Disruption of thyroid hormone homeostasis in Ugt1a-deficient Gunn rats by microsomal enzyme inducers is not due to enhanced thyroxine glucuronidation

Microsomal enzyme inducers (MEI) that increase UDP-glucuronosyltransferases (UGTs) are thought to increase glucuronidation of thyroxine (T₄), thus reducing serum T₄, and subsequently increasing thyroid stimulating hormone (TSH). Ugt1a1 and Ugt1a6 mediate T₄ glucuronidation. Therefore, this experiment determined the involvement of Ugt1a enzymes in increased T₄ glucuronidation, decreased serum T₄, and increased TSH after MEI treatment. Male Wistar and Ugt1a-deficient Wistar (Gunn) rats were fed a control diet or diet containing pregnenolone-16α-carbonitrile (PCN; 800 ppm), 3-methylcholanthrene (3-MC; 200 ppm), or Aroclor 1254 (PCB; 100 ppm) for 7 days. Serum T₄, triiodothyronine (T₃), and TSH concentrations, hepatic T₄/T₃ glucuronidation, and thyroid histology and follicular cell proliferation were investigated. PCN, 3-MC, and PCB treatments decreased serum T₄, whereas serum T₃ was maintained in both Gunn and Wistar rats (except for PCB treatment). TSH was increased in Wistar and Gunn rats after PCN (130 and 277%) or PCB treatment (72 and 60%). T₄ glucuronidation in Wistar rats was increased after PCN (298%), 3-MC (85%), and PCB (450%), but was extremely low in Gunn rats, and unchanged after MEI. T₃ glucuronidation was increased after PCN (121%) or PCB (58%) in Wistar rats, but only PCN increased T₃ glucuronidation in Gunn rats (43%). PCN treatment induced thyroid morphological changes and increased follicular cell proliferation in both strains. These data demonstrate that T₄ glucuronidation cannot be increased in Ugt1a-deficient Gunn rats. Thus, the decrease in serum T₄, increase in TSH, and increase in thyroid cell proliferation after MEI are not dependent on increased T₄ glucuronidation, and cannot be attributed to Ugt1a enzymes.     

The enantioselective glucuronidation of morphine in rats and humans. Evidence for the involvement of more than one UDP-glucuronosyltransferase isoenzyme

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.     

Drug metabolism in Gunn rats: inability to increase bilirubin glucuronidation by phenobarbital treatment

The components of the microsomal mixed-function oxidase system in Wistar and Gunn rats were the same, as were the overall hydroxylation reactions (p-nitroanisole O-demethylase, aryl hydrocarbon hydroxylase). The Gunn rat was, however, almost totally devoid of bilirubin glucuronidation activity. The UDP glucuronosyltransferase activity towards p-nitrophenol in the Gunn rat was in liver about 60 per cent and in the small intestinal mucosa about 10 per cent of that in the Wistar rat. The mono-oxygenation step in hepatic microsomal drug metabolism was induced in a similar way by phenobarbital treatment in both Wistar and Gunn strain rats, but no significant enhancement of the mono-oxygenase activity could be observed in the gut. The trypsin digestion of hepatic microsomes increased the specific UDPglucuronosyltransferase activity. Phenobarbital treatment enhanced the p-nitrophenol glucuronidation in livers of both Wistar (3-fold) and Gunn strain (2-fold) rats, but the bilirubin glucuronidation was enhanced (2-fold) only in Wistar rats. Phenobarbital-enhanced transferase activities could be detected only if the microsomes had been digested with trypsin before determination of the enzyme activity. The UDPglucuronosyltransferase activity in the small intestinal mucosa was not enhanced by phenobarbital. The results suggest that bilirubin and p-nitrophenol are conjugated by distinct enzymes and that the enzyme activities for mono-oxygenation and glucuronidation are controlled by different mechanisms. The microenvironment of the UDP glucuronosyltransferase is most probably similar in Gunn and Wistar rats.     

Comparison of the glucuronidation ability of liver microsomes from rats treated with 3-methylcholanthrene or phenolic antioxidants

1. UDP-glucuronyltransferase activity was studied in hepatic microsomal preparations isolated from female Sprague-Dawley rats treated with either 3-methylcholanthrene, butylated hydroxyanisole, propyl gallate, ethoxyquin or a combination of 3-methylcholanthrene and butylated hydroxyanisole. 2. The substrates p-nitrophenol, 1-naphthol and phenolphthalein were used. With p-nitrophenol all treatments, except propyl gallate, caused a significant increase in activity over the control value; the combined treatment of 3-methylcholanthrene and butylated hydroxyanisole gave an additive response when compared to each treatment used singly. With 1-naphthol as substrate, 3-methylcholanthrene and 3-methylcholanthrene + butylated hydroxyanisole caused 8-fold increases in activity compared to the 2-3 fold increased caused by the other treatments. When phenolphthalein was used, all treatments increased activity to the same extent. 3. When the assays were repeated with various concentrations of each substrate, the results revealed that, except where phenolphthalein was used, distinguishable differences in the activity of each microsomal preparation were discernible, the most significant being the bimodal response of each preparation to low and high concentrations of p-nitrophenol.     

Comparison of the glucuronidation ability of liver microsomes from rats treated with 3-methylcholanthrene or phenolic antioxidants

1. UDP-glucuronyltransferase activity was studied in hepatic microsomal preparations isolated from female Sprague-Dawley rats treated with either 3-methylcholanthrene, butylated hydroxyanisole, propyl gallate, ethoxyquin or a combination of 3-methylcholanthrene and butylated hydroxyanisole.

2. The substrates p-nitrophenol, 1-naphthol and phenolphthalein were used. With p-nitrophenol all treatments, except propyl gallate, caused a significant increase in activity over the control value; the combined treatment of 3-methylcholanthrene and butylated hydroxyanisole gave an additive response when compared to each treatment used singly. With 1-naphthol as substrate, 3-methylcholanthrene and 3-methylcholanthrene + butylated hydroxyanisole caused 8-fold increases in activity compared to the 2–3 fold increases caused by the other treatments. When phenolphthalein was used, all treatments increased activity to the same extent.

3. When the assays were repeated with various concentrations of each substrate, the results revealed that, except where phenolphthalein was used, distinguishable differences in the activity of each microsomal preparation were discernible, the most significant being the bimodal response of each preparation to low and high concentrations of p-nitrophenol.     

Persistent induction of hepatic and pulmonary phase II enzymes by 3-methylcholanthrene in rats.

We reported earlier that exposure of rats to 3-methylcholanthrene (MC) causes sustained induction of hepatic cytochrome P450 (CYP)1A expression for up to 45 days by mechanisms other than persistence of the parent MC (Moorthy, J. 2000. Pharmacology. Exp. Ther. 294, 313-322). The CYP1A genes are members of the Ah gene battery that also encode CYP1B1 and phase II enzymes such as glutathione S-transferase (GST-alpha), UDP glucuronyl transferase (UGT)1A, NAD(P)H (nicotinamide adenine dinucleotide phosphate, reduced):quinone oxidoreductase I (NQO1), aldehyde dehydrogenase (ALDH), etc. Therefore, in this investigation, we tested the hypothesis that MC elicits persistent induction of CYP1B1 and phase II genes, which are in part regulated by the Ah receptor (AHR). Female Sprague-Dawley rats were treated with MC (100 mumol/kg), ip, once daily for 4 days, and expression of CYP1B1 and several phase II (e.g., GST-alpha, NQO1) genes and their corresponding proteins were determined in lung and liver. The major finding was that MC persistently induced (3- to 10-fold) the expression of several phase II enzymes, including GST-alpha, NQO1, UGT1A1, ALDH, and epoxide hydrolase in both tissues for up to 28 days. However, MC did not elicit sustained induction of CYP1B1. Our results thus support the hypothesis that MC elicits coordinated and sustained induction of phase II genes presumably via persistent activation of the AHR, a phenomenon that may have implications for chemical-induced carcinogenesis and chemopreventive strategies in humans.     

Coordinate regulation of UDP-glucuronosyltransferase UGT1A6 induction by 3-methylcholanthrene and multidrug resistance protein MRP2 expression by dexamethasone in primary rat hepatocytes

Concentration-dependent regulation of 3-methylcholanthrene (MC) inducibility of UDP-glucuronosyltransferase UGT1A6 by the synthetic glucocorticoid, dexamethasone (DEX) was studied. Treatment of cultured rat hepatocytes with MC, 0.1, 1, and 10 microM DEX, and MC combined with DEX, resulted in different induction patterns measured in the intact cells compared to that observed in the microsomes prepared from the same cells. DEX treatment in various concentrations caused a concentration-dependent increase in p-nitrophenol (p-NP) conjugation in intact cells (3-, 4-, and 5-fold over control, respectively), and it positively regulated MC induction (4-, 5-, and 6-fold over control, respectively). In contrast, DEX had smaller effect on microsomal p-NP conjugation (115, 200, 220% of control, respectively) and although MC induction was increased significantly by 0.1 microM DEX (520% of control), but higher concentrations of DEX (10 microM) decreased the degree of induction to 410%. Similar results obtained from in vivo experiments showed that at high DEX concentration (100mg/kg), the rate of MC induction (540%) decreased (420%). Permeabilization of the plasma membrane resulted in a 15-fold increase of p-NP conjugation indicating the importance of transport in the rate of overall p-NP elimination, and the induction pattern was similar to that observed in microsomes isolated from cells. Hyper-osmolarity (405 mOsmol/L) led to a 3-fold decrease of p-NP conjugation, the loss of DEX inducibility and reduction of the MRP2 protein level. Our results suggest coordinated regulation of UGT1A6 inducibility and substrate or product transport by DEX.     

大鼠微粒体代谢酶对黄芩素葡萄糖醛酸化代谢物影响的实验研究

目的 观察大鼠微粒体代谢酶对黄芩素(治疗呼吸道感染中药)代谢作用的影响.方法 用体外微粒体药物代谢酶孵育法;用HPLC法测定黄芩素及其葡萄糖醛酸化代谢物的含量,在不同孵育时间和不同浓度下,观察大鼠肝肠不同微粒体对黄芩素葡萄糖醛酸化代谢产物生成速率的影响.结果 5种不同微粒体药物代谢酶对黄芩素均有代谢作用,且随孵育时间延长,代谢作用也相应增加,呈较好的时间和剂量依赖关系.在5种微粒体中,十二指肠微粒体代谢作用最强;而肝代谢作用最弱.结论 黄芩素主要代谢部位是肠道.     

Acyl glucuronidation of fluoroquinolone antibiotics by the UDP-glucuronosyltransferase 1A subfamily in human liver microsomes.

Acyl glucuronidation is an important metabolic pathway for fluoroquinolone antibiotics. However, it is unclear which human UDP-glucuronosyltransferase (UGT) enzymes are involved in the glucuronidation of the fluoroquinolones. The in vitro formation of levofloxacin (LVFX), grepafloxacin (GPFX), moxifloxacin (MFLX), and sitafloxacin (STFX) glucuronides was investigated in human liver microsomes and cDNA-expressed recombinant human UGT enzymes. The apparent Km values for human liver microsomes ranged from 1.9 to 10.0 mM, and the intrinsic clearance values (calculated as Vmax/Km) had a rank order of MFLX > GPFX > STFX > > LVFX. In a bank of human liver microsomes (n = 14), the glucuronidation activities of LVFX, MFLX, and STFX correlated highly with UGT1A1-selective beta-estradiol 3-glucuronidation activity, whereas the glucuronidation activity of GPFX correlated highly with UGT1A9-selective propofol glucuronidation activity. Among 12 recombinant UGT enzymes, UGT1A1, 1A3, 1A7, and 1A9 catalyzed the glucuronidation of these fluoroquinolones. Results of enzyme kinetics studies using the recombinant UGT enzymes indicated that UGT1A1 most efficiently glucuronidates MFLX, and UGT1A9 most efficiently glucuronidates GPFX. In addition, the glucuronidation activities of MFLX and STFX in human liver microsomes were potently inhibited by bilirubin with IC50 values of 4.9 microM and 4.7 microM, respectively; in contrast, the glucuronidation activity of GPFX was inhibited by mefenamic acid with an IC50 value of 9.8 microM. These results demonstrate that UGT1A1, 1A3, and 1A9 enzymes are involved in the glucuronidation of LVFX, GPFX, MFLX, and STFX in human liver microsomes, and that MFLX and STFX are predominantly glucuronidated by UGT1A1, whereas GPFX is mainly glucuronidated by UGT1A9.     

Male‐specific induction of CYP3A2 in rats by zolmitriptan

We report here a novel observation that zolmitriptan induced CYP3A2 in male but not female rats. As part of our research programme to evaluate sex differences in the response to zolmitriptan, we studied the effects of zolmitriptan on CYP3A activity, protein and gene expression in male and female rats. Zolmitriptan was found to induce CYP3A activity, measured as testosterone and diazepam metabolism in‐vitro, as well as midazolam pharmacokinetics in‐vivo, in male but not female rats. The sex difference in response to zolmitriptan was further evaluated by analysis of CYP3A1/2 mRNA levels using real‐time PCR, and CYP3A1/2 protein levels using immunoblotting. Zolmitriptan preferentially induced CYP3A2 in male but not female rats. No obvious effects on CYP3A1 were observed at any dose in either sex. Thus, we concluded that the observed sex‐dependent induction of CYP3A by zolmitriptan was largely due to induction of CYP3A2 in male rats.     

Methyl red azo-reductase and its induction by 3-methylcholanthrene in the liver of different species

1. Methyl red azo-reductase activity in the liver cytosol of mouse and sheep was considerably less than in the rat, guinea-pig, rabbit and hamster. In the mouse, the total activity of this soluble enzyme to reduce methyl red was only 4% of that found in rat.

2. Species variation in methyl red azo-reductase of microsomes was relatively small compared to that of the cytosol.

3. Azo-reductase in the cytosol of mouse liver also differed from that of the rat in its resistance to the inductive effects of 3-methylcholanthrene.     

Decreased exposure of atorvastatin in diabetic rats partly due to induction of hepatic Cyp3a and Oatp2

1. Atorvastatin is frequently prescribed for lowering blood cholesterol and for prevention of events associated with cardiovascular disease. The aim of this study was to investigate the pharmacokinetics of atorvastatin in diabetic rats.

2. Diabetes was induced in rats by combination of high-fat diet and low-dose streptozotocin (35?mg/kg). Plasma concentrations of atorvastatin following oral (10?mg/kg) and intravenous (2?mg/kg) administrations to rats were measured by LC-MS. Metabolism and uptake of atorvastatin in primary hepatocytes of experimental rats were assessed. Protein expressions and activities of hepatic Cyp3a and Oatp2 were further investigated.

3. Clearances of atorvastatin in diabetic rats following oral and intravenous administrations were remarkably increased, leading to marked decreases in area-under-the-plasma concentration–time curve (AUC). The estimated oral and systematic clearances of atorvastatin in diabetic rats were 4.5-fold and 2.0-fold of control rats, respectively. Metabolism and uptake of atorvastatin in primary hepatocytes isolated from diabetic rats were significantly increased, which were consistent with the up-regulated protein expressions and activities of hepatic Cyp3a and Oatp2.

4. All these results demonstrated that the plasma exposure of atorvastatin was significantly decreased in diabetic rats, which was partly due to the up-regulated activities and expressions of both hepatic Cyp3a and Oatp2.