Glucuronidation and sulfation of p-nitrophenol in isolated rat hepatocyte subpopulations. Effects of phenobarbital and 3-methylcholanthrene pretreatment

Parenchymal cells, isolated from untreated (control), phenobarbital(PB)-or 3-methylcholanthrene(3-MC)-treated rats, were separated into four subpopulations according to cell density, and glucuronidation and sulfation of p-nitrophenol (PNP) in the hepatocyte subpopulations were investigated. PB enhanced the glucuronidation almost 2-fold but not the sulfation, while 3-MC enhanced both glucuronidation (3-fold) and sulfation (2-fold) in the original cell suspensions. Some gradation trends were found in the conjugation activities among the hepatocyte subpopulations: In the control experiment, the extent of glucuronidation in four subpopulations was virtually the same but sulfation in high-density hepatocytes was slightly higher than in low-density ones. Both glucuronidation and sulfation were higher in low-density hepatocytes from PB-treated rats, though the gradation was very modest. Glucuronidation and sulfation tended to be slightly higher in middle-density hepatocytes in the 3-MC experiment. However, no definite correlation in conjugation activities vs. cell density, like those seen in cytochrome P-450s vs. cell density in the hepatocytes isolated from PB-treated rats, were found in the subpopulations from control or inducer-treated rats. Simultaneous studies on acetylation of p-aminobenzoic acid (PABA) revealed that the activities in the subpopulations were virtually the same and the inducers had little influence on the activity.     

Pivaloylcodeine,a new codeine derivative,for the inhibition of morphine glucuronidation. An in vitro study in the rat

We have previously found that phenanthrenic opioids, including codeine, modulate morphine glucuronidation in the rat. Here codeine and five of its derivatives were compared in their effects on the synthesis of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) from morphine by rat liver microsomal preparations, and by primary cultures of rat hepatocytes previously incubated for 72 h with either codeine or its derivatives. Acetylcodeine and pivaloylcodeine shared the capability of the parent compound of inhibiting the synthesis of M3G by liver microsomes through a noncompetitive mechanism of action. Their IC₅₀ were 3.25, 2.27, and 4.32 μM, respectively. Dihydrocodeine, acetyldihydrocodeine, and lauroylcodeine were ineffective. In all the experimental circumstances M6G was undetectable in the incubation medium. In primary hepatocyte cultures codeine only inhibited M3G formation, but with a lower efficacy than that observed with microsomes (IC₅₀ 20.91 vs 4.32 μM). Preliminary results show that at micromolar concentrations codeine derivatives exhibit a low rate of affinity for μ opiate receptors. In conclusion, acetyl and pivaloyl derivatives of codeine noncompetitively inhibit liver glucuronidation of morphine interacting with microsomes. This study further strengths the notion that phenanthrenic opioids can modulate morphine glucuronidation independently from their effects on μ opiate receptors.     

Practical one-step glucuronidation via biotransformation

We herein report a practical one-step glucuronidation method by biotransformation using Streptomyces sp. SANK 60895. This novel direct method of biotransformation has been shown to be more practical and scalable for glucuronidation than previously reported chemical and enzymatic procedures given its simplicity, high β-selectivity, cost-effectiveness, and reproducibility. We applied the present method to the synthesis of acyl glucuronide and hydroxy-β-glucuronide of mycophenolic acid and compound 4, respectively. This method was also shown to be applicable to the N-glucuronidation of various compounds.     

Glucuronidation of the green tea catechins, (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, by rat hepatic and intestinal microsomes

The flavonoids (-)-epigallocatechin-3-gallate (EGCg) and (-)-epicatechin-3-gallate (ECg) are major components of green tea and show numerous biological effects. We investigated the glucuronidation of these compounds and of quercetin by microsomes. Quercetin was almost fully glucuronidated by liver microsomes after 3 h, whereas ECg and ECGg were conjugated to a lesser extent ([Formula: See Text] and [Formula: See Text] respectively). The intestinal microsomes also glucuronidated quercetin much more efficiently than ECg and EGCg. Although the rates were lower than quercetin, intestinal microsomes exhibited higher activity on the galloyl group of ECg and EGCg compared to the flavonoid ring, whereas hepatic glucuronidation was higher on the flavonoid ring of EGCg and ECg compared to the galloyl groups. The low glucuronidation rates could partially explain why these flavanols are present in plasma as unconjugated forms.     

Ligand-dependent structural changes and limited proteolysis of Escherichia coli phosphofructokinase-2

An isoform (rhesus UGT1A01) orthologus to the human UGT1A1 was cloned and sequenced from female rhesus monkey liver cDNA using primers designed from the human nucleotide sequences. Open reading frame analysis of the PCR-generated product encodes a 533-amino acid protein with a proposed 27-residue signal peptide. Nucleotide sequence comparison of rhesus UGT1A01 to other rhesus UGT1A isoforms detected a single-transition mutation at nucleotide 1520 (T-->C), resulting in a neutral F to S substitution at position 507. Rhesus UGT1A01 was greater than 99 and 95% identical to cynomolgus UGT1A01 and human UGT1A1, respectively. The rhesus UGT1A01 was expressed in HK-293 cells for functional analysis. Catalytic activity of UGT1A01 was determined with 7-hydroxy-4-(trifluoromethyl)-coumarin and more specific human UGT1A1 substrates (1-naphthol, beta-estradiol, 17 alpha-ethinylestradiol, and bilirubin). Expression of UGT1A01 protein was also detected by a Western blot utilizing a polyclonal antibody developed against the human UGT1A family.     

Ethanol-dependent induction of bilirubin UDP-glucuronosyl-transferase in rat liver is mediated by Kupffer cells

Recently we have reported that bilirubin UDP-glucuronosyltransferase (UGT1A1) is induced in rat liver by chronic ethanol treatment. Several studies have shown that Kupffer cells play a central role in the mediation of various hepatic effects of chronic alcohol consumption. In the present work, the participation of Kupffer cells in the ethanol dependent induction of UGT1A1 was investigated. A group of rats was pretreated with gadolinium chloride, a known Kupffer-cell-depleting agent. We compared the effect of chronic ethanol ingestion on UGT1A1 expression in the liver of normal and gadolinium chloride treated rats. The effect of ethanol on bilirubin glucuronidation was completely prevented in Kupffer cell deficient rats. The western and northern blot analyses showed that the increase of both the protein and mRNA of UGT1A1 was prevented in these animals. These results suggest that Kupffer cells play a major role in the mediation of ethanol-stimulated induction of UGT1A1 in liver parenchymal cells.     

High glucuronidation activity of environmental estrogens in the carp (Cyprinus carpino) intestine

Many adverse effects on carp reproductive organs have been reported to be caused by exposure to environmental estrogens, such as nonylphenol and bisphenol A, which contaminate the aquatic environment. The glucuronidation activities of xenoestrogens (bisphenol A and diethylstilbestrol) and phytoestrogens (coumestrol, genistein and biochanin A), but not nonylphenol and octylphenol, were observed in microsomes prepared from carp organs. The highest levels of glucuronidation of environmental estrogens, for which the optimum temperature was 25-30 degrees C, were observed in the intestinal microsomes of 2-year-old carp. These activities in carp intestine increased developmentally, and the maximum levels corresponded to 5-10 % of that in rat liver microsomes. However, the glucuronidation of phytoestrogen by carp intestinal microsomes corresponded to that of rat liver microsomes. Only bisphenol A-glucuronide was excreted from the everted intestine, indicating that bisphenol A is metabolized in the carp intestine mainly as glucuronide.These results suggest that glucuronidation by carp intestine plays an important role for the detoxification of xenoestrogens and phytoestrogens, except for nonylphenol and octylphenol.     

Enzyme-assisted synthesis and characterization of glucuronide conjugates of neuroactive steroids

Synthesis of reference standards is needed to determine the presence and function of steroid glucuronides in the brain or other tissues, because commercial sources of steroid glucuronide standards are limited or unavailable. In the present study porcine, rat, and bovine liver microsomes were tested to evaluate their ability to glucuronidate eight neurosteroids and neuroactive steroids of various types: dehydroepiandrosterone, pregnenolone, isopregnanolone, 5alpha-tetrahydrodeoxycorticosterone, corticosterone, cortisol, beta-estradiol, and testosterone. In general, the glucuronidation efficiency of rat liver was rather poor compared with that of bovine and porcine liver microsomes. Since porcine liver apparently has a relatively large amount of dehydrogenase, its microsomes also produced dehydrogenated steroids and their glucuronides, as well as various regioisomers in which the site of glucuronidation varied. In contrast, bovine liver microsomes produced mainly a single major glucuronidation product and few dehydrogenation products and gave the best overall yield for two-third of the steroids tested. The enzymatic synthesis of five glucuronides of four steroids was carried out and the conditions, purification, and analytical methods for the glucuronidation products were optimized. The steroid glucuronides synthesized were characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography-mass spectrometry (LC-MS). The stereochemically pure steroid glucuronide conjugates were recovered in milligram amounts (yield 10-78%) and good purity (>85-90%), which is sufficient for LC-MS/MS method development and analyses of steroid glucuronides in biological matrices such as brain, urine, or plasma.     

Human and animal hepatocytes in vitro with extrapolation in vivo

Human and animal hepatocytes are now being used as an in vitro technique to aid drug discovery by predicting the in vivo metabolic pathways of drugs or new chemical entities (NCEs), identifying drug-metabolizing enzymes and predicting their in vivo induction. Because of the difficulty of establishing whether the cytotoxic susceptibility of human hepatocytes to xenobiotics/drugs in vitro could be used to predict in vivo human hepatotoxicity, a comparison of the susceptibility of the hepatocytes of human and animal models to six chemical classes of drugs/xenobiotics in vitro have been related to their in vivo hepatotoxicity and the corresponding activity of their metabolizing enzymes. This study showed that the cytotoxic effectiveness of 16 halobenzenes towards rat hepatocytes in vitro using higher doses and short incubation times correlated well with rat hepatotoxic effectiveness in vivo with lower doses/longer times. The hepatic/hepatocyte xenobiotic metabolizing enzyme activities of various animal species and human have been reviewed for use by veterinarians and research scientists. Where possible, recommendations have been made regarding which animal hepatocyte model is most applicable for modeling the susceptibility to xenobiotic induced hepatotoxicity of those humans with slow versus rapid metabolizing enzyme polymorphisms. These recommendations are based on the best human fit for animal drug/xenobiotic metabolizing enzymes in terms of activity, kinetics and substrate/inhibitor specificity. The use of human hepatocytes from slow versus rapid metabolizing individuals for drug metabolism/cytotoxicity studies; and the research use of freshly isolated rat hepatocytes and "Accelerated Cytotoxicity Mechanism Screening" (ACMS) techniques for identifying drug/xenobiotic reactive metabolites are also described. Using these techniques the molecular hepatocytotoxic mechanisms found in vitro for seven classes of xenobiotics/drugs were found to be similar to the rat hepatotoxic mechanisms reported in vivo.     

Glucuronidation of carboxylic acid containing compounds by UDP-glucuronosyltransferase isoforms

Glucuronide conjugation of xenobiotics containing a carboxylic acid moiety represents an important metabolic pathway for these compounds in humans. Several human UDP-glucuronosyltransferases (UGTs) have been shown to catalyze the formation of acyl-glucuronides, including UGT2B7, UGT1A3, and UGT1A9. In this study, recombinant expressed UGT isoforms were investigated with many structurally related carboxylic acid analogues, and the UGT rank order for catalyzing the glucuronidation of carboxylic acids was UGT2B7?UGT1A3 approximately UGT1A9. Despite being a poor substrate with UGT1A3, coumarin-3-carboxylic acid was not a substrate for any other UGT isoform tested in this study, suggesting that it could be a specific substrate for UGT1A3. Interestingly, UGT1A7 and UGT1A10 also react with several carboxylic acid aglycones. Kinetic analysis showed that UGT2B7 exhibits much higher glucuronidation efficiency (Vmax/Km) with ibuprofen, ketoprofen, and others, compared to UGT1A3. These data indicate that UGT2B7 could be the major isoform involved in the glucuronidation of carboxylic acid compounds in humans.