UDP-glucuronosyltransferase Activity Research Articles

Comparison of the hepatic and thyroid gland effects of sodium phenobarbital and pregnenolone-16α-carbonitrile in wild-type and constitutive androstane receptor (CAR)/pregnane X receptor (PXR) knockout rats

The hepatic and thyroid gland effects of the constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) and the pregnane X receptor (PXR) activator pregnenolone-16α-carbonitrile (PCN) were examined in male Sprague-Dawley wild-type (WT) and knockout (KO) rats lacking both hepatic CAR and PXR receptors (CAR KO/PXR KO rats).The treatment of WT rats for 7 d with 500 ppm NaPB in the diet and 100 mg/kg/d PCN by gavage resulted in increased relative liver weight, hepatocyte hypertrophy, increased hepatocyte replicative DNA synthesis (RDS) and induction of cytochrome P450 CYP2B and CYP3A subfamily enzymes. NaPB and PCN also induced thyroid gland follicular cell RDS and hepatic microsomal UDP-glucuronosyltransferase activity towards thyroxine as substrate. These effects were not observed in the liver and thyroid gland of CAR KO/PXR KO rats.Male C57BL/6 J (WT) and CAR KO/PXR KO mice were given 1000 ppm NaPB in the diet for 7 d. In WT, but not in CAR KO/PXR KO, mice NaPB treatment resulted in liver hypertrophy and induction of hepatocyte RDS and Cyp2b enzymes.These results suggest that the CAR KO/PXR KO rat and mouse models are useful experimental models for mode of action studies with rodent CAR activators.

Identification of Intestinal UDP-Glucuronosyltransferase Inhibitors in Green Tea (Camellia sinensis) Using a Biochemometric Approach: Application to Raloxifene as a Test Drug via In Vitro to In Vivo Extrapolation

Green tea (Camellia sinensis) is a popular beverage worldwide, raising concern for adverse interactions when co-consumed with conventional drugs. Like many botanical natural products, green tea contains numerous polyphenolic constituents that undergo extensive glucuronidation. As such, the UDP-glucuronosyltransferases (UGTs), particularly intestinal UGTs, represent potential first-pass targets for green tea-drug interactions. Candidate intestinal UGT inhibitors were identified using a biochemometrics approach, which combines bioassay and chemometric data. Extracts and fractions prepared from four widely consumed teas were screened (20-180 μg/ml) as inhibitors of UGT activity (4-methylumbelliferone glucuronidation) in human intestinal microsomes; all demonstrated concentration-dependent inhibition. A biochemometrics-identified fraction rich in UGT inhibitors from a representative tea was purified further and subjected to second-stage biochemometric analysis. Five catechins were identified as major constituents in the bioactive subfractions and prioritized for further evaluation. Of these catechins, (-)-epicatechin gallate and (-)-epigallocatechin gallate showed concentration-dependent inhibition, with IC50 values (105 and 59 μM, respectively) near or below concentrations measured in a cup (240 ml) of tea (66 and 240 μM, respectively). Using the clinical intestinal UGT substrate raloxifene, the Ki values were ∼1.0 and 2.0 μM, respectively. Using estimated intestinal lumen and enterocyte inhibitor concentrations, a mechanistic static model predicted green tea to increase the raloxifene plasma area under the curve up to 6.1- and 1.3-fold, respectively. Application of this novel approach, which combines biochemometrics with in vitro-in vivo extrapolation, to other natural product-drug combinations will refine these procedures, informing the need for further evaluation via dynamic modeling and clinical testing.

Endocrine Disruption Activity of 30-day Dietary Exposure to Decabromodiphenyl Ethane in Balb/C Mouse

ObjectiveThis study aimed to evaluate the hepatotoxicity, metabolic disturbance activity and endocrine disrupting activity of mice treated by Decabromodiphenyl ethane (DBDPE). MethodsIn this study, Balb/C mice were treated orally by gavage with various doses of DBDPE. After 30 days of treatment, mice were sacrificed; blood, livers and thyroid glands were obtained, and hepatic microsomes were isolated. Biochemical parameters including 8 clinical chemistry parameters, blood glucose and hormone levels including insulin and thyroid hormone were assayed. The effects of DBDPE on hepatic cytochrome P450 (CYP) levels and activities and uridinediphosphate-glucuronosyltransferase (UDPGT) activities were investigated. Liver and thyroid glands were observed. ResultsThere were no obvious signs of toxicity and no significant treatment effect on body weight, or liver-to-body weight ratios between treatment groups. The levels of ALT and AST of higher dose treatment groups were markedly increased. Blood glucose levels of treatment groups were higher than those of control group. There was also an induction in TSH, T3, and fT3. UDPGT, PROD, and EROD activities were found to have been increased significantly in the high dose group. Histopathologic liver changes were characterized by hepatocyte hypertrophy and cytoplasmic vacuolization. Our findings suggest that DBDPE can cause a certain degree of mouse liver damage and insufficiency. ConclusionDBDPE has the activity of endocrine disruptors in Bal/C mice, which may induce drug-metabolizing enzymes including CYPs and UDPGT, and interfere with thyroid hormone levels mediated by AhR and CAR signaling pathways. Endocrine disrupting activity of DBDPE could also affect the glucose metabolism homeostasis.

Endometrial blood perfusion as assessed using a novel laser Doppler technique in Angus cows

Previous studies have characterized ovarian steroid synthesis which directly affects uterine environment and blood flow. Clearance of steroids occurs primarily in hepatic tissues, however, it was discovered that there is an abundant activity of the phase II steroid metabolizing enzyme UDP-glucuronosyltransferase (UGT) in uterine biopsies. No minimally invasive techniques for collecting endometrial perfusion, which is affected by steroids and indicative of reproductive health, have been developed for livestock. The objective of the present study was to characterize UGT activity and endometrial blood perfusion during a normal estrous cycle of cattle. It was hypothesized that there would be increased steroid metabolism during the luteal phase of the estrous cycle and in the uterine horn ipsilateral to the corpus luteum (CL). During the first synchronized estrous cycle, progesterone and UGT activity increased on Day 6 compared with 0 and 3, with the first day of estrus being considered Day 0 of the study. Endometrial perfusion was greater ipsilateral to the CL compared with contralateral on Day 12, and was less ipsilateral to the CL compared with contralateral on Day 18. Similar to perfusion results, nitric oxide metabolites (nitrites) were greatest in the endometrium ipsilateral as compared with contralateral to the CL. Moreover, there was a positive correlation (r = 0.28; P = .04) between endometrial perfusion and nitrite concentration. It is concluded that activity of UGT within the endometrium is affected by the contralateral or ipsilateral location of the CL, and collection of endometrial perfusion data using a laser Doppler probe could be a viable measurement technique as indicated by associated nitrite concentrations in the present study.

Toxicological responses on cytochrome P450 and metabolic transferases in liver of goldfish (Carassius auratus) exposed to lead and paraquat

As the producer of reactive oxygen species (ROS), both lead (Pb) and paraquat (PQ) can generate serious oxidative stress in target organs which result in irreversible toxic effects on organisms. They can disturb the normal catalytic activities of many enzymes by means of different toxicity mechanism. The changed responses of enzymes are frequently used as the biomarkers for indicating the relationship between toxicological effects and exposure levels. In this work, goldfish was exposed to a series of test groups containing lead and paraquat in the range of 0.05–10mg/L, respectively. Four hepatic enzyme activities, including 7-ethoxyresorufinO-deethylase (EROD), 7-benzyloxy-4-trifluoromethyl-coumarin-O-debenzyloxylase (BFCOD), glutathione S-transferase (GST) and UDP-glucuronosyltransferase (UGT) were determined after 1, 7, 14, 28 days exposure. The results showed that the activities of EROD and BFCOD in fish were significantly inhibited in response to paraquat at all exposure levels during the whole experiment. Similarly, the inhibitory effects of lead exposure on BFCOD activity were found in our study, while different responses of lead on EROD were observed. There were no significant differences on EROD activity under lower concentrations of lead (less than 0.1mg/L) before 14 days until an obvious increase was occurred for the 0.5mg/L lead treatment group at day 14. Furthermore, lead showed stronger inhibition on GST activity than paraquat when the concentrations of the two toxicants were more than 0.5mg/L. However, the similar dose and time-dependent manners of UGT activity were found under lead and paraquat exposure. Our results indicated that higher exposure levels and longer accumulations caused inhibitory effects on the four enzymes regardless of lead or paraquat stress. In addition, the responses of phase I enzymes were more sensitive than that of phase II enzymes and they may be served as the acceptable biomarkers for evaluating the toxicity effects of both lead and paraquat.

Inhibitory Effects of Endogenous Linoleic Acid and Glutaric Acid on the Renal Glucuronidation of Berberrubine in Mice and on Recombinant Human UGT1A7, 1A8, and 1A9.

Berberrubine (BRB) has a strong lipid-lowering effect and can be extensively metabolized into berberrubine-9-O-β-d-glucuronide (BRBG) in vivo. Recently, pharmacokinetics studies showed that the production of BRBG was significantly decreased in the urine of mice fed with a high-fat diet (HFD), indicating a decreased glucuronidation capacity. Based on the UDP-glucuronosyltransferase (UGT) isoform identification, hepatic and renal microsomal incubation, glucuronidation was examined to suggest the metabolism of BRB in liver and kidneys. The results showed that the renal UGT activity for metabolizing BRB markedly decreased, which may be highly related to the decreased expression and activity of renal Ugt1a7c. Surprisingly, in vitro studies revealed neither BRB nor BRBG inhibited the renal UGT activity. By employing an integrated strategy of metabolomics and pharmacokinetics, we identified and confirmed for the first time the inhibitory effect of some potential endogenous molecules on the renal glucuronidation of C57BL/6J mice, such as glutaric acid (GA) and linoleic acid (LA). By employing recombinant human UGTs, we found that GA and LA efficiently affect the activity of recombinant human UGT1A7, 1A9, and 1A8 at their normal or abnormal physiologic levels in vivo. GA (2 mM) markedly inhibited the activity of UGT1A7 by 89.4% and UGT1A9 by 32.8%. The inhibition rates reached 99.3% for UGT1A9, 48.3% for UGT1A7, and 46.8% for UGT1A8 with LA at 200 μM. It has been suggested that the endogenous molecules have the potential to affect the efficiency of glucuronidation, which might be a key factor contributing to individual differences in drug metabolism.

Human UDP-Glucuronosyltransferase 2B4 and 2B7 Are Responsible for Naftopidil Glucuronidation in Vitro.

Naftopidil (NAF) is widely used for the treatment of benign prostatic hyperplasia and prevention of prostate cancer in elderly men. These patients receive a combination of drugs, which involves high risk for drug–drug interaction. NAF exhibits superior efficacy but must be administered at a much higher dosage than other therapeutic drugs. We previously showed that extensive glucuronidation of NAF enantiomers caused poor bioavailability. However, the metabolic pathway and mechanism of action of NAF enantiomer remain to be elucidated. The present study was performed to identify the human UDP-glucuronosyltransferases (UGTs) responsible for the glucuronidation of NAF enantiomers and to investigate the potential inhibition of UGT activity by NAF. The major metabolic sites examined were liver and kidney, which were compared with intestine. Screening of 12 recombinant UGTs showed that UGT2B7 primarily contributed to the metabolism of both enantiomers. Moreover, enzyme kinetics for R(+)-NAF, UGT2B7 (mean Km, 21 μM; mean Vmax, 1043 pmol/min/mg) showed significantly higher activity than observed for UGT2B4 and UGT1A9. UGT2B4 (mean Km, 55 μM; mean Vmax, 1976 pmol/min/mg) and UGT2B7 (mean Km, 38 μM; mean Vmax, 1331 pmol/min/mg) showed significantly higher catalysis of glucuronidation of S(-)-NAF than UGT1A9. In human liver microsomes, R(+)-NAF and S(-)-NAF also inhibited UGT1A9: mean Ki values for R(+)-NAF and S(-)-NAF were 10.0 μM and 11.5 μM, respectively. These data indicate that UGT2B7 was the principal enzyme mediating glucuronidation of R(+)-NAF and S(-)-NAF. UGT2B4 plays the key role in the stereoselective metabolism of NAF enantiomers. R(+)-NAF and S(-)-NAF may inhibit UGT1A9. Understanding the metabolism of NAF enantiomers, especially their interactions with metabolic enzymes, will help to elucidate potential drug–drug interactions and to optimize the administration of this medicine.

Alleviating Effect of Mitragynine on Microsomal UDP-Glucuronosyltransferase Activity and UGT1A6 Expression in STZ-Induced Diabetic Rat

Alleviating Effect of Mitragynine on Microsomal UDP-Glucuronosyltransferase Activity and UGT1A6 Expression in STZ-Induced Diabetic Rat

Effects of Ganoderma, Rhodiola and grape seed extracts on the glucuronidation and oral bioavailability of resveratrol in rats

Herb extracts were shown to inhibit the activity of UDP-glucuronosyltransferases (UGTs) in vitro. However, the actual in vivo effect of the inhibitory ability on oral bioavailability is yet verified. In this study, resveratrol (RES) was used as a model drug to study the effect of three Chinese herb extracts, Ganoderma, Rhodiola and grape seed, on the in vitro and in vivo inhibition of glucuronidation and the in vivo bioavailability of RES. Overall, although herb extracts might show inhibition on glucuronidation of RES in vitro and in vivo, the inhibition of glucuronidation did not necessarily mean to improve the in vivo bioavailability of RES.

Modulation of UDP-glucuronidation by acridinone antitumor agents C-1305 and C-1311 in HepG2 and HT29 cell lines, despite slight impact in noncellular systems.

Among the studied antitumor acridinone derivatives developed in our laboratory, 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) and 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) exhibited cytotoxic and antitumor properties against several cancer types and were selected to be evaluated in preclinical and early-phase clinical trials. In the present work, we investigated the impact of C-1305 and C-1311 on UDP-glucuronosyltransferase (UGT) activity. Enzyme activity modulation was studied using HPLC by analyzing standard UGT substrate metabolism in the presence and absence of antitumor drugs. The investigations were performed in two model systems: (i) under noncellular conditions, including human liver microsomes (HLM) and recombinant UGT1A1, 1A9 and 1A10 isoenzymes and (ii) in tumor cells. There was observed a slight impact of studied drugs on enzyme activity. Only UGT1A1 action was altered by both compounds. The modulatory effects of UGT activity in cellular systems depended on the tumor cell type. In the case of HepG2, C-1305 and C-1311 strongly induced UGT activity, particularly for C-1311, at concentrations significantly lower than the EC50. This effect contradicted irinotecan mediated UGT inhibition. HT29 colon tumor cells were less sensitive than HepG2 to enzyme modulation in the presence of the studied compounds, particularly C-1305, where enzymatic inhibition similar to that of irinotecan was observed. The results demonstrated that UGT activity modulation should be expected in the case of antitumor therapy with C-1305 or/and C-1311. Analysis of the results indicated that these modulations would occur via cellular regulatory pathways not by direct drug-enzyme interactions.

The effects of UDP-sugars, UDP and Mg2+on uridine diphosphate glucuronosyltransferase activity in human liver microsomes

1. The UDP-glucuronosyltransferase (UGT) enzymes are important in the metabolism, elimination and detoxification of many xenobiotics and endogenous compounds. As extrapolation of in vitro kinetics of drug metabolizing enzymes to predict in vivo clearance rates becomes more sophisticated, it is important to ensure proper optimization of enzyme assays. The luminal location of the enzyme active site (i.e. latency), and the complexity of UGT kinetics, results in consistent under-prediction of clearance of drugs metabolized by glucuronidation.2. We examined inhibition of UGT activity in alamethicin-disrupted human liver microsomes (HLM) by uridine diphosphate (UDP), a UGT reaction product, and its reversal by Mg2+ ions. We also determined whether UDP-sugars other than the co-substrate UDP-glucuronic acid (UDP-GlcA) affected glucuronidation.3. We show that other UDP-sugars do not significantly influence glucuronidation. We also demonstrate that UDP inhibits HLM UGT activity and that this is reversed by including Mg2+ in the assay. The Mg2+ effect can be offset using EDTA, and is dependent on the concentration of UDP-GlcA in the assay.4. We propose that formation of a Mg2+–UDP complex prevents UDP from affecting the enzyme. Our results suggest that 5 mM UDP-GlcA and 10 mM Mg2+ be used for UGT assays in fully disrupted HLM.

In vitro inhibition of human UGT isoforms by ritonavir and cobicistat

1. Ritonavir and cobicistat are pharmacokinetic boosting agents used to increase systemic exposure to other antiretroviral therapies. The manufacturer’s data suggests that cobicistat is a more selective CYP3A4 inhibitor than ritonavir. However, the inhibitory effect of ritonavir and cobicistat on human UDP glucuronosyltransferase (UGT) enzymes in Phase II metabolism is not established. This study evaluated the inhibition of human UGT isoforms by ritonavir versus cobicistat.2. Acetaminophen and ibuprofen were used as substrates to evaluate the metabolic activity of the principal human UGTs. Metabolite formation rates were determined by HPLC analysis of incubates following in vitro incubation of index substrates with human liver microsomes (HLMs) at different concentrations of ritonavir or cobicistat. Probenecid and estradiol served as positive control inhibitors.3. The 50% inhibitory concentrations (IC50) of cobicistat and ritonavir were at least 50 µM, which substantially exceeds usual clinical plasma concentrations. Probenecid inhibited the glucuronidation of acetaminophen (IC50 0.7 mM), but not glucuronidation of ibuprofen. At relatively high concentrations, estradiol inhibited ibuprofen glucuronidation (IC50 17 µM).4. Ritonavir and cobicistat are unlikely to produce clinically important drug interactions involving drugs metabolized to glucuronide conjugates by UGT1A1, 1A3, 1A6, 1A9, 2B4 and 2B7.

Revisiting the Latency of Uridine Diphosphate-Glucuronosyltransferases (UGTs)-How Does the Endoplasmic Reticulum Membrane Influence Their Function?

Uridine diphosphate-glucuronosyltransferases (UGTs) are phase 2 conjugation enzymes mainly located in the endoplasmic reticulum (ER) of the liver and many other tissues, and can be recovered in artificial ER membrane preparations (microsomes). They catalyze glucuronidation reactions in various aglycone substrates, contributing significantly to the body’s chemical defense mechanism. There has been controversy over the last 50 years in the UGT field with respect to the explanation for the phenomenon of latency: full UGT activity revealed by chemical or physical disruption of the microsomal membrane. Because latency can lead to inaccurate measurements of UGT activity in vitro, and subsequent underprediction of drug clearance in vivo, it is important to understand the mechanisms behind this phenomenon. Three major hypotheses have been advanced to explain UGT latency: compartmentation, conformation, and adenine nucleotide inhibition. In this review, we discuss the evidence behind each hypothesis in depth, and suggest some additional studies that may reveal more information on this intriguing phenomenon.

Direct comparison of UDP-glucuronosyltransferase and cytochrome P450 activities in human liver microsomes, plated and suspended primary human hepatocytes from five liver donors

UDP-glucuronosyltransferases (UGTs) and cytochrome P450s (CYPs) are the major enzymes involved in hepatic metabolism of drugs. Hepatic drug metabolism is commonly investigated using human liver microsomes (HLM) or primary human hepatocytes (PHH). We describe the development of a sensitive assay to phenotype activities of six major hepatic UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7) in intact PHH by analysis of glucuronidation of selective probe substrates. The non-selective, general substrate 7-hydroxycoumarin was included for comparison. For each liver donor preparation (five donors) UGT activities in cryopreserved suspended and plated PHH were compared to HLM prepared from the same donors. Standard CYP reaction phenotyping of seven major isoforms was performed in parallel. For all donors, CYP- and UGT-isoforms activity profiles were comparable in PHH and HLM, indicating that reaction phenotyping with selective probe substrates in intact cells primarily reflects respective CYP or UGT activity. System-dependent effects on UGT and CYP isoform activity were still found. While UGT activity of UGT1A1 was equivalent in plated and suspended PHH, UGT1A3, UGT1A6 and UGT2B7 activity was higher in suspended PHH and UGT1A9 and UGT1A4 activity was higher in plated PHH. The well-known decrease in activity of most CYP isoforms in plated compared to suspended PHH was confirmed. Importantly, we found a significant loss in CYP2C19 and CYP2B6 in HLM, activity being lower than in intact cells. Taken together, these findings implicate that, dependent on the UGT or CYP isoforms involved in the metabolism of a given compound, the outcome of metabolic assays is strongly dependent on the choice of the in vitro system. The currently described UGT- and CYP- activity profiling method can be used as a standard assay in intact cells and can especially aid in reaction phenotyping of in vitro systems for which a limited number of cells are available.

Human Biliary Amount Prediction Using Simple, Bile Flow-Rate Corrected and Uridine Diphosphate Glucuronosyltransferase Activity Corrected Allometric Methods

Aim: Retrospective scaling of human biliary amounts has been performed using allometry. Methods: Human biliary excretory data for 14 drugs were predicted using simple, bile flow-rate corrected and uridine diphosphate glucuronosyltransferase (UDPGT) activity corrected allometry methods. Allometry was performed using Y = aXb relationship with correction factors. Statistical tests consisting of fold difference (predicted/observed) and root-mean-square error (RMSE) computation were carried out. Results: UDPGT activity corrected allometry predicted higher biliary amounts in humans as compared with other methods. The RMSE values were 38, 32 and 81 for simple, bile flow-rate corrected and UDPGT activity corrected allometry, respectively. Conclusion: Although data showed the usefulness of the approaches for human biliary predictions in the decision process for nominating drug candidate(s) based on % RMSE and fold differences, for some drugs prediction appeared not satisfactory using any of the methods.

Everolimus-inhibited multiple isoforms of UDP-glucuronosyltransferases (UGTs)

1. Everolimus is an inhibitor of mammalian target of rapamycin (mTOR) and has been clinically utilized to prevent the rejection of organ transplants. This study aims to determine the inhibition of everolimus on the activity of phase-II drug-metabolizing enzymes UDP-glucuronosyltransferases (UGTs).2. The results showed that 100 μM of everolimus exerted more than 80% inhibition toward UGT1A1, UGT-1A3 and UGT-2B7. UGT1A3 and UGT2B7 were selected to elucidate the inhibition mechanism, and in silico docking showed that hydrogen bonds and hydrophobic interactions mainly contributed to the strong binding of everolimus toward the activity cavity of UGT1A3 and UGT2B7. Inhibition kinetic-type analysis using Lineweaver–Burk plot showed competitive inhibition toward all these UGT isoforms. The inhibition kinetic parameters (Ki) were calculated to be 2.3, 0.07 and 4.4 μM for the inhibition of everolimus toward UGT1A1, UGT-1A3 and UGT-2B7, respectively.3. In vitro–in vivo extrapolation (IVIVE) showed that [I]/Ki value was calculated to be 0.004, 0.14 and 0.002 for UGT1A1, UGT-1A3 and UGT-2B7, respectively. Therefore, high DDI potential existed between everolimus and clinical drugs mainly undergoing UGT1A3-catalyzed glucuronidation.

Disturbance of Mammary UDP-Glucuronosyltransferase Represses Estrogen Metabolism and Exacerbates Experimental Breast Cancer

The progression of breast cancer is closely related to the levels of estrogens within the body. UDP-glucuronosyltransferase (UGT) is an important class of phase II metabolizing enzymes, playing a pivotal role in detoxifying steroid hormone. In the present study, we aim at uncovering the potential dysregulation pattern of UGT and its role in estrogen metabolism and in the pathogenesis of breast cancer. Female Sprague-Dawley rats were treated with 100 mg/kg dimethylbenz(a)anthracene (DMBA) to induce breast cancer. Our results showed that the expression and activity of UGT in mammary tissues were downregulated significantly in DMBA rats. Consistent with this, levels of estradiol, 4-hydroxylated estradiol, and 2-hydroxylated estradiol were increased in both mammary tissues and serum, supporting a notable accumulation of toxic estrogen species in the target tissue of breast cancer. In addition, we also observed the decreased cell migration, cell proliferation, and DNA damage in UGT-transfected MCF-7 cells, suggesting a protective role of UGT against estrogen-induced mammary carcinogenesis. Taken together, these results indicated that accumulation of estrogens induced by UGT deficiency is a critical factor to induce the development of breast cancer. UGT contributes to estrogen elimination, and its glucuronidation capacity influences the estrogen signaling pathway and the pathogenesis of breast cancer.

Long-Term Stability of Cryopreserved Human Hepatocytes: Evaluation of Phase I and II Drug-Metabolizing Enzyme Activities and CYP3A4/5 Induction for More than a Decade

We evaluated the long-term stability of hepatocytes stored in the vapor phase of liquid nitrogen for their viability, cytochrome P450 (CYP) 1A2 activity, CYP3A4/5 activity, uridine diphosphate-glucuronosyl transferase (UGT) activity, sulfotransferase (SULT) activity, and CYP3A4/5 induction during 14 years of preservation. No substantial degradation of viability, CYP1A2 activity, UGT activity, or CYP3A4/5 induction was observed. CYP3A4/5 activity showed a slight decrease after 7 years of storage, and SULT activity gradually decreased during storage, although substantial activities remained even after 14 years. These results indicate that cryopreserved human hepatocytes can be stored stably for more than a decade with little or no change in viability, activity of drug-metabolizing enzymes, or CYP3A4/5 induction, and can be widely applicable to qualitative research in drug metabolism.

Phylogenetic signals in detoxification pathways in Cyprinid and Centrarchid species in relation to sensitivity to environmental pollutants

Observations in a previous study on biomarker responses in fish collected from urban creeks in Greenville, SC, indicated that there might be considerable differences in the expression of biotransformation enzymes in chub and sunfish species. To further investigate these species differences a dosing experiment was performed in which bluehead and creek chub (Nocomis leptocephalus and Semotilus atromaculatus), and redbreast sunfish, pumpkinseed, and bluegill (Lepomis auritus, L. gibbosus, and L. macrochirus) were injected with benzo[a]pyrene (BaP) as a model compound for common pollutants in urban creeks. Fish were injected with BaP doses of 0, 25 and 50mg/kg, and after 3days BaP metabolites in bile, and enzymatic activities of cytochrome P450-1A (CYP1A), UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) were measured. CYP1A activity was significantly increased after BaP dosing in both species groups, but chubs had significantly lower levels than were observed in the dosed sunfish. The UGT activity in unexposed animals was comparable in both species groups, and significantly increased in both groups as a result of BaP dosage. Finally, GST activity was significantly higher in chubs, but did not change in either species group as a result of BaP exposure. There were no significant differences between species within each species group, and the results confirmed that unexposed chubs have much lower CYP1A activity, but a much higher GST activity than unexposed sunfish. The metabolized BaP was excreted in both species groups, but at the time of sampling there were no differences in the amount of BaP metabolites in the bile of dosed animals. The differences in baseline enzyme activity and induction capacity between both species groups are an example of phylogenetically determined differences between fish families, and may explain why chubs are in general more sensitive to exposure to environmental pollutants than sunfish. This conclusion was corroborated by the observation that the highest BaP dose of 50mg/kg was close to the apparent LC50 for chub, while no mortality was observed in the sunfish at this dose.

The time-dependent effects of St John’s wort on cytochrome P450, uridine diphosphate-glucuronosyltransferase, glutathione S-transferase, and NAD(P)H-quinone oxidoreductase in mice

Hypericum perforatum [St. John’s wort (SJW)] is known to cause a drug interaction with the substrates of cytochrome P450 (P450, CYP) isoforms, mainly CYP3A. This study aims to determine the dose response and time course of the effects of SJW extract on P450s, UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), and NAD(P)H-quinone oxidoreductase (NQO) in mice. The oral administration of SJW extract to male mice at 0.6 g/kg/d for 21 days increased hepatic oxidation activity toward a Cyp3a substrate nifedipine. By extending the SJW treatment to 28 days, hepatic nifedipine oxidation (NFO) and warfarin 7-hydroxylation (WOH) (Cyp2c) activities were increased by 95% and 34%, respectively. Immunoblot analysis of liver microsomal proteins revealed that the Cyp2c protein level was elevated by the 28-day treatment. However, the liver microsomal activities of the oxidation of the respective substrates of Cyp1a, Cyp2a, Cyp2b, Cyp2d, and Cyp2e1 remained unchanged. In the kidney, SJW increased the NFO, but not the WOH activity. The extended 28-day treatment did not alter mouse hepatic and renal UGT, GST, and NQO activities. These findings demonstrate that SJW stimulates hepatic and renal Cyp3a activity and hepatic Cyp2c activity and expression. The induction of hepatic Cyp2c requires repeated treatment for a period longer than the initial induction of Cyp3a.