatants were isolated from liver. Detoxication enzyme activities were measured by radiochemical and spectrophotometric assays, and immunoreactive protein amounts were measured by Western blot analysis. Total cytochrome P450, as measured by the CO-difference spectrum, and activities of superoxide dismutase (EC 1.15.1.1), epoxide hydrolase (EC 4.2.1.63), catalase (EC 1.11.1.6), glutathione disulfide reductase (EC 1.6.4.2), and glutathione (GSH) S-transferase (EC 2.5.1.18) were not affected by this extent of hypoxia. In contrast, 10 days of hypoxia decreased activities or immunoreactivities (% of aerobic) of GSH peroxidase (EC 1.11.1.9) (54%), cytochrome P450EtOH2 (42%), CYP3A1 (53%), sulfotransferase (EC 2.8.2.1) (77%) and UDP-glucuronosyltransferase (EC 2.4.1.17) (65%). Activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), an important enzyme in NADPH production was also decreased to 56% of the aerobic value, but Western blot analysis showed that the amount of protein reactive with antibodies to glucose-6-phosphate dehydrogenase was not affected by hypoxia. Thus, hypoxia may decrease activity of enzymes by regulatory mechanisms even though the amount of immuno-detectable enzyme is unchanged. Liver cells isolated from rats exposed to hypoxia also gave lower GSH synthetic rates than cells from normoxic rats. This result, together with the effect of hypoxia on glucose-6-phosphate dehydrogenase, indicates that the GSH supply for GSH-dependent detoxication reactions may be limited due to chronic hypoxia. To test directly whether chronic hypoxia increased sensitivity to a compound normally detoxified by a GSH-dependent reaction, sensitivity to tert-butyl hydroperoxide (t-BuOOH) of hepatocytes from rats exposed to in vivo hypoxia was compared to that from normoxic rats. The results showed that the cells from the hypoxic rats were much more sensitive to injury. Taken together, these results suggest that decreases in amounts and/or activities of detoxication enzymes during chronic hypoxia may result in increased susceptibility of cells to chemical injury.

(1) Sensitive fluorimetric assays were developed for the determination of microsomal UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine and 4-aminobiphenyl. (2) In rat liver microsomes, enzyme activity towards 1-naphthylamine was orders of magnitude higher than the activities towards 2-naphthylamine, 4-aminobiphenyl or aniline. The differences were less marked with human liver microsomes. (3) Glucuronidation of aniline and 4-aminobiphenyl was not appreciably altered in rat liver microsomes from 3-methylcholanthrene- or phenobarbital-treated rats. UDP-glucuronosyltransferase activities towards 1- and 2-naphthylamine were selectively increased (about 2-fold) by 3-methylcholanthrene-treatment. However the increases were less marked than those observed with representative substrates of the 3-methylcholanthrene-inducible enzyme form. The results suggest that the arylamines investigated are predominantly conjugated by constitutive enzyme forms in rat liver. (4) Arylamine N-glucuronides were found to be susceptible to hydrolysis by E. coli beta-glucuronidase suggesting the release of carcinogenic arylamines in the gut and their enterohepatic circulation.

The glucuronidation kinetics of 4-methylumbelliferone (4MU) and 1-naphthol (1NP) have been investigated in human liver microsomes to determine the validity of using these compounds as probes for specific UDP-glucuronosyltransferase (GT) activities in human liver. 4MU glucuronidation followed Michaelis-Menten kinetics, whereas 1NP glucuronidation kinetics were biphasic. Cross inhibition studies were performed with 4MU and 1NP to determine the relationship between 4MU glucuronidation and the two phases of 1NP glucuronidation. 4MU glucuronidation was competitively inhibited by 1NP but 4MU inhibited only the high affinity component of 1NP glucuronidation. There was good agreement between the apparent Km values for 4MU and the high affinity component of 1NP glucuronidation and their respective apparent K1 values determined in the cross inhibition studies. These data suggest that the same form(s) of human liver GT is involved in 4MU glucuronidation and the high affinity component of 1NP glucuronidation. A number of compounds known to be specific substrates for purified rat liver GTs were screened for inhibitory effects on 4MU glucuronidation in human liver microsomes. 4-Nitrophenol, 2-aminophenol and androsterone inhibited 4MU glucuronidation whereas bilirubin, chloramphenicol, digitoxigenin monodigitoxoside, morphine, oestrone and testosterone had no effect. 4-Nitrophenol and 2-aminophenol were competitive inhibitors of 4MU glucuronidation but the inhibition of 4MU glucuronidation by androsterone followed atypical kinetics. Overall, the substrate specificity of the human liver 4MU/high affinity 1NP-GT activity appears to be broadly similar to that of the 3-methylcholanthrene inducible rat hepatic microsomal GT.

Hepatic UDP glucuronosyltransferase (EC 2.4.1.17) (GT) enzymes in control, phenobarbital- and 3-methylcholanthrene-induced microsomes from C57BL/6N mice have been fractionated according to charge heterogeneity on a chromatofocusing system using a pH 9.5 to 6 gradient. Transferase activities for eleven different substrates were determined on column fractions. Activities toward 3-hydroxybenzo[a]pyrene, phenolphthalein and estrone (type 1 substrates) were enhanced by both effector compounds and always eluted primarily at pH 8.5. In control and phenobarbital-induced microsomes, activities toward testosterone, 4-hydroxybiphenyl, morphine, naphthol and 9-hydroxybenzo[a]pyrene (type 2 substrates) eluted primarily at about pH 6.7. Activities toward p-nitrophenol, 4-methylumbelliferone and 2-hydroxybiphenyl (type 3 substrates) in control and phenobarbital-induced microsomes exhibited two peaks which eluted at pH 8.5 and 6.7. 3-Methylcholanthrene treatment increased almost exclusively activities which eluted at pH 8.5 for each of the three types of substrates. The pH value of elution corresponds to the approximate isoelectric point of the eluted protein. Immunoabsorption studies with an antibody preparation raised against a purified low pI form confirmed that a 51,000-dalton transferase form, GTM1, eluted primarily at pH 6.7 and that a 54,000-dalton form, GTM2, eluted at pH 8.5. A mathematical treatment of the ratios of activity after 3-methylcholanthrene treatment to that after phenobarbital treatment versus pH produced six patterns of activity. A minimum of two enzymes at the low pH region and one enzyme at the high pH region, all with broad-substrate specificity, could account for these patterns.

The formation of morphine glucuronides is enantio- and regioselective in rats and humans. In rat liver microsomes, natural (-)-morphine formed only the 3-O-glucuronide, whereas the unnatural (+)-morphine formed glucuronides at both the 3-OH and 6-OH positions, with the 6-O-glucuronide being the principal product. In human liver microsomes, both the 3-OH-and 6-OH positions were glucuronidated with each of the enantiomers, with the 3-O-glucuronide being the major product with (-)-morphine, and the 6-OH position preferred with the (+)-enantiomer. By using a series of biochemical and biological situations such as induction by xenobiotics, ontogeny, selective inhibition and genetic deficiencies, which are considered to be diagnostic of UDP-glucuronosyltransferase heterogeneity, we determined that two UDP-glucuronosyltransferase isoenzymes were responsible for the glucuronidation of morphine in rat liver. One isoenzyme (the so-called "morphine UDP-glucuronosyltransferase") was responsible for the glucoronidation at the (-)-3-OH and (+)-6-OH positions of morphine, whereas the other formed only the (+)-morphine-3-glucuronide. Evidence from enzyme induction and the genetically deficient deficient Gunn rat suggested that bilirubin UDPGT may be responsible for the (+)-morphine-3-UDP-glucuronosyltransferase activity. In human kidney, glucuronidation of both (-)- and (+)-enantiomers at the 6-OH position was deficient, whereas the activity at the 3-OH positions was still present, which indicated the involvement of two UDP-glucuronosyltransferases in the glucuronidation of morphine in man, as well as rats.

Morphine 3-glucuronidation kinetics and the inhibitory effects of a number of xenobiotics on morphine glucuronidation in human liver microsomes have been investigated. In both native and detergent-activated microsomes morphine glucuronidation exhibited biphasic kinetics, with a high affinity, low capacity component and a low affinity, high capacity component. These data suggest the involvement of at least two forms of human liver UDP-glucuronosyltransferase (UDPGT) in morphine glucuronidation. The high affinity morphine-UDPGT activity is likely to be of most importance in morphine glucuronidation in vivo. Chloramphenicol, 4-hydroxybiphenyl, 4-methylumbelliferone, 1-naphthol and 4-nitrophenol were all shown to inhibit the low affinity morphine-UDPGT activity, but only chloramphenicol and 1-naphthol were competitive inhibitors. Each of these xenobiotics were shown to be a non-inhibitor of the high affinity morphine-UDPGT activity, or at least to have considerably lower affinity for this enzyme form(s) than morphine itself. Overall the results provide further evidence for the heterogeneity of human liver UDPGT and, in conjunction with other recent studies (Miners JO et al., Kinetic and inhibitor studies of 4-methylumbelliferone and 1-naphthol glucuronidation in human liver microsomes, Biochem Pharmacol 37: 665-671, 1988) of the kinetics of human liver glucuronidation reactions, indicate that xenobiotic substrates such as morphine, 4-methylumbelliferone and 1-naphthol may be used to differentiate UDPGT isozyme activities in human liver microsomes.

The mechanism by which an acute fast decreases the glucuronidation of hepatotoxic doses of acetaminophen in the rat was examined. Fasting did not depress the level of the enzyme, glucuronyl transferase, or the basal level of the co-substrate, UDP-glucuronic acid (UDPGA). Administration of a hepatotoxic dose of acetaminophen rapidly depleted UDPGA levels in both fed and fasted rats to the same nadir. Fed and fasted rats differed in that the rate of repletion of UDPGA levels was markedly slower in fasted rats. The total hepatic levels of UDP-glucose dehydrogenase and its cofactor, NAD+, were not decreased by fasting. In fasted rats, hepatic levels of the UDPGA precursor, UDP-glucose, were approximately 60% those of fed rats both before and after a hepatotoxic dose of acetaminophen. In fed rats, acetaminophen induced a marked depletion of hepatic glycogen levels and a dramatic increase in blood glucose levels. Acetaminophen induced a similar marked increase in blood glucose levels in fasted rats in spite of the fact that they lacked hepatic glycogen. It is concluded that the fasting-induced decrease in the glucuronidation of hepatotoxic doses of acetaminophen results from decreased production of UDPGA. The decreased synthetic capacity for UDPGA does not appear to be due to the inability of the liver to produce glucose units per se, but rather to the fasting-induced altered activities of the enzymes of carbohydrate metabolism which, in turn, alter the fate of glucose-6-phosphate derived from gluconeogenesis.

We have studied the effects of prochlorperazine on the activities of UDP-glucuronosyltransferase and glucose-6-phosphatase (glucose-6-P'ase) in rat liver microsomes. The activity of UDP-glucuronosyltransferase was increased in a graded fashion by addition of prochlorperazine. Maximal stimulation occurred at 1 mg prochlorperazine to 2 mg microsomal protein, which resulted in a 6-fold increase in activity. However, with smaller concentrations of drug, there was a time-dependent increase in the activity of UDP-glucuronosyltransferase. Sensitivity of UDP-glucuronosyltransferase to activation by UDP-N-acetylglucosamine was lost after treatment of microsomes with prochlorperazine. These results indicate that prochlorperazine causes a profound reorganization of the interactions between lipids and enzyme since the activity and allosteric properties of UDP-glucuronosyltransferase are known to depend on interactions with lipids in a gel phase. Glucose-6-P'ase also was activated in a graded fashion by prochlorperazine; 1 mg of drug/2 mg microsomal protein resulted in a 60% increase in activity. The temperature-dependent instability of glucose-6-P'ase was increased by treatment of microsomes with prochlorperazine and could be prevented only partially by substrate. We conclude that prochlorperazine disrupts the structural organization between lipids and proteins in microsomal membranes, altering thereby the activity and regulation of at least two different integral membrane proteins.

In this report we present evidence that the heterogeneity of hepatic microsomal UDP-glucuronosyltransferase(s) (UDPGT) activities depends on the chemical structures of the aglycones as well as their biophysical constants. Three animal models were used: Wistar rats, which have active UDPGTs; Gunn rats, in which some of the UDPGT activities are reduced, but which can be induced by phenobarbital; and guinea pigs. In Wistar rats, we found that some coumarins were poor substrates of UDPGT (GT1) and that twenty monoterpenoid alcohol activities showed typical phenobarbital-inducible behavior. In Gunn rats, we showed that substitution of the phenolic aglycone by bulky (alkyl- or methoxy-) groups in the 2-position of the phenolic ring decreased UDPGT (GT1) activity, whereas substitution in the 4-position resulted in an increase in this activity. We also showed that, in this particular strain, activities toward terpenes were less affected than activities toward flat (aromatic) aglycones. Induction by phenobarbital in Gunn rats increased the activity and limited the deficiency for monoterpenoid alcohols. In guinea pigs, we confirmed that phenobarbital selectively increased the activities of UDPGT towards twenty monoterpenoid alcohols without affecting other typical phenobarbital-induced activities such as those for conjugation of morphine. Finally, we showed that orientation of the aglycone molecule in the active site was apparently related to its dipole moment and that the distance between "acceptor-oxygen" (hydroxyl) and the carbons out of the general plane of the molecule was an important factor. These studies clearly suggest that rat and guinea pig contain a UDPGT(monoterpenoid alcohols) with restricted specificities and also that UDPGT(GT1) comprises at least two or three different isoenzymes, each with a slightly different restricted specificity towards flat aromatic aglycones.

Since 2-acetylaminofluorene (2-AAF), 4-acetylaminobiphenyl (4-AABP) and 2-aminonaphthalene (2-AN) display varying degrees of carcinogenicity in the rat, which is capable of N-acetylating arylamines, an attempt was made to correlate the difference in carcinogenicity of these compounds with the ease of O-glucuronidation of their hydroxamic acids by rat hepatic microsomes, a reaction believed to be a detoxification mechanism. UDP-glucuronosyltransferase activity of rat hepatic microsomes was activated by Triton X-100. Glucuronidation by Triton X-100 activated microsomes of the N-hydroxy derivative of 2-AN was approximately 1.5 and 1.8 times faster than the corresponding derivatives of 2-aminofluorene (2-AF) and 4-aminobiphenyl (4-ABP) respectively. However, glucuronidation of the N-hydroxy-N-acetyl derivative of 2-AN was 40 and 17 times faster than the corresponding derivatives of 2-AF and 4-ABP respectively. Aroclor 1254 and 3-methylcholanthrene, but not phenobarbital, acetanilide and butylated hydroxytoluene, induced the enzyme for the glucuronidation of 2-AN derivatives. The present study (1) demonstrates an inverse relationship between the carcinogenicity of 2-AN, 4-AABP and 2-AAF and the ease of glucuronidation of their hydroxamic acid derivatives, and (2) suggests that, in addition to N- and C-hydroxylation, glucuronidation may play an important role in determining the carcinogenicity of arylamines and arylacetamides in the rat.

The expression of xenobiotic-metabolizing enzymes was studied in the human lung tumour cell lines NCI-H322 and NCI-H358. These cells are derived from adenocarcinomas and exhibit features of Clara cells and alveolar type II cells, respectively. Examination of the in vitro activities showed that both cell lines lack UDP-glucuronosyltransferase against the substrates 3-hydroxybenzo[a]pyrene (3-OH-BaP) and 4-hydroxybiphenyl (4-OH-Bph) and that in vitro conjugation of sulfate with 3-OH-BaP was only just detectable. In contrast, both cell lines showed fairly high levels of glutathione-S transferase activity with the substrate 1-chloro-2,4-dinitro-benzene (54.4 and 83.0 nmol/min X mg protein, respectively) and of glutathione (81 and 41 nmole/mg protein, respectively). The metabolic capacity of intact NCI-H322 and NCI-H358 cells was examined using benzo[a]pyrene (BaP) and 3-OH-BaP as substrates. The cell lines formed sulfate conjugates from 3-OH-BaP (4.5 and 0.4 pmol/min X mg protein, respectively) but did not produce any detectable glucuronides. When cultures of the two cell lines were exposed to BaP, phenolic products accumulated in the growth medium. NCI-H322 cells also formed some sulfate conjugates, whereas such conjugates were barely detectable in the medium of NCI-H358 cells. In contrast A549, a human pulmonary adenocarcinoma cell line known to contain UDP-glucuronosyltransferase activity, efficiently conjugated 3-OH-BaP to glucuronic acid and converted the primary phenolic products formed from BaP to glucuronides. Thus the NCI-H322 and NCI-H358 cells are exceptional in that they possess no or very little glucuronosyltransferase activity but exhibit appreciable monooxygenase activity. The cell lines may therefore be of interest for examining the biological effects of potentially toxic chemicals which are otherwise detoxified by glucuronic acid conjugation. The cells may also be useful as test systems for evaluating the potential cytotoxicity and genotoxicity of chemicals to human lung.

The ability of phenobarbital (PB) to neonatally "imprint" or "program" the hepatic microsomal cytochrome P-450-dependent monooxygenase system (MOS) was investigated. Phenobarbital (30 mg/kg) was administered subcutaneously to neonatal rats of both sexes on days 1-5 postpartum. Various hepatic MOS activities were measured at 6, 22, 50 and 140 days of age. Six-day-old animals of both sexes displayed the increased hepatic microsomal protein levels and enzyme activities expected from the action of phenobarbital as a transitory MOS inducer. Most of these increases dissipated by 22 and 50 days of age. However, at 140 days of age rats of both sexes that had received neonatal phenobarbital showed increased levels of cytochrome P-450, as well as both P-450 and cytochrome c reductase, ethoxycoumarin O-deethylation, glucuronyl transferase activity, in vitro covalent binding of benzo[a]pyrene to DNA and in vivo covalent binding of aflatoxin B1 to hepatic macromolecular fractions. Neonatal phenobarbital administration can alter the metabolic profile of rats in adulthood, apparently by a mechanism different from that responsible for either transitory PB induction or testosterone imprinting.