Role of microsomal metabolism in bromfenac-induced cytotoxicity.

This study delves into the intricate mechanisms underlying drug-induced liver injury (DILI) with a specific focus on bromfenac, the withdrawn nonsteroidal anti-inflammatory drug. DILI is a pervasive concern in drug development, prompting market withdrawals and posing significant challenges to healthcare. Despite the withdrawal of bromfenac due to DILI, the exact role of its microsomal metabolism in inducing hepatotoxicity remains unclear. Herein, employing HepG2 cells with human liver microsomes and UDP-glucuronic acid (UDPGA), our investigation revealed a substantial increase in bromfenac-induced cytotoxicity in the presence of UDPGA, pointing to the significance of UDP-glucuronosyltransferase (UGT)-dependent metabolism in augmenting toxicity. Notably, among the recombinant UGTs examined, UGT2B7 emerged as a pivotal enzyme in the metabolic activation of bromfenac. Metabolite identification studies disclosed the formation of reactive intermediates, with bromfenac indolinone (lactam) identified as a potential mediator of hepatotoxic effects. Moreover, in cytotoxicity experiments, the toxicity of bromfenac lactam exhibited a 34-fold increase, relative to bromfenac. The toxicity of bromfenac lactam was mitigated by nicotinamide adenine dinucleotide phosphate-dependent metabolism. This finding underscores the role of UGT-dependent metabolism in generating reactive metabolites that contribute to the observed hepatotoxicity associated with bromfenac. Understanding these metabolic pathways and the involvement of specific enzymes, such as UGT2B7, provides crucial insights into the mechanisms of bromfenac-induced liver injury. In conclusion, this research sheds light on the metabolic intricacies leading to cytotoxicity induced by bromfenac, especially emphasizing the role of UGT-dependent metabolism and the formation of reactive intermediates like bromfenac lactam. These findings offer insight into the mechanistic basis of DILI and emphasize the importance of understanding metabolism-mediated toxicity.

Continuous exposure to bisphenol S increases the accumulation of endogenous metabolic toxicants by obstructing the glucuronic acid pathway.

Uridine diphosphate glucuronic acid (UDPGA) is an essential substrate in the glucuronidation of exogenous and endogenous lipophilic compounds via the liver glucuronic acid pathway, and its synthesis depends on glucose and energy in the body. Bisphenol S (BPS), as a lipophilic environmental pollutant, has been widely utilized in the manufacturing of daily necessities. The biological effect of BPS in interference with liver energy metabolism might affect UDPGA synthesis and the excretion of lipophilic compounds, but this was not clearly revealed. Here, female zebrafish that were exposed to BPS for 35 days exhibited a significant decrease in UDPGA in the liver with significant accumulation of exogenous BPS and endogenous bilirubin in the body. One vital reason may be that the exposure to BPS for 35 days promoted the lipid formation through PPARg signaling and reduced energy levels in the liver, resulting in the decreased raw materials for UDPGA production in glucuronic acid pathway. Meanwhile, transcriptome analysis showed that BPS inhibited the mRNA expression levels of genes related to the glucuronic acid pathway. The accumulation of endogenous and exogenous lipophilic compounds can trigger a variety of toxicological effect. Thus, weakened liver detoxification might be the primary cause of the toxicological effects of lipophilic pollutants.

Acyl Glucuronide Metabolites of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1 H-indole-3-carboxylic Acid (PF-06409577) and Related Indole-3-carboxylic Acid Derivatives are Direct Activators of Adenosine Monophosphate-Activated Protein Kinase (AMPK).

Studies on indole-3-carboxylic acid derivatives as direct activators of human adenosine monophosphate-activated protein kinase (AMPK) α1ß1γ1 isoform have culminated in the identification of PF-06409577 (1), PF-06885249 (2), and PF-06679142 (3) as potential clinical candidates. Compounds 1-3 are primarily cleared in animals and humans via glucuronidation. Herein, we describe the biosynthetic preparation, purification, and structural characterization of the glucuronide conjugates of 1-3. Spectral characterization of the purified glucuronides M1, M2, and M3 indicated that they were acyl glucuronide derivatives. In vitro pharmacological evaluation revealed that all three acyl glucuronides retained selective activation of ß1-containing AMPK isoforms. Inhibition of de novo lipogenesis with representative parent carboxylic acids and their respective acyl glucuronide conjugates in human hepatocytes demonstrated their propensity to activate cellular AMPK. Cocrystallization of the AMPK α1ß1γ1 isoform with 1-3 and M1-M3 provided molecular insights into the structural basis for AMPK activation by the glucuronide conjugates.

[Investigation of metabolic kinetics and reaction phenotyping of ligustrazin by using liver microsomes and recombinant human enzymes].

The metabolic characteristics of ligustrazin (TMPz) in liver microsomes were investigated in the present study. The reaction phenotyping of TMPz metabolism was also identified by in vitro assessment using recombinant human cytochrome P450 enzymes (CYP) and UDP glucuronosyltransferases (UGT). TMPz was incubated at 37 degrees C with human (HLM) and rat liver microsomes (RLM) in the presence of different co-factors. The metabolic stability and enzyme kinetics of TMPz were studied by determining its remaining concentrations with a LC-MS/MS method. TMPz was only metabolically eliminated in the microsomes with NADPH or NADPH+UDPGA. In the HLM and RLM with NADPH+UDPGA, t1/2, K(m) and V(max) of TMPz were 94.24 +/- 4.53 and 105.07 +/- 9.44 min, 22.74 +/- 1.89 and 33.09 +/- 2.74 micromol x L(-1), 253.50 +/- 10.06 and 190.40 +/- 8.35 nmol x min(-1) x mg(-1) (protein), respectively. TMPz showed a slightly higher metabolic rate in HLM than that in RLM. Its primary oxidative metabolites, 2-hydroxymethyl-3, 5, 6-trimethylpyrazine (HTMP), could undergo glucuronide conjugation. The CYP reaction phenotyping of TMPz metabolism was identified using a panel of recombinant CYP isoforms (rCYP) and specific CYP inhibitors in HLM. CYP1A2, 2C9 and 3A4 were found to be the major CYP isoforms involved in TMPz metabolism. Their individual contributions were assessed b) using the method of the total normalized rate to be 19.32%, 27.79% and 52.90%, respectively. It was observed that these CYP isoforms mediated the formation of HTMP in rCYP incubation. The UGT reaction phenotyping of HTMP glucuronidation was also investigated preliminarily by using a panel of 6 UGT isoforms (rUGT). UGT1A1, 1A4 and 1A6 were the predominant isoforms mediated the HTMP glucuronidation. The results above indicate that the metabolism of TMPz involves multiple enzymes mediated phase I and phase II reactions.

Bilirubin glucuronidation revisited: proper assay conditions to estimate enzyme kinetics with recombinant UGT1A1.

Bilirubin, an end product of heme catabolism, is primarily eliminated via glucuronic acid conjugation by UGT1A1. Impaired bilirubin conjugation, caused by inhibition of UGT1A1, can result in clinical consequences, including jaundice and kernicterus. Thus, evaluation of the ability of new drug candidates to inhibit UGT1A1-catalyzed bilirubin glucuronidation in vitro has become common practice. However, the instability of bilirubin and its glucuronides presents substantial technical challenges to conduct in vitro bilirubin glucuronidation assays. Furthermore, because bilirubin can be diglucuronidated through a sequential reaction, establishment of initial rate conditions can be problematic. To address these issues, a robust high-performance liquid chromatography assay to measure both bilirubin mono- and diglucuronide conjugates was developed, and the incubation conditions for bilirubin glucuronidation by human embryonic kidney 293-expressed UGT1A1 were carefully characterized. Our results indicated that bilirubin glucuronidation should be assessed at very low protein concentrations (0.05 mg/ml protein) and over a short incubation time (5 min) to assure initial rate conditions. Under these conditions, bilirubin total glucuronide formation exhibited a hyperbolic (Michaelis-Menten) kinetic profile with a K(m) of ∼0.2 µM. In addition, under these initial rate conditions, the relative proportions between the total monoglucuronide and the diglucuronide product were constant across the range of bilirubin concentration evaluated (0.05-2 µM), with the monoglucuronide being the predominant species (∼70%). In conclusion, establishment of appropriate incubation conditions (i.e., very low protein concentrations and short incubation times) is necessary to properly characterize the kinetics of bilirubin glucuronidation in a recombinant UGT1A1 system.

Enhanced potency of nucleotide-dendrimer conjugates as agonists of the P2Y14 receptor: multivalent effect in G protein-coupled receptor recognition.

The P2Y(14) receptor is a G protein-coupled receptor activated by uridine-5'-diphosphoglucose and other nucleotide sugars that modulates immune function. Covalent conjugation of P2Y(14) receptor agonists to PAMAM (polyamidoamine) dendrimers enhanced pharmacological activity. Uridine-5'-diphosphoglucuronic acid (UDPGA) and its ethylenediamine adduct were suitable functionalized congeners for coupling to several generations (G2.5-6) of dendrimers (both terminal carboxy and amino). Prosthetic groups, including biotin for avidin complexation, a chelating group for metal complexation (and eventual magnetic resonance imaging), and a fluorescent moiety, also were attached with the eventual goals of molecular detection and characterization of the P2Y(14) receptor. The activities of conjugates were assayed in HEK293 cells stably expressing the human P2Y(14) receptor. A G3 PAMAM conjugate containing 20 bound nucleotide moieties (UDPGA) was 100-fold more potent (EC(50) 2.4 nM) than the native agonist uridine-5'-diphosphoglucose. A molecular model of this conjugate docked in the human P2Y(14) receptor showed that the nucleotide-substituted branches could extend far beyond the dimensions of the receptor and be available for multivalent docking to receptor aggregates. Larger dendrimer carriers and greater loading favored higher potency. A similar conjugate of G6 with 147 out of 256 amino groups substituted with UDPGA displayed an EC(50) value of 0.8 nM. Thus, biological activity was either retained or dramatically enhanced in the multivalent dendrimer conjugates in comparison with monomeric P2Y(14) receptor agonists, depending on size, degree of substitution, terminal functionality, and attached prosthetic groups.

Characterization of drug metabolites and cytotoxicity assay simultaneously using an integrated microfluidic device.

An integrated microfluidic device was developed for the characterization of drug metabolites and a cytotoxicity assay simultaneously. The multi-layer device was composed of a quartz substrate with embedded separation microchannels and a perforated three-microwell array containing sol-gel bioreactors of human liver microsome (HLM), and two PDMS layers. By aligning the microwell array on the quartz substrate with cell culture chambers on the bottom PDMS layer, drug metabolism studies related to functional units, including metabolite generation, detection and incubation with cultured cells to assess metabolism induced cytotoxicity, were all integrated into the microfluidic device. To validate the feasibility of drug metabolism study on the microfluidic chip, UDP-glucuronosyltransferase (UGT) metabolism of acetaminophen (AP) and its effect on hepG2 cytotoxicity were studied first. Then metabolism based drug-drug interaction between AP and phenytoin (PH), which resulted in increased hepG2 cytotoxicity, was proved on this device. All this demonstrated that the developed microfluidic device could be a potential useful tool for drug metabolism and metabolism based drug-drug interaction research.

Characterization of 1'-hydroxymidazolam glucuronidation in human liver microsomes.

Midazolam is a potent benzodiazepine derivative with sedative, hypnotic, anticonvulsant, muscle-relaxant, and anxiolytic activities. It undergoes oxidative metabolism catalyzed almost exclusively by the CYP3A subfamily to a major metabolite, 1'-hydroxymidazolam, which is equipotent to midazolam. 1'-Hydroxymidazolam is subject to glucuronidation followed by renal excretion. To date, the glucuronidation of 1'-hydroxymidazolam has not been evaluated in detail. In the current study, we identified an unreported quaternary N-glucuronide, as well as the known O-glucuronide, from incubations of 1'-hydroxymidazolam in human liver microsomes enriched with uridine 5'-diphosphoglucuronic acid (UDPGA). The structure of the N-glucuronide was confirmed by nuclear magnetic resonance analysis, which showed that glucuronidation had occurred at N-2 (the imidazole nitrogen that is not a part of the benzodiazepine ring). In a separate study, in which midazolam was used as the substrate, an analogous N-glucuronide also was detected from incubations with human liver microsomes in the presence of UDPGA. Investigation of the kinetics of 1'-hydroxymidazolam glucuronidation in human liver microsomes indicated autoactivation kinetics (Hill coefficient, n = 1.2-1.5). The apparent S(50) values for the formation of O- and N-glucuronides were 43 and 18 microM, respectively, and the corresponding apparent V(max) values were 363 and 21 pmol/mg of microsomal protein/min. Incubations with recombinant human uridine diphosphate glucuronosyltransferases (UGTs) indicated that the O-glucuronidation was catalyzed by UGT2B4 and UGT2B7, whereas the N-glucuronidation was catalyzed by UGT1A4. Consistent with these observations, hecogenin, a selective inhibitor of UGT1A4, selectively inhibited the N-glucuronidation, whereas diclofenac, a potent inhibitor of UGT2B7, had a greater inhibitory effect on the O-glucuronidation than on the N-glucuronidation. In summary, our study provides the first demonstration of N-glucuronidation of 1'-hydroxymidazolam in human liver microsomes.

Prediction of metabolic clearance of bisphenol A (4,4 '-dihydroxy-2,2-diphenylpropane) using cryopreserved human hepatocytes.

This study investigated the kinetics of glucuronidation of bisphenol A (BPA; 4,4'-dihydroxy-2,2-diphenylpropane) in cryopreserved human hepatocytes (HCs). Incubation conditions were developed using Sprague-Dawley rat HCs. For determination of the kinetic constants of BPA glucuronidation rates with human HCs, viable HCs (0.125 x 10(6)) were incubated with [(14)C]BPA (1.3-52 microM) for 10 min. The glucuronidation reaction demonstrated Michaelis-Menten kinetics and yielded a mean K(m) for males and females of 9 +/- 3 and 8 +/- 2 microM, respectively. The V(max) values of these reactions were 438 +/- 129 pmol/min/10(6) for male HCs and 480 +/- 208 pmol/min/10(6) for female HCs. The scaled intrinsic clearance (CL(int)) for male human HCs was 149 +/- 67 ml/min/kg (range 53-246) and for female HCs was 165 +/- 89 ml/min/kg (range 73-336). Overall, there are no apparent gender differences in the glucuronidation of BPA. These CL(int) values were then extrapolated to estimate total hepatic metabolic clearance (CL(met)) using a nonrestrictive well stirred model. The estimated CL(met) value for both male and female HCs was 6 ml/min/kg, which represents 30% of hepatic blood flow. Thus, in vivo clearance seems to depend highly on plasma protein binding. These in vitro results correlate well with in vivo studies in humans, which report extensive glucuronidation of BPA.

Functional expression of the P2Y14 receptor in human neutrophils.

Previous studies using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis have shown that the P2Y(14) receptor is expressed at high levels in human neutrophils. Therefore the primary aim of this study was to determine whether the P2Y(14) receptor is functionally expressed in human neutrophils. In agreement with previous studies RT-PCR analysis detected the expression of P2Y(14) receptor mRNA in human neutrophils. UDP-glucose (IC(50)=1 microM) induced a small but significant inhibition (circa 30%) of forskolin-stimulated cAMP accumulation suggesting functional coupling of endogenously expressed P2Y(14) receptors to the inhibition of adenylyl cyclase activity in human neutrophils. In contrast, the other putative P2Y(14) receptor agonists UDP-galactose and UDP-glucuronic acid (at concentrations up to 100 microM) had no significant effect, whereas 100 microM UDP-N-acetylglucosamine-induced a small but significant inhibition of forskolin-stimulated cAMP accumulation (20% inhibition). UDP-galactose, UDP-glucuronic acid and UDP-N-acetylglucosamine behaved as partial agonists by blocking UDP-glucose mediated inhibition of forskolin-induced cAMP accumulation. Treatment of neutrophils with pertussis toxin (G(i/o) blocker) abolished the inhibitory effects of UDP-glucose on forskolin-stimulated cAMP accumulation. UDP-glucose (100 microM) also induced a modest increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, whereas the other sugar nucleotides had no effect on ERK1/2 activation. Finally, UDP-glucose and related sugar nucleotides had no significant effect on N-formyl-methionyl-leucyl-phenylalanine-induced elastase release from neutrophils. In summary, although we have shown that the P2Y(14) receptor is functionally expressed in human neutrophils (coupling to inhibition of forskolin-induced cAMP and ERK1/2 activation) it does not modulate neutrophil degranulation (assessed by monitoring elastase release). Clearly further studies are required in order to establish the functional role of the P2Y(14) receptor expressed in human neutrophils.

Glucuronidation converts gemfibrozil to a potent, metabolism-dependent inhibitor of CYP2C8: implications for drug-drug interactions.

Gemfibrozil more potently inhibits CYP2C9 than CYP2C8 in vitro, and yet the opposite inhibitory potency is observed in the clinic. To investigate this apparent paradox, we evaluated both gemfibrozil and its major metabolite, an acyl-glucuronide (gemfibrozil 1-O-beta-glucuronide) as direct-acting and metabolism-dependent inhibitors of the major drug-metabolizing cytochrome P450 enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) in human liver microsomes. Gemfibrozil most potently inhibited CYP2C9 (IC50 of 30 microM), whereas gemfibrozil glucuronide most potently inhibited CYP2C8 (IC50 of 24 microM). Unexpectedly, gemfibrozil glucuronide, but not gemfibrozil, was found to be a metabolism-dependent inhibitor of CYP2C8 only. The IC50 for inhibition of CYP2C8 by gemfibrozil glucuronide decreased from 24 microM to 1.8 microM after a 30-min incubation with human liver microsomes and NADPH. Inactivation of CYP2C8 by gemfibrozil glucuronide required NADPH, and proceeded with a K(I) (inhibitor concentration that supports half the maximal rate of enzyme inactivation) of 20 to 52 microM and a k(inact) (maximal rate of inactivation) of 0.21 min(-1). Potent inhibition of CYP2C8 was also achieved by first incubating gemfibrozil with alamethicin-activated human liver microsomes and UDP-glucuronic acid (to form gemfibrozil glucuronide), followed by a second incubation with NADPH. Liquid chromatography-tandem mass spectrometry analysis established that human liver microsomes and recombinant CYP2C8 both convert gemfibrozil glucuronide to a hydroxylated metabolite, with oxidative metabolism occurring on the dimethylphenoxy moiety (the group furthest from the glucuronide moiety). The results described have important implications for the mechanism of the clinical interaction reported between gemfibrozil and CYP2C8 substrates such as cerivastatin, repaglinide, rosiglitazone, and pioglitazone.

Quaternary ammonium-linked glucuronidation of tamoxifen by human liver microsomes and UDP-glucuronosyltransferase 1A4.

Tamoxifen (TAM), a nonsteroidal antiestrogen, is the most widely used drug for chemotherapy of hormone-dependent breast cancer in women. In the present study, we found a new potential metabolic pathway of TAM via N-linked glucuronic acid conjugation for excretion in humans. TAM N(+)-glucuronide was isolated from a reaction mixture consisting of TAM and human liver microsomes fortified with UDP-glucuronic acid (UDPGA) and identified with a synthetic specimen by high-performance liquid chromatography-electrospray ionization-mass spectrometry. However, no TAM-glucuronidating activity was detected in microsomes from rat, mouse, monkey, dog, and guinea pig livers. A strong correlation (r(2) =0.92 ) was observed between N-glucuronidating activities toward TAM and trifluoperazine, a probe substrate for human UDP-glucuronosyltransferase (UGT) 1A4, in human liver microsomes from eight donors (five females, three males). However, no correlation ( (r(2) =0.02 )) was observed in the activities between 7-hydroxy-4-(trifluoromethyl)coumarin and TAM. Only UGT1A4 catalyzed the N-linked glucuronidation of TAM among recombinant UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) expressed in insect cells. Apparent K(m) values for TAM N-glucuronidation by human liver microsomes and recombinant UGT1A4 were 35.8 and 32.4 microM, respectively. These results strongly suggested that UGT1A4 could play a role in metabolism and excretion of TAM without Phase I metabolism in human liver. TAM N(+)-glucuronide still had binding affinity similar to TAM itself for human estrogen receptors, ERalpha and ERbeta, suggesting that TAM N(+)-glucuronide might contribute to the biological activity of TAM in vivo.

Metabolic fate of pitavastatin, a new inhibitor of HMG-CoA reductase: similarities and difference in the metabolism of pitavastatin in monkeys and humans.

1. To elucidate any potential species differences, the in vitro metabolism of pitavastatin and its lactone was studied with hepatic and renal microsomes from rats, dogs, rabbits, monkeys and humans. 2. With the addition of UDP-glucuronic acid to hepatic microsomes, pitavastatin lactone was identified as the main metabolite in several animals, including humans. 3. Metabolic clearances of pitavastatin and its lactone in monkey hepatic microsome were much greater than in humans. 4. M4, a metabolite of pitavastatin with a 3-dehydroxy structure, was converted to its lactone form in monkey hepatic microsomes in the presence of UDP-glucuronic acid as well as to pitavastatin. These results implied that lactonization is a common pathway for drugs such as 5-hydroxy pentanoic acid derivatives. 5. The acid forms were metabolized to their lactone forms because of their structural characteristics. 6. UDP-glucuronosyltransferase is the key enzyme responsible for the lactonization of pitavastatin, and overall metabolism is different compared with humans owing to the extensive oxidative metabolism of pitavastatin and its lactone in monkey.

Purification and kinetic properties of UDP-glucose dehydrogenase from sugarcane.

In this study, UDP-glucose dehydrogenase has been purified to electrophoretic homogeneity from sugarcane (Saccharum spp. hybrid) culm. The enzyme had a pH optimum of 8.4 and a subunit molecular mass of 52 kDa. Specific activity of the final preparation was 2.17 micromol/min/mg protein. Apparent K(m) values of 18.7+/-0.75 and 72.2+/-2.7 microM were determined for UDP-glucose and NAD(+), respectively. The reaction catalyzed by UDP-glucose dehydrogenase was irreversible with two equivalents of NADH produced for each UDP-glucose oxidized. Stiochiometry was not altered in the presence of carbonyl-trapping reagents. With respect to UDP-glucose, UDP-glucuronic acid, and UDP-xylose were competitive inhibitors of UDP-glucose dehydrogenase with K(i) values of 292 and 17.1 microM, respectively. The kinetic data are consistent with a bi-uni-uni-bi substituted enzyme mechanism for sugarcane UDP-glucose dehydrogenase. Oxidation of the alternative nucleotide sugars CTP-glucose and TDP-glucose was observed with rates of 8 and 2%, respectively, compared to UDP-glucose. The nucleotide sugar ADP-glucose was not oxidized by UDP-glucose dehydrogenase. This is of significance as it demonstrates carbon, destined for starch synthesis in tissues that synthesize cytosolic AGP-glucose, will not be partitioned toward cell wall biosynthesis.

Differential regulation of UDP-GlcUA transport in endoplasmic reticulum and in Golgi membranes.

BACKGROUND: In the endoplasmic reticulum (ER), the stimulation of UDP-glucuronosyltransferase (UGT) by UDP-GlcNAc is based on the interaction of transport across the ER membrane of UDP-GlcUA with UDP-GlcNAc. Intramicrosomal UDP-GlcNAc stimulates influx of UDP-GlcUA and thereby enhances delivery of UDP-GlcUA to the catalytic center of UGT in the ER lumen. AIM: The aim of this study is to investigate whether the interactions between nucleotide sugars for transport across the ER membrane also occur in the Golgi apparatus, and thereby affect UGT activity in Golgi membranes. RESULTS: We found that Golgi membrane preparations display UGT activity which, unlike in ER membranes, is not stimulated by UDP-GlcNAc. Efflux of intravesicular UDP-GlcNAc and UDP-Xyl marginally enhanced uptake of UDP-GlcUA in Golgi vesicles; such trans-stimulation was much more pronounced in the ER. Efflux of intravesicular UDP-GlcNAc was strongly trans-stimulated by cytosolic UDP-GlcUA in ER-derived vesicles but less so in Golgi-derived vesicles. CONCLUSION: The interaction between transport of UDP-GlcUA and transport of UDP-GlcNAc or UDP-Xyl is different in Golgi vesicles compared with ER vesicles. This finding is consistent with the different effects of UDP-GlcNAc on glucuronidation in Golgi and ER.

Inhibitory effect of azole antifungal agents on the glucuronidation of lorazepam using rabbit liver microsomes in vitro.

Azole antifungal agents (azoles) have inhibitory effects on the cytochrome P450. However, the effect of azoles on conjugative metabolism has not been given much attention. Lorazepam (LZP), a benzodiazepine sedative agent, is known to be metabolized by uridine 5'-diphosphate (UDP)-glucuronyltransferase. Herein we report investigation of the effect of azoles on the enzyme-kinetics of glucuronidation of lorazepam using rabbit liver microsomes in vitro. The Km and Vmax for LZP glucuronidation were determined to be 0.26+/-0.08 mM and 1.25+/-0.21 nmol/min/mg protein, respectively, when evaluated in the presence of a detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) (0.8 mg/mg protein). Azoles fluconazole, miconazole, and ketoconazole competitively inhibited the glucuronidation of LZP, with Ki values of 7.17+/-4.78 mM, 0.17+/-0.08 mM, and 0.092+/-0.026 mM, respectively. These results are comparable to the previously reported Ki values of azoles with zidovudine (AZT) glucuronidation (1.4, 0.18, and 0.08 mM for fluconazole, miconazole, and ketoconazole, respectively) [Sampol et al., Br. J. Clin. Pharmacol., 40, 83-86, 1995]. Therefore, in order to avoid possible side effects of LZP, the concomitant administration of LZP and azoles should be carefully evaluated.

Interaction of periodate-oxidized UDP-glucuronic acid with recombinant human liver UDP-glucuronosyltransferase 1A6.

Sodium periodate reacts with UDP-glucuronic acid (UDP-GlcUA) to generate a reactive derivative [periodate-oxidized UDP-GlcUA (o-UDP-GlcUA)]. The ability of this analog of UDP-GlcUA to inactivate and label the human recombinant UDP-glucuronosyltransferase (UGT) UGT1A6 via the UDP-GlcUA binding site was investigated. At an o-UDP-GlcUA concentration of 20 mM, the enzymatic activity of UGT1A6 was totally inactivated after 30 min of incubation at pH 7.4. Inhibition was irreversible, time-dependent, and concentration-dependent and exhibited pseudo-first order kinetics (kinact = 4.0 M-1.min-1). Cosubstrate protection with UDP-GlcUA was biphasic, with no protection in the first phase and almost total protection in the second phase, suggesting that at least 65% of the cross-linking occurs at the cosubstrate binding site. Partial inactivation by o-UDP-GlcUA led to a decrease in Vmax, suggesting that o-UDP-GlcUA can act as an active site-directed inhibitor. Furthermore, proteins, including the UGTs, from membrane fractions of a recombinant V79 cell line expressing the UGT1A6 enzyme and from rat liver microsomes were cross-linked by in situ periodate oxidation of [beta-32P]UDP-GlcUA. The present results suggest that periodate-oxidized UDP-GlcUA, which inactivates UGT1A6 by the possible formation of a Schiff base adduct with active site lysyl residues, can be used as a new affinity label for the UDP-GlcUA binding site.

Monometoxytrityl derivatives of uridine as inhibitors of a human recombinant UDP-glucuronosyltransferase: UGT1*6.

A series of inhibitors of the human liver recombinant UDP-glucuronosyltransferase 1*6 derived from uridine were synthetized as probes of the binding site of the cosubstrate, UDP-glucuronic acid. If triphenylmethanol or uridine alone failed to inhibit the glucuronidation of 4-methylumbelliferone, the trityl derivatives of uridine were found to be very effective inhibitors of the enzyme (Ki 4.4 to 73 microM). The type of inhibition (competitive or mixed) varied with the substitutions on the uracile or on the triphenylmethyl moiety by halogen atoms or methyl groups. Structural features for the binding of the cofactor are postulated.

Alterations in activation and deactivation of mutagens in aging rat liver.

Age-associated alternations in activation and deactivation of benzo[a]pyrene (BP), furylfuramide (AF2), and 2-nitrofluorene (NF) in rat liver were investigated. A modified Ames mutagenicity test system used liver 9000 g supernatant (S-9) from male Fischer 344 rats aged 3, 6, 12, and 24 months fortified with NADPH generating system alone or together with cofactors of conjugating enzymes. The numbers of revertant colonies due to mutagenic activation of BP during preincubation were markedly high in young rats and decreased with aging. They were decreased by the addition of UDP-glucuronic acid (15 mM) or glutathione (30 mM), the cofactors of UDP-glucuronyl transferase and glutathione S-transferase, respectively, in the preincubation mixture. The difference in the BP activation by liver S-9 from different age groups almost disappeared by the addition of reduced glutathione. A direct mutagen, AF2, was not metabolized during preincubation in the absence of cofactors of conjugating enzymes, but detoxified up to about 50% by the addition of glutathione to the preincubation mixture containing liver S-9 from rats of any age group. Another direct mutagen, NF, was partly detoxified during preincubation by liver S-9 from 3-month-old rats more than by that from 24-month-old rats. It is suggested that incidence of chemical carcinogenesis may increase along with aging due to the altered xenobiotics metabolism.

Paradoxical effect of Sudan III on the in vivo and in vitro genotoxicity elicited by 7,12-dimethylbenz(a)anthracene.

Effect of the induction of drug metabolizing enzymes by Sudan III on the in vivo and in vitro genotoxicity elicited by 7,12-dimethyl-benz(a)anthracene (DMBA) was investigated. A significant suppression of DMBA-induced micronucleated reticulocytes was observed in C57BL/6 mice treated with Sudan III intraperitoneally for 3 or 5 days before injection of the DMBA. However, the preincubation of DMBA with hepatic microsomes from Sudan III-treated rats caused a marked increase in the in vitro mutagenicity in the Ames assay, paradoxically. Sudan III was found to induce CYP 1A1, 7-ethoxycoumarin O-deethylase activity as well as both UDP-glucuronyl transferase and glutathione S-transferase activities. The increase of mutagenicity of DMBA observed in the Ames assay using hepatic microsomes from Sudan III-treated rats was inhibited by the addition of uridine 5'-diphosphoglucuronic acid or reduced glutathione with cytosol. Mutagenic metabolites of DMBA formed by CYP1A1 appeared to be effectively detoxified by these phase II enzymes. The results of this study suggest that Sudan III-induced prevention of in vivo mutagenesis is due to the induction of both CYP 1A1 and detoxifying phase II enzymes. The induced CYP1A1 may accelerate formation of active metabolic intermediates, but phase II enzymes are also induced and detoxify these intermediates to inactive metabolites. This would reduce residence time of the carcinogen in the body and the time of exposure to active metabolites for target organs.