Flavin monooxygenases, FMO1 and FMO3, not cytochrome P450 isoenzymes, contribute to metabolism of anti-tumour triazoloacridinone, C-1305, in liver microsomes and HepG2 cells

5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, being the close structural analogue of the clinically tested imidazoacridinone anti-tumour agent, C-1311, expressed high activity against experimental tumours and is expected to have more advantageous pharmacological properties than C-1311. The aim of this study was to elucidate the role of selected liver enzymes in the metabolism of C-1305. We demonstrated that the studied triazoloacridinone was transformed with rat and human liver microsomes, HepG2 hepatoma cells and with human recombinant flavin-containing monooxygenases FMO1, FMO3 but not with CYPs. Furthermore, this compound was an effective inhibitor of CYP1A2 and CYP3A4. The product of FMO catalysed metabolism was shown to be identical to the main metabolite from liver microsomes and HepG2 cells. It was identified as an N-oxide derivative and, under hypoxia, it underwent retroreduction back to C-1305, what was extremely effective with participation of CYP3A4. In summary, this work revealed that the involvement of the P450 enzymatic system in microsomal and cellular metabolism of C-1305 was negligible, whereas this agent was an inhibitor of CYP1A2 and CYP3A4. In contrast, FMO1 and FMO3 were crucial for metabolism of C-1305 by liver microsomes and in HepG2 cells, which makes C-1305 an attractive potent anti-tumour agent.     

Flavin monooxygenases,FMO1 and FMO3, not cytochrome P450 isoenzymes,contribute to metabolism of anti-tumour triazoloacridinone,C-1305, in liver microsomes and HepG2 cells

1. 5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, being the close structural analogue of the clinically tested imidazoacridinone anti-tumour agent, C-1311, expressed high activity against experimental tumours and is expected to have more advantageous pharmacological properties than C-1311.

2. The aim of this study was to elucidate the role of selected liver enzymes in the metabolism of C-1305.

3. We demonstrated that the studied triazoloacridinone was transformed with rat and human liver microsomes, HepG2 hepatoma cells and with human recombinant flavin-containing monooxygenases FMO1, FMO3 but not with CYPs. Furthermore, this compound was an effective inhibitor of CYP1A2 and CYP3A4. The product of FMO catalysed metabolism was shown to be identical to the main metabolite from liver microsomes and HepG2 cells. It was identified as an N-oxide derivative and, under hypoxia, it underwent retroreduction back to C-1305, what was extremely effective with participation of CYP3A4.

4. In summary, this work revealed that the involvement of the P450 enzymatic system in microsomal and cellular metabolism of C-1305 was negligible, whereas this agent was an inhibitor of CYP1A2 and CYP3A4. In contrast, FMO1 and FMO3 were crucial for metabolism of C-1305 by liver microsomes and in HepG2 cells, which makes C-1305 an attractive potent anti-tumour agent.

     

Biotransformation of geldanamycin and 17-allylamino-17-demethoxygeldanamycin by human liver microsomes: reductive versus oxidative metabolism and implications.

Comparative metabolite profiling of geldanamycin and 17-allylamino-17-demethoxygeldanamycin (17AAG) using human liver microsomes in normoxia and hypoxia was conducted to understand their differential metabolic fates. Geldanamycin bearing a 17-methoxy group primarily underwent reductive metabolism, generating the corresponding hydroquinone under both conditions. The formed hydroquinone resists further metabolism and serves as a reservoir. On exposure to oxygen, this hydroquinone slowly reverts to geldanamycin. In the presence of glutathione, geldanamycin was rapidly converted to 19-glutathionyl geldanamycin hydroquinone, suggesting its reactive nature. In contrast, the counterpart (17AAG) preferentially remained as its quinone form, which underwent extensive oxidative metabolism on both the 17-allylamino sidechain and the ansa ring. Only a small amount (<1%) of 19-glutathione conjugate of 17AAG was detected in the incubation of 17AAG with glutathione at 37 degrees C for 60 min. To confirm the differential nature of quinone-hydroquinone conversion between the two compounds, hypoxic incubations with human cytochrome P450 reductase at 37 degrees C and direct injection analysis were performed. Approximately 89% of hydroquinone, 5% of quinone, and 6% of 17-O-demethylgeldanamycin were observed after 1-min incubation of geldanamycin, whereas about 1% of hydroquinone and 99% of quinone were found in the 60-min incubation of 17AAG. The results provide direct evidence for understanding the 17-substituent effects of these benzoquinone ansamycins on their phase I metabolism, reactivity with glutathione, and acute hepatotoxicity.     

Analysis of the key networks,metabolic pathways,and regulation substances of hypoxia based on the omics and zebrafish model

OBJECTIVE Hypoxia is associated with many complicated pathophysiological and biochemical processes that integrated and regulated via the key gene,protein and endogenous metabolite levels.Up to date,the exact molecular mechanism of hypoxia still remains unclear.In this work,we further explore the molecular mechanism of hypoxia and adaption to attenuate the damage in zebrafish model that have potential to resist hypoxic environment.METHODS The hypoxic zebrafish model was established in different concentration of oxygen with 3%,5%,10%,21%in water.The brain tissue was separated and the RNA-seq was used to identify the differentially expressed genes.The related endogenous metabolites profiles were obtained by LC-HDMS,and the multivariate statistics was applied to discover the important metabolites candidates in hypoxic zebrafish.The candidates were searched in HMDB,KEGG and Lipid Maps databases.RESULTS The zebrafish hypoxic model was successfully constructed via the different concentration of oxygen,temperature and hypoxic time.The activities of the related hypoxic metabolic enzymes and factors including HIF-1a,actate dehydrogenase(LDH)and citrate synthase(CS)were evaluated.Significant differences(P<0.05 and fold change>2)in the expression of 422 genes were observed between the normal and 3% hypoxic model.Among them,201 genes increased depended on the lower concentration of oxygen.53 metabolites were identified that had significant difference between the hypoxia and control groups(P<0.05,fold change>1.5 and VIP>1.5).The ten key metabolites were increased gradually while six compounds were decreased.The endogenous hypoxic metabolites of phenylalanine,D-glucosamine-6P and several important lipids with the relevant hub genes had similar change in hypoxic model.In addition,the metabolic pathways of phenylalanine,glutamine and glycolipid were influenced in both the levels of genes and metabolites.CONCLUSION The up-regulation of phenylalanine,D-glucosamine-6P and lipid may have further understanding of protective effect in hypoxia.Our data provided an insight to further reveal the hypoxia and adaptation mechanism.     

Metabolic studies of ornidazole in the rat, in the dog and in man

1. Ornidazole, labelled with 14C in the imidazole ring, administered orally to rats, dogs and men was largely excreted in the urine, predominantly as metabolites, with less than 4% of the drug being excreted unchanged. Free and conjugated metabolites were found in the ratio of approx. 1 : 2. 2. The pattern of free ornidazole and metabolites was different in the three species: while ornidazole predominated in man, ornidazole and metabolite M1 in the dog, the most extensive metabolic pattern was found in the rat. 3. The following metabolites were identified: M1, 1-chlorlo-3-(2-hydroxymethyl-5-nitro-1-imidazolyl)-2-propanol; M2, 2-methyl-5-nitroimidazole; M3, N-(3-chloro-2-hydroxypropyl)acetamide: M4, 3-(-2-methyl-5-nitro-1-imidazolyl)-1, 2-propanediol; M5, acetamide. 4. The formation of metabolite M3 and M5 indicated cleavage of the imidazole ring between N-1/C-5 and C-2/C-3. Other ring scissions were not observed. Metabolites carrying a free amino group were not detected. On the basis of the structures identified, a scheme is suggested for the metabolism of ornidazole.     

Comparative biodisposition and metabolism of 14C-(+/-)-fenfluramine in mouse, rat, dog and man.

1. The comparative metabolism of fenfluramine was investigated in mouse, rat, dog and man following a single oral dose of 14C-(+/-)-fenfluramine hydrochloride (1 mg/kg), and also in rat after eight consecutive 12-h subcutaneous doses (24 mg/kg). 2. Main route of excretion of radioactivity in all species and at all doses was into urine (> 80%), with only minor amounts of radioactivity found in faeces. 3. From all species examined a total of 11 metabolites were observed in urine and plasma by t.l.c. and h.p.l.c. analysis and no metabolite was present in the plasma which was not present in urine. 4. All species dealkylate fenfluramine to the active metabolite norfenfluramine, to a relative greater or lesser extent, with plasma metabolic ratios (norfenfluramine/fenfluramine) showing inter-animal variation (rat > dog > mouse = man). 5. These differences are due to the efficient deamination of both compounds to polar inactive metabolites in man, with less dealkylation and lower plasma levels of norfenfluramine compared with the other species studied. 6. In conclusion, major species differences in the metabolism of (+/-)-fenfluramine, both qualitative and quantitative were observed, and no one species had a similar metabolic profile to that found in man.     

Preclinical toxicological examination of a putative prostate cancer-specific 4-methyl-1-nitroacridine derivative in rodents

Nitroacridines are potent DNA-binding and cytotoxic agents in cancer cells, but could not be developed clinically due to high systemic toxicities. We are developing a 1-nitroacridine derivative, 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748), as an effective chemotherapeutic agent for prostate cancer. C-1748 demonstrates high antitumor efficacy against human prostate cancer xenografts with markedly low mutagenicity and toxicity in dogs compared with its parent 9-(2'-hydroxyethylamino)-1-nitroacridine (C-857). A surprising feature of C-1748 is the 40-fold difference in 50% inhibitory concentration between DU145 prostate cancer and HL-60 leukemia cells. In this study, we report the preclinical toxicity study of a single acute dose of C-1748 in Copenhagen rats and BALB/c mice, intraperitoneally and intravenously for 24 h and 7 days. The effect of C-1748 on hematology, cardiac and liver enzymes, and renal electrolytes was assessed by blood and serum analysis. The LD50 (lethal dose, 50%) for C-1748 was 9 and 13.42 mg/kg compared with 2.2 and 3 mg/kg for C-857 intraperitoneally and intravenously, respectively, in mice. In Copenhagen rats, LD50 was 15 and 14.4 mg/kg intraperitoneally and intravenously, respectively, compared to 4 and 1.3 mg/kg for C-857. No changes in blood cell counts were observed, which were in the normal range for rodents. No changes were observed in clinical chemistries of enzymes such as aspartate aminotransferase, alkaline phosphatase and creatine phosphokinase, which were within the normal range of values. No genome alterations were seen in prostate cancer cell lines by comparative genomic hybridization together with a lack of systemic toxicity, making it a unique cancer cell-type-specific drug that needs further clinical evaluation for toxicity and synergy in combination chemotherapy regimens.     

Metabolism of diphenyl sulfoxide in perfused guinea pig liver. Involvement of aldehyde oxidase as a sulfoxide reductase

To evaluate the metabolic capacity of intact guinea pig liver under normoxic and hypoxic conditions, oxidative and reductive metabolism of diphenyl sulfoxide (DPSO) was studied by the nonrecirculating perfusion method in situ. DPSO was exclusively converted into diphenyl sulfone (DPSO2), an oxidative metabolite, under normoxia. When diphenyl sulfide (DPS) was infused, DPSO was eliminated as a predominant metabolite. Judging from the susceptibility toward selective inhibitors of cytochrome P-450, both oxidative steps appear to be catalyzed by cytochrome P-450-dependent monooxygenase rather than flavin adenine dinucleotide-containing monooxygenase. Under hypoxic conditions, however, DPSO2 formation was decreased in parallel with reduced oxygen concentration in the influent perfusate, whereas only a trace amount of DPS, a reductive metabolite, was detected. On the other hand, coinfusion of an electron donor for aldehyde oxidase such as 2-hydroxypyrimidine and benzaldehyde, but not xanthine, markedly stimulated the formation of DPS during hypoxia. These results indicate that the oxidative pathway catalyzed by cytochrome P-450-dependent monooxygenase is predominant in DPSO metabolism under normoxic conditions, whereas only under hypoxia does the reductive pathway become the major one if an electron donor for aldehyde oxidase exists in intact guinea pig liver.     

A UPLC-MS/MS method reveals the pharmacokinetics and metabolism characteristics of kaempferol in rats under hypoxia

As a natural flavonoid, kaempferol is widely distributed in natural medicines. Our study was aimed at analyzing and comparing the pharmacokinetic differences of kaempferol between normoxia and hypoxia in rats, to further explore the effect of hypoxia on drug metabolism enzymes. A sensitive UPLC-MS/MS method was established and validated for the determination of kaempferol in rat plasma. The results indicated that AUC, MRT, t_1/2 and C_max of kaempferol significantly increased and the clearance reduced in hypoxic rats. Based on the comparison of pharmacokinetics, the metabolites of kaempferol in hypoxic rats were identified by using UPLC-QTOF-MS and UNIFI 1.8 software. Then we explored the effect of hypoxia on the mRNA and protein expression of CYP1A2 and UGT1A9. The study revealed that hypoxia could markedly reduce the mRNA and protein expression of CYP1A2 and UGT1A9, resulting in the reduction of metabolic rate and enhancement of systematic exposure. Our data also indicated that we should pay attention to adjusting the dosage regimen and reducing drug interactions when drugs metabolized by CYP1A2 and UGT1A9 are used in combination with kaempferol. Our findings suggested the potential requirement for dose adjustment of kaempferol or its structural analogs in hypoxic condition.     

Aluminum toxicity triggers the nuclear translocation of HIF-1alpha and promotes anaerobiosis in hepatocytes.

Although aluminum (Al) is known to be toxic, the exact molecular events that enable this trivalent metal to be involved in various diseases have not been fully delineated. In this report, we show that Al promotes the translocation of the HIF-1alpha (hypoxia inducible factor) to the nucleus and activates the anaerobic metabolism of D-glucose. Al-exposed hepatocytes (HepG2 cells) showed a marked increase in HIF-1alpha that was associated with nuclear extracts. D-Glucose consumption in these Al-stressed cells was rapid as more GLUT-1 transporter was expressed. Furthermore, these Al-treated HepG2 cells were characterized with enhanced activities of such metabolic enzymes as hexokinase (HK), pyruvate kinase (PK), lactate dehydrogenase (LDH) and glucose 6-phosphate dehydrogenase (G6PDH). (13)C- NMR studies pointed to a metabolic profile in Al-stressed cells that favored enhanced glycolysis. HPLC analyses confirmed increased glycolytic ATP production in Al-exposed hepatocytes. These findings reveal the ability of Al to create a hypoxic environment that promotes the translocation of HIF-1alpha to the nucleus and stimulates the anaerobic metabolism of D-glucose.     

尼莫地平在人肝微粒体内的代谢

:采用人肝微粒体在体外研究尼莫地平 (Nim)在人体内的代谢物及代谢途径 . Nim在人肝微粒体内被迅速代谢成 3个代谢物 ,分别是 Nim二氢吡啶环脱氢代谢物 M1,二氢吡啶环侧链脱甲基代谢物M2 ,二氢吡啶环脱氢及其侧链脱甲基代谢物 M3.Nim在人肝微粒体中的最初的两步代谢反应是其二氢吡啶环脱氢氧化及其侧链脱甲基反应 ,两者的代谢产物可以被进一步代谢为代谢物 M3.CYP3A的特异性抑制剂醋竹桃霉素和酮康唑可以抑制Nim的二氢吡啶环脱氢氧化及其侧链脱甲基反应 ,使 Nim的代谢速率明显下降 ,结果提示 CYP3A参与了 Nim在人肝微粒体内的代谢     

Drug-drug interaction potential of antitumor acridine agent C-1748: The substrate of UDP-glucuronosyltransferases 2B7, 2B17 and the inhibitor of 1A9 and 2B7

Background

The compound 9-(2′-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748), the promising antitumor agent developed in our laboratory was determined to undergo phase I metabolic pathways. The present studies aimed to know its biotransformation with phase II enzymes – UDP-glucuronosyltransferases (UGTs) and its potential to be engaged in drug-drug interactions arising from the modulation of UGT activity.

Fluorescent markers for hypoxic cells: a study of nitroaromatic compounds, with fluorescent heterocyclic side chains, that undergo bioreductive binding

Several novel compounds having both a 2-nitroimidazole nucleus and a fluorescent ring system in their molecular structure were prepared and evaluated as potential fluorescent probes for hypoxia. Bioreduction of nitroimidazoles, which is inhibited by oxygen, is known to lead to binding of bioreductive metabolites to cellular macromolecules and this provides a mechanism for binding the fluorescent moiety to hypoxic cells. These compounds can incorporate a wide range of fluorophors and can therefore be designed to suit the laser-line wavelengths available for excitation of fluorescence in the flow cytometer. Several nitroimidazoles with naphthalimide side chains were rapidly taken up into cells and became concentrated in the cells, thus reducing their concentration in the extracellular medium. This suggests a potential microscopic bioavailability problem with probes of this type when used in vivo as they would become progressively depleted in the extracellular fluid as they diffused from blood vessels, through layers of packed cells in tumors, to the hypoxic cells where they could undergo hypoxia-specific metabolism. Synthesis of nitroimidazoles with coumarin fluorophors led to several potentially useful probes for hypoxia; substituents on the coumarin fluorophor had a marked effect on the cellular fluorescence of these compounds.     

Antitumor 1-nitroacridine derivative C-1748, induces apoptosis, necrosis or senescence in human colon carcinoma HCT8 and HT29 cells

C-1748 is a DNA-binding agent with potent antitumor activity, especially towards prostate and colon carcinoma xenografts in mice. Here, we elucidated the nature of cellular response of human colon carcinoma HCT8 and HT29 cells to C-1748 treatment, at biologically relevant concentrations (EC₉₀ and their multiplicity). Cell cycle analysis showed gradual increase in HCT8 cells with sub-G1 DNA content (25% after 72 h) considered as apoptotic. Hypodiploid cell population increased up to 60% upon treatment with 4× EC₉₀ concentration of the drug. Compared with HCT8 cells, the fraction of sub-G1 HT29 cells did not exceed 14%, even following 4-fold dose escalation. Morphological changes and biochemical markers such as: phosphatydylserine externalization, apoptotic DNA breaks, mitochondrial dysfunction and caspase activation confirmed the presence of considerable amount of apoptotic HCT8 cells but only a low amount of apoptotic HT29 cells. Next, we demonstrated that HCT8 cells surviving after exposure to C-1748 were in the state of senescence, based on altered cell morphology and expression of a pH 6-dependent β-galactosidase. On the contrary, no β-galactosidase staining was observed in HT29 cells after C-1748 treatment. Moreover, prolonged drug incubation (up to 168 h) resulted in massive detachment of cells from culture plates, which together with Annexin V/PI results, indicated that necrosis was the main response of HT29 cells to C-1748 treatment. We also determined the ability of C-1748 to induce reactive oxygen species (ROS) in colon cancer cells and demonstrated, that generation of ROS was not essential for C-1748-induced apoptosis and cytotoxic activity of this drug.     

Synthesis, structure and hypoxic cytotoxicity of 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives

A series of novel 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives were synthesized and screened for their in vitro cytotoxicity against promyelocytic leukemia HL-60, androgen-independent prostate tumor PC3, hepatocellular carcinoma Bel-7402, human esophagus tumor ECA-109, and human breast tumor MCF-7 cell lines in hypoxia and in normoxia. Most compounds showed higher cytotoxic activity both in hypoxia and in normoxia. Among them, compounds 61 and 62 showed more potent cytotoxic activity and hypoxic selectivity when compared to tirapazamine.     

Anthracycline metabolism and toxicity in human myocardium: comparisons between doxorubicin, epirubicin, and a novel disaccharide analogue with a reduced level of formation and [4Fe-4S] reactivity of its secondary alcohol metabolite

Secondary alcohol metabolites have been proposed to mediate chronic cardiotoxicity induced by doxorubicin (DOX) and other anticancer anthracyclines. In this study, NADPH-supplemented human cardiac cytosol was found to reduce the carbonyl group in the side chain of the tetracyclic ring of DOX, producing the secondary alcohol metabolite doxorubicinol (DOXol). A decrease in the level of alcohol metabolite formation was observed by replacing DOX with epirubicin (EPI), a less cardiotoxic analogue characterized by an axial-to-equatorial epimerization of the hydroxyl group at C-4 in the amino sugar bound to the tetracyclic ring (daunosamine). A similar decrease was observed by replacing DOX with MEN 10755, a novel anthracycline with preclinical evidence of reduced cardiotoxicity. MEN 10755 is characterized by the lack of a methoxy group at C-4 in the tetracyclic ring and by intercalation of 2, 6-dideoxy-L-fucose between daunosamine and the aglycone. Multiple comparisons with methoxy- or 4-demethoxyaglycones, and a number of mono- or disaccharide 4-demethoxyanthracyclines, showed that both the lack of the methoxy group and the presence of a disaccharide moiety limited alcohol metabolite formation by MEN 10755. Studies with enzymatically generated or purified anthracycline secondary alcohols also showed that the presence of a disaccharide moiety, but not the lack of a methoxy group, made the metabolite of MEN 10755 less reactive with the [4Fe-4S] cluster of cytoplasmic aconitase, as evidenced by its limited reoxidation to the parent carbonyl anthracycline and by a reduced level of delocalization of Fe(II) from the cluster. Collectively, these studies (i) characterize the different influence of methoxy and sugar substituents on the formation and [4Fe-4S] reactivity of anthracycline secondary alcohols, (ii) lend support to the role of alcohol metabolites in anthracycline-induced cardiotoxicity, as they demonstrate that the less cardiotoxic EPI and MEN 10755 share a reduction in the level of formation of such metabolites, and (iii) suggest that the cardiotoxicity of MEN 10755 might be further decreased by the reduced [4Fe-4S] reactivity of its alcohol metabolite.     

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

Background

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.

In vivo metabolism of isoxicam in rats, dogs, and monkeys

Isoxicam is a long half-life nonsteroidal anti-inflammatory agent which undergoes extensive metabolism prior to elimination in animals and man. The major route of isoxicam transformation is hydroxylation of the methylisoxazole functionality to form hydroxymethylisoxicam, and cleavage of its benzothiazine moiety to give an oxoacetic acid metabolite. The metabolic pathway for scission of the benzothiazine moiety to the oxoacetic acid metabolite and to other potential metabolites is not known. To gain additional information on the metabolic fate of isoxicam, 14C-isoxicam labeled on the N-methyl group was administered to rats, dogs, and monkeys with urine and feces collected for metabolic profiling and identification. Identified as new metabolites of isoxicam were an open-ring sulfonamide, N-methylsaccharin, and saccharin. The formation of these metabolites suggests that isoxicam undergoes direct oxidative scission of its benzothiazine ring at carbon atom 3 to generate the observed open-ring sulfonamide, N-methylsaccharin, and oxoacetic acid metabolites.     

Phenacetin and acetaminophen metabolism in the isolated perfused rat liver. Precursor concentration influences the selection of kinetic parameters to assess hypoxic impairment.

Subnormal oxygen concentrations affect a host of cellular functions and can exist under normal or pathological conditions. The present study examined the effects of hypoxia on phenacetin O-deethylation by the mixed-function oxidase system and the subsequent sulfation and glucuronidation of the generated metabolite acetaminophen, compared to hypoxic alterations in conjugation of administered acetaminophen, in isolated perfused rat livers. A recirculating perfusion system containing either 20% (normoxic conditions) or 2.5% (hypoxic conditions) donor rat blood delivered 4.46 and 1.47 mumol/min/g liver oxygen, respectively, resulting in a 44% reduction in oxygen consumption during hypoxia. The total hepatic clearance of phenacetin was diminished significantly during hypoxia, due to a 60% decrease in the formation clearance of acetaminophen. Hypoxia did not influence significantly the total hepatic clearance of either administered or generated acetaminophen. Although the formation clearance for acetaminophen sulfate (AS) remained unchanged, the Vmax for sulfate formation was diminished 35%. The formation clearance (mean +/- SD; N = 4/group) of acetaminophen glucuronide (AG) was greater from administered compared to generated acetaminophen during normoxia (0.47 +/- 0.15 vs. 0.22 +/- 0.06 ml/min, p less than 0.05), and was decreased 2- to 3-fold during hypoxia (0.14 +/- 0.08 vs. 0.11 +/- 0.07 ml/min). Hypoxic conditions did not affect differentially the time lag for the appearance of AG in perfusate, and did not appear to alter the diffusional barrier for AS and AG from perfusate into the hepatocyte.