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This work describes a new analytical method useful for monitoring the human exposure to the endocrine-disrupting plasticizer triphenyl phosphate (TPP) via nail polish use. The method allows trace determination of this parent compound and its main metabolite, namely diphenyl phosphate (DPP), in urine samples of nail polish users. The method is based on a novel microextraction technique termed stir bar sorptive-dispersive microextraction (SBSDME) using a magnetic composite made of CoFe 2 O 4 magnetic nanoparticles embedded into a mixed-mode weak anion exchange polymer (Strata ™ -X-AW), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The main parameters involved in the extraction procedure were evaluated and optimized. Under the optimized conditions, the method was successfully validated showing good linearity (at least up to 100 ng mL -1 ) and enrichment factors (17 and 30), limits of detection and quantification in the low ng L -1 range (1.9-17.1 ng L -1 and 6.3-57.1 ng L -1 , respectively) and good intra- and inter-day precision (RSD < 8%). Excellent recoveries (81-112 %) were achieved by using matrix-matched calibration for quantification. Finally, the method was applied to the determination of both the parent compound and the metabolite in human urine samples from volunteers who applied themselves a nail polish containing TPP. Detectable amounts of the parent compound were found just in the first urination, whereas quantifiable amounts of DPP were found in the low ng mL -1 range even 24 h after application of the nail polish, thus suggesting a rapid biotransformation and a low excretion, and showing DPP as excellent biomarker of human exposure to TPP. This work expands the analytical potential of the novel SBSDME, and the proposed methodology contributes to the study of the absorption/excretion levels of endocrine disruptors present in cosmetic products., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The correlation of in vitro inhibition of cathepsin K (CatK) activity and in vivo suppression of collagen I biomarkers was examined with three selective CatK inhibitors to explore the potential translatability from animal species to human. These inhibitors exhibited good in vitro potencies toward recombinant CatK enzymes across species, with IC 50 values ranging from 0.20 to 6.1 nM. In vivo studies were conducted in animal species following multiple-day dosing of the CatK inhibitors to achieve steady-state plasma drug concentration-time profiles. Measurement of urinary bone resorption biomarkers (cross-linked N-terminal telopeptide and helical peptide of type I collagen) revealed drug concentration-dependent suppression of biomarkers, with EC 50 values estimated to be 12 to 160 nM. Marked improvement in the correlation between in vitro and in vivo CatK activities was observed with the application of unbound (free) fraction in plasma, consistent with the conditions stipulated by the free-drug hypothesis. These results indicate that the in vitro-in vivo translation of CatK inhibition observed in animal species can translate to humans when the unbound fraction of the inhibitor is considered. Interestingly, residual levels of urinary bone resorption marker were detected as the suppression reached saturation (at an average of 82% inhibition), an apparent phenomenon observed regardless of the species, biomarker, or compound examined. Since cathepsin enzymes other than CatK were reported to catalyze cleavage of collagen I, it is hypothesized that CatK-mediated degradation of collagen I in bone represents ~82% of overall collagen I turnover in the body.

Aims: The purpose of this study was to identify predictive markers of irbesartan (an angiotensin receptor blocker) treatment response in immunoglobulin A nephropathy (IgAN) patients., Methods: Urine samples were collected both before and after irbesartan treatment in IgAN patients and compared with urine from healthy volunteers. The total urinary protein produced in 24 h was measured to determine therapeutic response. The urinary proteome was evaluated by two-dimensional gel electrophoresis coupled with MALDI-TOF-MS/MS analysis. Western blotting was used to verify protein expression. A receiver operating characteristic curve was used to evaluate the sensitivity and specificity of candidate biomarkers., Results: Four differentially expressed proteins were identified as vitamin D-binding proteins (VDPs). Western blot showed that urinary VDPs were significantly elevated in nonresponsive versus responsive IgAN patients. The sensitivity, specificity, and accuracy of urinary VDP as a predictive biomarker of irbesartan nonresponsiveness in IgAN were 65%, 85%, and 75%, respectively., Conclusion: Our results revealed that urinary VDP might be a useful biomarker for predicting irbesartan treatment response.

We developed a fast, selective and sensitive on-line LC/LC-MS/MS method for the simultaneous determination of nine parabens and seven environmental phenols in urine. Parabens are widely used as antimicrobial preservatives. Bisphenol A, triclosan, triclocarban, 2-phenylphenol, and benzophenones are used inter alia in disinfectants, sunscreens and in polymers. Some of these substances are suspected endocrine disruptors. Limits of quantification and analytical quality criteria fully met the needs for determining exposure levels occurring in the general population. We analyzed 157 spot urine samples from the general German population (59 females, 39 males and 59 children). For the parabens, we found methyl, ethyl and n-propyl paraben with high detection rates (77-98%), followed by n-butyl (36%), iso-butyl (17%), iso-propyl (3%) and benzyl paraben (3%). We detected no pentyl and heptyl paraben. Urinary concentrations were highest for methyl paraben (median 24.5 μg/L; 95th percentile 379 μg/L) followed by ethyl (1.4 μg/L; 35.2 μg/L) and n-propyl paraben (1.2 μg/L; 68.1 μg/L). Other environmental phenols with high detection rates were BPA (95%), triclosan (45%) and benzophenone 1 and 3 (26%). For most of the parabens/environmental phenols we found higher urinary levels in females than in males or children, probably due to differences in (personal care) product use. However, high levels (in the mg/L range) were also observed in children. Exposure to the above substances is occurring worldwide. Differences between countries do seem to exist and might be caused by different product compositions or different use habits. Human metabolism data is urgently needed to extrapolate from urinary biomarker levels to doses actually taken up., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Irbesartan (IRB) and hydrochlorothiazide (HCT) are angiotensin-II receptor antagonist and thiazide-class diuretic compounds, respectively, which are in use in the treatment of hypertension. A novel dilute-and-shoot HPLC assay method for simultaneous quantification of IRB and HCT in fixed-dose combination tablets and urine samples was described. The separation of IRB, HCT and agomelatine (internal standard) was carried out using a second generation C18-bonded monolithic silica column (Chromolith(®) High Resolution RP-18e, 100×4.6mm, Merck KGaA), utilizing both mobile phase and flow rate gradient elution programs. The analytes were detected at 230 nm wavelength using photodiode array detector within 24 minutes with high resolution, observing about 50 percent more peak capacity when using second generation C18-bonded monolithic silica column. Urine samples were introduced into the system effortlessly, with only filtration and subsequent dilution. Validation studies were performed according to the official recommendations of USP and ICH, and the developed method was successfully applied to pharmaceutical tablets and urine samples., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Purpose: The absorption, distribution, metabolism, and excretion of the hedgehog pathway inhibitor sonidegib (LDE225) were determined in healthy male subjects., Methods: Six subjects received a single oral dose of 800 mg ¹⁴C-sonidegib (74 kBq, 2.0 µCi) under fasting conditions. Blood, plasma, urine, and fecal samples were collected predose, postdose in-house (days 1-22), and during 24-h visits (weekly, days 29-43; biweekly, days 57-99). Radioactivity was determined in all samples using accelerator mass spectrometry (AMS). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine concentrations of sonidegib and its main circulating metabolite in plasma. Metabolite profiles and structures were determined in pooled plasma, urine, and fecal samples using high-performance LC-AMS and LC-MS/MS, respectively., Results: A single dose of ¹⁴C-sonidegib was well tolerated in healthy subjects. Unchanged sonidegib and total radioactivity reached peak concentration in plasma by 2 and 3 h, respectively, and demonstrated similarly long half-lives of 319 and 331 h, respectively. Absorbed sonidegib (estimated 6-7 %) was extensively distributed, and the approximate terminal volume of distribution was 2,500 L. Unchanged sonidegib and a metabolite resulting from amide hydrolysis were the major circulating components (36.4 and 15.4 % of radioactivity area under the curve, respectively). Absorbed sonidegib was eliminated predominantly through oxidative metabolism of the morpholine part and amide hydrolysis. Unabsorbed sonidegib was excreted through the feces. Metabolites in excreta accounted for 4.49 % of the dose (1.20 % in urine, 3.29 % in feces). The recovery of radioactivity in urine and feces was essentially complete (95.3 ± 1.93 % of the dose in five subjects; 56.9 % of the dose in one subject with incomplete feces collection suspected)., Conclusions: Sonidegib exhibited low absorption, was extensively distributed, and was slowly metabolized. Elimination of absorbed sonidegib occurred largely by oxidative and hydrolytic metabolism.

Odanacatib is a selective inhibitor of the cathepsin K enzyme that is expressed in osteoclasts involved in the degradation of bone organic matrix, and is being developed as a novel treatment of osteoporosis. Odanacatib has demonstrated increases in bone mineral density in postmenopausal women and is undergoing a pivotal phase III trial. The absorption, metabolism, and excretion of [(14)C]odanacatib were studied in healthy male volunteers (n = 6) after a single oral dose of 25 mg (100 µCi). Plasma, urine, and fecal samples were collected at intervals up to 34 days postdose. The pharmacokinetics of odanacatib were characterized by slow absorption (mean time to achieve maximum plasma concentration of 14.2 hours) and long apparent elimination half-life (mean t1/2 96.7 hours); 74.5% of the dose was recovered in feces and 16.9% in urine, resulting in a total recovery of 91.4%. Seven metabolites were identified in urine; the major pathway (methyl hydroxylation producing M8 and its derivatives) was largely dependent on CYP3A. Metabolites and odanacatib accounted for 77% and 23% of urinary radioactivity, respectively. In fecal extracts, the only radioactive components identified were odanacatib (60.9%) and M8 (9.5%). The fraction of odanacatib in feces derived from absorbed drug was estimated using a bioavailability value obtained from the results of a separate intravenous study. Collectively, the data indicate that odanacatib has a long t1/2 on account of its low metabolic intrinsic clearance, and that metabolism (principally mediated by CYP3A) and excretion of intact parent compound account for ∼70% and ∼30% of the clearance of odanacatib in humans.

PCBs are contaminants in the air of older buildings and cities, which raises the concern of inhalation exposure. No reliable biomarker of such exposure is available. We exposed rats to air containing 2 mg/m(3) PCB3 via nose-only inhalation for 2 h, collected urine, and analyzed it by LC/MS. Each rat inhaled an estimated dose of 35 μg PCB3, and excreted 27 ± 2% of it as sulfates within 24 h. Peak excretion occurred within 6 h. PCB sulfates were stable in urine for at least three days at room temperature without chemical preservatives. These data support the use of PCB sulfate conjugates as suitable urinary biomarkers of PCB3 and other airborne PCBs.

An environmentally friendly ionic liquids dispersive liquid-liquid microextraction (IL-DLLME) method coupled with high-performance liquid chromatography (HPLC) for the determination of antihypertensive drugs irbesartan and valsartan in human urine samples was developed. The HPLC separations were accomplished in less than 10 min using a reversed-phase C(18) column (250 × 4.60 mm i.d., 5 µm) with a mobile phase containing 0.3 % formic acid solution and methanol (v/v, 3:7; flow rate, 1.0 mL/min). UV absorption responses at 236 nm were linear over a wide concentration range from 50 µg/mL to the detection limits of 3.3 µg/L for valsartan and 1.5 µg/L for irbesartan. The effective parameters on IL-DLLME, such as ionic liquid types and their amounts, disperser solvent types and their volume, pH of the sample and extraction time were studied and optimized. The developed IL-DLLME-HPLC was successfully applied for evaluation of the urine irbesartan and valsartan profile following oral capsules administration., (Copyright © 2012 John Wiley & Sons, Ltd.)

Polychlorinated biphenyls (PCBs) are legacy pollutants that exert toxicities through various mechanisms. In recent years exposure to PCBs via inhalation has been recognized as a hazard. Those PCBs with lower numbers of chlorine atoms (LC-PCBs) are semivolatile and have been reported in urban air, as well as in the indoor air of older buildings. LC-PCBs are bioactivated to phenols and further to quinone electrophiles with genotoxic/carcinogenic potential. We hypothesized that phenolic LC-PCBs are subject to conjugation and excretion in the urine. PCB3, often present in high concentrations in air, is a prototypical congener for the study of the metabolism and toxicity of LC-PCBs. Our objective was to identify metabolites of PCB3 in urine that could be potentially employed in the estimation of exposure to LC-PCBs. Male Sprague-Dawley rats (150-175 g) were housed in metabolism cages and received a single intraperitoneal injection of 600 μmol/kg body weight of PCB3. Urine was collected every 4 h; rats were euthanized at 36 h; and serum was collected. LC/MS analysis of urine before and after incubation with β-glucuronidase and sulfatase showed that sulfate conjugates were in higher concentrations than glucuronide conjugates and free phenolic forms. At least two major metabolites and two minor metabolites were identified in urine that could be attributed to mercapturic acid metabolites of PCB3. Quantitation by authentic standards confirmed that approximately 3% of the dose was excreted in the urine as sulfates over 36 h, with peak excretion occurring at 10-20 h after exposure. The major metabolites were 4'PCB3sulfate, 3'PCB3 sulfate, 2'PCB3 sulfate, and presumably a catechol sulfate. The serum concentration of 4'PCB3 sulfate was 6.18 ± 2.16 μg/mL. This is the first report that sulfated metabolites of PCBs are formed in vivo. These findings suggest a prospective approach for exposure assessment of LC-PCBs by analysis of phase II metabolites in urine.

Aim: Fimasartan is a selective angiotensin II receptor blocker. Hydrochlorothiazide (HCTZ), which is used to treat hypertension and edematous conditions, is coadministered with many antihypertensive agents., Methods: An open-label, randomized, multiple-dosing, 2-arm, 1-sequence, 2-period study was conducted to assess the effects of fimasartan (240 mg) on HCTZ (25 mg) or vice versa in 18 and 14 healthy male volunteers, respectively. During each drug administration period, drugs were given once daily for 7 days, with a 7-day washout period between the 2 administration periods., Results: The respective geometric mean ratios of fimasartan for AUC τ,ss and C max,ss with HCTZ were 1.30 [90% confidence interval (CI), 0.84-2.01] and 1.17 (90% CI, 0.93-1.47) compared with fimasartan alone. The respective geometric mean ratios of HCTZ for AUC τ,ss and C max,ss with fimasartan were 0.94 (90% CI, 0.84-1.04) and 0.88 (90% CI, 0.80-0.97) compared with HCTZ alone. Plasma renin activity indicated no significant differences between fimasartan monotherapy and coadministered treatment., Conclusions: Fimasartan administered alone or in combination with HCTZ was well tolerated at the described dosages. Coadministration of fimasartan increased the urinary excretion of HCTZ and urine volume, but these observations are unlikely to have any clinical relevance.

Background and Objectives: Fimasartan (BR-A-657) is a novel, non-peptide angiotensin II receptor antagonist with a selective type I receptor blockade effect. Two first-in-human studies investigated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of fimasartan., Methods: Fasted single oral tablet doses of fimasartan 20-480 mg or placebo were administered to 40 healthy male subjects (aged 19-54 years) in a double-blind, randomized, sequential-group design. Subjects receiving fimasartan 240 mg also received the same treatment in the fed state after an interval of 7 days. In another study, oral tablet doses of fimasartan 120 and 360 mg or placebo were given once daily for 7 days to groups of eight fasted healthy male subjects (aged 20-55 years) in a double-blind, randomized, sequential-group design. Safety and tolerability were assessed. The PK and PD of fimasartan were also evaluated and compared for the different doses., Results: Fimasartan was safe and well tolerated, but with an increased incidence of low BP and postural dizziness for the 360 mg dose after repeated administration. Fimasartan produced increases in plasma renin activity, angiotensin I and II, which were not dose dependent. Maximal increases occurred between 6 and 8 hours post-dose, lasting up to 48 hours. Fimasartan was absorbed rapidly after all doses and had a multiphasic distribution. Two peaks in the plasma concentration-time profile were observed in most subjects. Steady state was achieved after three doses, and accumulation was minimal after repeated doses for 7 days (24-30%). The effective half-life ranged from 9.84 to 13.2 hours. The systemic exposure of fimasartan was dose proportional, and no marked food effect was noted after administration of 240 mg in the fed state. Urinary excretion of fimasartan was very low (1.74-2.51%), suggesting non-renal elimination., Conclusion: Fimasartan had a good safety profile and was well tolerated after fasted single oral doses of 20-480 mg, a fed single oral dose of 240 mg, and fasted repeated oral doses of 120 and 360 mg in healthy subjects. In addition, the PK and PD of fimasartan in this population were well characterized. Further studies are needed to evaluate the safety, efficacy, and dose-response relationship of fimasartan in patients with hypertension.

Bacterial bioreporters have substantial potential for contaminant assessment but their real world application is currently impaired by a lack of sensitivity. Here, we exploit the bioconcentration of chemicals in the urine of animals to facilitate pollutant detection. The shore crab Carcinus maenas was exposed to the organic contaminant 2-hydroxybiphenyl, and urine was screened using an Escherichia coli-based luciferase gene (luxAB) reporter assay specific to this compound. Bioassay measurements differentiated between the original contaminant and its metabolites, quantifying bioconcentration factors of up to one hundred-fold in crab urine. Our results reveal the substantial potential of using bacterial bioreporter assays in real-time monitoring of biological matricesto determine exposure histories, with wide ranging potential for the in situ measurement of xenobiotics in risk assessments and epidemiology.

In this work, an SPE-HPLC method coupled to photodiode array detection was validated in human urine matrix, in order to monitor four antihypertensive angiotensin II receptor antagonist drugs in patients under cardiovascular treatment. For that purpose, experimental design was used. Quantitation was accomplished by the internal standard method. The obtained LOQs were 95, 113, 125, and 85 ng/mL for eprosartan, telmisartan, irbesartan, and valsartan, respectively. The intraday and interday precision and accuracy at four concentration levels in the working range (LOQ-15 microg/mL) were always lower than 11% RSD and 8% relative error. The urine samples proved to be stable during 4 h at room temperature, after three thaw-freeze cycles, and for 2 months at -20 degrees C. No interferences from other endogenous compounds or co-administered drugs were found. The method has been successfully applied to monitor the renal elimination of eprosartan and valsartan during 24 h.

Novel polymer monolith microextraction (PMME) using a poly(methacrylic acid-ethylene glycol dimethacrylate) (poly(MAA-EGDMA)) monolith in conjunction with capillary zone electrophoresis (CZE) was developed for the determination of several angiotensin II receptor antagonists (ARA-IIs) in human urine. The extraction device consisted of a regular plastic syringe (1 mL), a poly(MAA-EGDMA) monolithic capillary (2 cm x 530 microm I.D.) and a plastic pinhead connecting the former two components seamlessly. The extraction was achieved by driving the sample solution through the monolithic capillary tube using a syringe infusion pump, and for the desorption step, an aliquot of organic solvent was injected via the monolithic capillary and collected into a vial for subsequent analysis by CZE. The best separation was realized at 25 kV using a buffer that consisted of 50% acetonitrile and 50% buffer solution (v/v) containing 10 mM disodium hydrogenphosphate (adjusted to pH 2.3 with 1M hydrochloric acid). The method was successfully applied to the determination of telmisartan (T), irbesartan (I) and losartan (L) in urine samples with candesartan (C) as internal standard, yielding the detection limit of 15-20 ng/mL. Close correlation coefficients (R>0.999) and excellent method reproducibility were obtained for all the analytes over a linear range of 0.08-3 microg/mL.

Unlabelled: 4,4'-Bis-1-hydroxy-2-(4-methylpiperidin-1-yl)ethyl-biphenyl (A-4), a tertiary amine analog of hemicholinium-3 (HC-3), is an inhibitor of the sodium-dependent high-affinity choline uptake (HACU) system. We have evaluated 4-[1-hydroxy-2-(4-(18)F-fluoromethylpiperidin-1-yl)ethyl]-4'-[1-hydroxy-2-(4-methylpiperidin-1-yl)ethyl]biphenyl ((18)F-FA-4) and 4-[1-hydroxy-2-(4-(11)C-methylpiperazin-1-yl)ethyl]-4'-[1-hydroxy-2-(4-methylpiperidin-1-yl)ethyl]biphenyl ((11)C-pipzA-4), an (18)F- and a (11)C-labeled derivative of A-4 as potential in vivo tracers for the HACU system., Methods: The biodistribution of both compounds was determined in mice, and the intracerebral distribution was visualized by ex vivo and in vitro autoradiography. The in vitro affinity of the compounds was determined by a displacement study with (3)H-HC-3 on mice brain slices., Results: In mice, both tracers show a high and persistent brain uptake. In vitro autoradiography shows binding to the striatum, whereas ex vivo autoradiography shows homogeneous binding throughout the brain. FA-4 and pipzA-4 inhibited the (3)H-HC-3 binding with a 50% inhibitory concentration of 57 nmol/L and 320 nmol/L, respectively., Conclusion: The evaluated compounds have affinity for HACU and show high uptake in the brain. In vitro binding of the compounds to the striatum cannot be inhibited by the presence of HC-3, whereas binding of HC-3 was inhibited by the presence of both FA-4 and pipzA-4, suggesting allosteric binding.

Coadministration of biphenyl and KHCO3 in the diet of male rats for 13 weeks produced urine crystals, which, by means of LC-MS/MS analyses, were determined to be composed of the potassium salt of 4-hydroxy-biphenyl-O-sulfate (4-HBPOSK). Biphenyl alone or biphenyl with KCl or NaHCO3 in the diet did not produce urine crystals. It was found that the higher concentration of potassium in the urine and the alkaline pH induced by feeding KHCO3 to rats resulted in the formation of urine crystals of 4-HBPOSK due to 4-HBPOSK solubility being lower in urine than in plasma. Urine crystals of 4-HBPOSK produced hyperplasia of the transitional epithelium of the ureter, ureteral obstruction, and hydronephrosis in the urinary tract., (Copyright 2001 Academic Press.)

A reversed-phase high performance liquid chromatographic method for simultaneous determination of magnolol and honokiol in serum and urine of rat has been established. Two drugs were determined within 15 minutes by the method on the column with spherisorb C18, by using a mobile phase consisted of methanol-water-glacial acetic acid (70:30:1, V/V) at 1 mL/min, monitored at 294 nm and with a sensitivity of 0.005 AUFS. After 0.25, 1 and 8 hour of administration of the drugs, protein in serum and urine of Wistar rat was precipitated by methanol and magnolol and honokiol in acidified body fluid were determined after being extracted by a mixture of ethyl acetate and ether. Good linear relationship between concentration in serum and urine and peak area in the ranges of 0.05-2 mg/L for magnolol and 0.025-1 mg/L for honokiol was obtained. Good precision and reproducibility were found too. The average recoveries of the two drugs were 95.6% (RSD = 3.85%), 93.8% (RSD = 3.95%) in serum and 96.0% (RSD = 3.83%), 94.9% (RSD = 3.54%) for urine respectively. The lower limit of the method was 0.02 mg/L of magnolol and 0.04 mg/L of honokiol respectively. The results showed that this method is suitable for the determination of magnolol and honokiol in body fluids.

Metabolism and disposition of irbesartan, an angiotensin II AT(1) receptor antagonist, were investigated in mice, rats, rabbits, and macaques. In both rats and macaques, irbesartan was characterized by a rapid oral absorption, a large volume of distribution, a low plasma clearance, and a long terminal half-life. The oral bioavailability in macaques was notably higher than in rats. Irbesartan was highly protein bound in rats and macaques. A lower binding rate was found in mice and rabbits. In distribution studies performed in rats, mice, and rabbits, irbesartan was rapidly distributed into most organs and tissues including brain, intrauterine area, and milk. No retention of radioactivity in tissues other than liver and kidney was noted. Irbesartan was the main circulating compound in rats, rabbits, and macaques representing a maximum of 67, 68, and 80% of plasma radioactivity, respectively. The drug was metabolized mainly by glucuronidation (primarily on the tetrazole ring), hydroxylation, and additional oxidation. The overall pathways within the different species generated 18 metabolites identified from bile, urine, and feces samples. Irbesartan did not significantly induce or inhibit most of the isoenzymes commonly associated with drug metabolism in either rats or macaques after oral administration for 1 month. In most species irbesartan and its metabolites were mainly excreted in feces with more than 80% of a radioactive dose recovered within 24 or 48 h. Enterohepatic circulation was demonstrated in rats and macaques.

Liquid chromatographic methods are described for the determination of a new effective anti-hypertensive drug candesartan (CV-11974), its prodrug candesartan cilexetil (TCV-116) and a metabolite, CV-15959 in human plasma and urine. The assays comprise liquid-liquid extraction and separation on a phenyl column with fluorometric detection. The methods give absolute recoveries of 70, 83 and 78% for candesartan cilexetil, candesartan and CV-15959, respectively, and the limit of quantification is 5, 1 and 3 nM of plasma (RSD < 20%), respectively. The methods were applied to plasma and urine samples from biopharmaceutical and clinical studies in man.

1. The biotransformation of pentachlorophenol (PCP), aniline and biphenyl in rainbow trout (Oncorhynchus mykiss) isolated liver cells was investigated to examine if fish hepatocytes represent a suitable alternative to the in vivo approach for studying the biotransformation of chemicals. Each compound was incubated at two concentrations (10 and 60 microM) for 2 h. For comparison, the metabolic profile of these xenobiotics was also studied in urine and bile of trout orally exposed to 1.8-4.0 mg/kg wet wt of each compound. 2. In vitro as in vivo, PCP glucuronide and to a lesser extent PCP sulphate were the metabolites formed by trout from PCP. 3. Aniline was mainly metabolized to acetanilide and to a lesser extent to 2-aminophenol by isolated hepatocytes, but neither hydroxylated acetanilide nor conjugates were found in vitro whereas they were present in bile and urine of trout treated with this chemical. 4. Trout hepatocytes metabolized biphenyl to hydroxylated and dihydroxylated products and the corresponding glucuronides. These results correlated well with the metabolic profile obtained from the bile of trout exposed to this pesticide. 5. It is concluded that although hepatocytes are well suited for several types of biotransformation studies, the fact that this system may in some cases produce a different metabolic pattern than in vivo should be considered when attempting to extrapolate in vitro to in vivo data.

ortho-Phenylphenol (OPP), a fungicide and antibacterial agent with food residues, is carcinogenic to rat bladder. The present studies provide information on changes in urinary composition and urinary metabolites, urothelial cytotoxicity and regenerative hyperplasia, and DNA adducts in male F344 rats fed OPP. An initial experiment evaluated dietary doses of 0, 1,000, 4,000, and 12,500 ppm OPP fed for 13 weeks. There was no evidence of urinary calculi, microcrystalluria, or calcium phosphate-containing precipitate, but urothelial cytotoxicity and hyperplasia occurred at the highest dose only. In a second experiment, rats were fed dietary OPP levels of 0, 800, 4,000, 8,000, and 12,500 ppm. Urinary pH was > 7 in all groups. Urinary volume was increased at the 2 highest doses with consequent decreases in osmolality, creatinine, and other solutes. Total urinary OPP metabolite excretions were increased, mostly excreted as conjugates of OPP and of phenylhydroquinone. Free OPP or free metabolites accounted for less than 2% excreted in the urine without a dose response. Urothelial toxicity and hyperplasia occurred only at doses of 8,000 and 12,500 ppm. OPP-DNA adducts were not detected in the urothelium at any dose. In summary, OPP produces cytotoxicity and proliferation of the urothelium at dietary doses > or = 8,000 ppm without formation of urinary solids. The paucity of unconjugated metabolites and the lack of OPP-DNA adducts suggests that OPP is acting as a bladder carcinogen in male rats by inducing cytotoxicity and hyperplasia without it or its metabolites directly binding to DNA.

1. Ortho-phenylphenol (OPP) was well absorbed in the male B6C3F1 mouse, with 84 and 98% of the administered radioactivity recovered in the 0-48-h urine of animals administered a single oral dose of 15 or 800 mg/kg respectively. High absorption and rapid elimination were also seen in the female and male F344 rat with 86 and 89% respectively of a single oral dose (27-28 mg/kg) found in the urine in 24 h. OPP was also rapidly eliminated from human volunteers following dermal exposure for 8 h (0.006 mg/kg), with 99% of the absorbed dose in the urine in 48 h. 2. Sulphation of OPP was found to be the major metabolic pathway at low doses in all three species, accounting for 57, 82 and 69% of the urinary radioactivity in the male mouse (15 mg/kg, p.o.), male rat (28 mg/kg, p.o.) and male human volunteers (0.006 mg/kg, dermal). OPP-glucuronide was also present in all species, representing 29, 7 and 4% of the total urinary metabolites in the low dose groups of mouse, rat and human volunteers respectively. 3. Conjugates of 2-phenylhydroquinone (PHQ) in these single-dose studies accounted for 12, 5 and 15% of the dose in mouse, rat and human, respectively. Little or no free OPP was found in any species. No free PHQ or PBQ was found in the mouse, rat or human (LOD = 0.1-0.6%). 4. A novel metabolite, the sulphate conjugate of 2,4'-dihydroxybiphenyl, was identified in rat and man, comprising 3 and 13% of the low dose respectively. 5. Dose-dependent shifts in metabolism were seen in the mouse for conjugation of parent OPP, indicating saturation of the sulphation pathway. Dose-dependent increases in total PHQ were also observed in mouse. 6. This study was initiated to elucidate a mechanistic basis for the difference in carcinogenic potential for OPP between rat and mouse. However, the minor differences seen in the metabolism of OPP in these two species do not appear to account for the differences in urinary bladder toxicity and tumour response between mouse and rat.

The metabolism of irbesartan, a highly selective and potent nonpeptide angiotensin II receptor antagonist, has been investigated in humans. An aliquot of pooled urine from healthy subjects given a 50-mg oral dose of [14C]irbesartan was added as a tracer to urine from healthy subjects that received multiple, 900-mg nonradiolabeled doses of irbesartan. Urinary metabolites were isolated, and structures were elucidated by mass spectroscopy, proton NMR, and high-performance liquid chromatography (HPLC) retention times. Irbesartan and the following eight metabolites were identified in human urine: (1) a tetrazole N2-beta-glucuronide conjugate of irbesartan, (2) a monohydroxylated metabolite resulting from omega-1 oxidation of the butyl side chain, (3, 4) two different monohydroxylated metabolites resulting from oxidation of the spirocyclopentane ring, (5) a diol resulting from omega-1 oxidation of the butyl side chain and oxidation of the spirocyclopentane ring, (6) a keto metabolite resulting from further oxidation of the omega-1 monohydroxy metabolite, (7) a keto-alcohol resulting from further oxidation of the omega-1 hydroxyl of the diol, and (8) a carboxylic acid metabolite resulting from oxidation of the terminal methyl group of the butyl side chain. Biotransformation profiles of pooled urine, feces, and plasma samples from healthy male volunteers given doses of [14C]irbesartan were determined by HPLC. The predominant drug-related component in plasma was irbesartan (76-88% of the plasma radioactivity). None of the metabolites exceeded 9% of the plasma radioactivity. Radioactivity in urine accounted for about 20% of the radiolabeled dose. In urine, irbesartan and its glucuronide each accounted for about 5 to 10% of the urinary radioactivity. The predominant metabolite in urine was the omega-1 hydroxylated metabolite, which constituted about 25% of the urinary radioactivity. In feces, irbesartan was the predominant drug-related component (about 30% of the radioactivity), and the primary metabolites were monohydroxylated metabolites and the carboxylic acid metabolite. Irbesartan and these identified metabolites constituted 90% of the recovered urinary and fecal radioactivity from human subjects given oral doses of [14C]irbesartan.

A sensitive gas chromatographic-mass spectrometric method was developed to quantitate total o-phenylphenol (OPP) (free plus conjugates) in human urine. Conjugates of OPP were acid-hydrolyzed to free OPP, derivatized to the pentafluorobenzoyl ester derivative and analyzed via negative-ion chemical ionization gas chromatography-mass spectrometry. Two stable isotope analogs of OPP were shown to be suitable as internal standards for this method (D2-phenol ring, 13C6-phenyl ring). A synthetic method is presented for the preparation of the D2-OPP internal standard. The 13C6-OPP analog was also shown to be useful as an alternate test material for laboratory-based exposure studies. The limit of quantitation for this method was 1 ng OPP/ml urine. Calibration curves were linear for the analyte over the concentration range of 0.5-1117 ng OPP/ml urine. Relative recovery of OPP from urine ranged from 97.0 to 104.7%. Low levels of OPP (mean=6+/-7 ng/ml; n=22) were found in control human urine samples. The method was validated with urine samples obtained from human volunteers undergoing a dermal exposure study with 12C-/13C6-/14C-OPP. This method was developed to aid in assessments of human exposure to OPP during a variety of uses of the compound.

A sensitive high-performance liquid chromatographic (HPLC) method is described for the determination of a new potent antihypertensive agent, TCV-116, and its two metabolites (M-I and M-II) in human serum or urine. After pre-treatment of the specimens, the analytes were determined using a column switching technique, except for the metabolites in urine which were determined by gradient elution mode HPLC. The quantitation limits for TCV-116, M-I and M-II were all 0.5 ng/ml in serum, and 0.5, 10 and 110 ng/ml in urine, respectively. The methods were applied to clinical trials of TCV-116.

Column-switching HPLC methods have been developed and validated for the determination of a new antihypertensive prodrug, TCV-116 (I), and its metabolites, CV-11974 (II) and CV-15959 (III), in human serum and urine. Initial sample cleanup was achieved by extracting the analytes into an organic solvent. After chromatographing on an ODS column with a mobile phase consisting of acetonitrile and an acidic phosphate buffer, the zone of the analyte's retention was heart-cut onto a second ODS column with a mobile phase of acetonitrile and a phosphate buffer at a higher pH. Complete separation of the analytes and the endogenous peaks was accomplished by the two-dimensional chromatography. Good precision and linearity of the calibration standards, as well as the inter-day and intra-day precision and accuracy of quality control samples, were achieved. The limit of quantitation (LOQ), using 0.5 ml of serum, was 2 ng/ml for I, 0.8 ng/ml for II, and 0.5 ng/ml for III. The LOQ for urine sample was 10 ng/ml for II and III. Stability of the analytes during storage, extraction, and chromatography processes was established. The results illustrate the versatile application of column switching to method development of multiple analytes in various biological matrices. The methods have been successfully used for the analyses of I and its metabolites in thousands of clinical samples to provide pharmacokinetic data.

Saiboku-To is an anti-asthmatic herbal remedy which consists of ten herbal extracts. To investigate the clinical relationship between the effects and chemical components of Saiboku-To, a simple and sensitive high-performance liquid chromatographic method (HPLC) for determination of magnolol, one of the major urinary products, was developed. Organic solvent extraction of urinary magnolol was conducted by diatomaceous earth column rapid-flow fractionation using ethanol/dichloromethane (8/92, v/v). Recovery rates of magnolol were more than 99% with coefficient of variations less than 6% in the concentration range 9.7-970 ng mL-1. Subsequent HPLC determination of magnolol was achieved using a conventional silica-gel column, a mobile phase mixture of acetic acid/diethyl ether/n-hexane (0.2/17.0/82.8, v/v), and a UV-absorption detector set at 290 nm. Calibration was on the basis of peak height ratio between magnolol and flavone as an internal standard. The method was used to demonstrate excretion profiles of magnolol in healthy and asthmatic subjects following single administration of Saiboku-To.

Saiboku-To, a mixture of ten different herbal extracts, has been used in Japan and Czechoslovakia for corticosteroid-dependent severe asthma to reduce the maintenance doses of corticosteroid. Magnolol has been considered to be an active component of Saiboku-To as an inhibitor of 11 beta-hydroxysteroid dehydrogenase and T-lymphocyte proliferation resulting in corticosteroid-sparing. To investigate the relationship between magnolol and the clinical effects of Saiboku-To, urinary magnolol excretion was compared in responders and non-responders under long-term Saiboku-To treatment. The clinical outcome of the Saiboku-To treatment was evaluated in nine asthmatic patients at 52 weeks after the onset of the treatment, using individual fluctuation of asthmatic points obtained from the patients' diary cards. Three patients whose clinical conditions were improved by the treatment were termed responders and six others were termed non-responders. The difference in the amounts of the total magnolol excreted were not significant; however, free (or non-conjugated) amounts of magnolol excreted in the responders were 7 times those in the non-responders (P < 0.05). These results suggest that the magnolol might be responsible for the therapeutic effect of Saiboku-To, indicating practical bioavailability in the responders.

1. The pharmacokinetics and biochemical efficacy of losartan, an orally active nonpeptide angiotensin II (AII) receptor antagonist, were evaluated in healthy male volunteers after single and multiple oral administration. 2. Plasma and urinary concentrations of losartan and its active metabolite, E-3174, were determined by a specific high performance liquid chromatographic (h.p.l.c.) method. 3. Plasma concentrations of losartan were proportional to dose over the range of 25 to 200 mg and the terminal half-lives (t1/2,z) ranged from 1.5 to 2.5 h. The mean values of Cmax and AUC0-infinity increased in a dose-dependent manner. 4. Plasma concentrations of E-3174 were higher than those of losartan at all dose levels. The values of Cmax and AUC0-infinity for E-3174 were approximately 2 and 5-8 times higher than those for losartan, respectively. Also the value of t1/2,z was 2 times longer than that of losartan. 5. After multiple dosing for 7 days, the pharmacokinetics of losartan and E-3174 each did not change significantly between day 1 and day 7. 6. Plasma renin activity (PRA) and plasma concentrations of AII increased markedly at all dose levels. Plasma aldosterone levels were slightly reduced, but a similar decrease was also observed with placebo. 7. No clinically significant adverse reaction was observed in any of the volunteers during either study. Blood counts, routine laboratory tests, urine analyses, and electrocardiograms were also not modified by losartan. 8. Losartan appears to be a potent orally active angiotensin II antagonist with a relatively long duration of action.

A sensitive and selective high-performance liquid chromatographic method for the simultaneous determination of a new angiotensin II receptor blocking agent, losartan (DuP 753, MK-954, I), and its active metabolite, EXP3174 (II), in human plasma or urine is described. The two analytes and internal standard are extracted from plasma and urine at pH 2.5 by liquid-liquid extraction and analyzed on a cyano column with ultraviolet detection at 254 nm. The mobile phase is composed of acetonitrile and phosphate buffer at pH 2.5. The limit of quantification for both compounds in plasma is 5 ng/ml. The limit in urine is 20 and 10 ng/ml for I and II, respectively. The assay described has been successfully applied to samples from pharmacokinetic studies.

A selective thromboxane A2 (TXA2) receptor blocking agent, vapiprost, was orally administered to healthy male Japanese volunteers to investigate the pharmacokinetic and pharmacodynamic properties. The time-profile of vapiprost concentration in plasma was determined and the effects of the drug on platelet aggregation in platelet-rich plasma (PRP) induced by a stable TXA2 receptor agonist U-46619, adenosine diphosphate (ADP) and collagen, and platelet aggregation in whole blood induced by U-46619 ex vivo were simultaneously examined and compared. In the single-dose study (5, 10, and 20 mg/man) the plasma concentrations of the drug were fitted well to a one-compartment open model with a first-order absorption. The area under plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) showed dose-related increases, whereas the mean elimination half-lives (t1/2) remained approximately constant within the range of 0.99-1.1 hour. The drug was hardly recovered unchanged in urine. The platelet aggregation in PRP induced by collagen or U-46619 and the secondary aggregation by ADP were inhibited; that induced by U-46619 was the most specifically and completely inhibited at 2 hours after administration of any dose. The duration for maintaining the significant inhibition tended to depend on the dose and ranged from 24 to 36 hours after administration, which was much longer than expected from the plasma concentration of drug. The time-profile of inhibiting whole blood platelet aggregation that was induced by U-46619 was almost parallel to that of platelet aggregation in PRP by the same aggregant. The bleeding time was slightly prolonged 2 and 8 hours after administrations of 10 and 20 mg, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A study was carried out in a nylon producing plant to determine exposure to Dowtherm A from frequently occurring leaks in the heating system. The daily exposure to both diphenyl and diphenylether of 20 workers was determined over seven consecutive days. Urine samples before and after the daily eight-hour workshift were obtained and analysed spectrofluorimetrically on hydroxydiphenyl. Urine concentration of creatinin was used as a correction factor for dilution. No correlation was found between the increase of urinary hydroxydiphenyl during the workshift and the eight-hour average exposure to diphenyl for the individual workers. This could be explained by the small variance of the daily exposure of each worker. Averaging, however, for each worker both exposure values and increases of urinary hydroxydiphenyl over 7 d, resulted in an explained variance of 72.5% using regression techniques. No influence of smoking was found.

The phenolic metabolites of biphenyl in guinea pigs and rabbits were qualitatively and quantitatively analysed as trimethylsilyl (TMS) ethers by combined gas chromatography/mass spectrometry and gas chromatography, respectively. The parent compound was hydroxylated to monohydroxylated biphenyls and minor amounts of dihydroxylated derivatives, and the main route of body clearance appeared to be by the urine in both species. Thus, in the urine of guinea pigs 32.9% of the dose was detected 96 hrs after dosing, while the major part (29.5%) was eliminated during the first day as conjugates. The main metabolite was 4-hydroxybiphenyl (25.5%). During the first 24 hrs faecal recovery was 20.3% of the dose, and most of this (14.3%) consisted of biphenyl itself. Biliary excretion of the metabolites of biphenyl origin amounted to 3.3% of the dose during the first day, and 4-hydroxybiphenyl was the major metabolite. In the urine of rabbits 49.1% of the dose was recovered 96 hrs after dosing, and most of this (25.4 and 15.9%, respectively) was eliminated during the first two days as conjugates. The major metabolite was 4-hydroxybiphenyl (35.3%). On the first day faecal recovery was 1.6%, of which 1.4% was detected as biphenyl itself. Less than 1% of the dose was found in the 7 hrs rabbit bile, and exclusively as 4-hydroxybiphenyl. The experiments thus show that both qualitative and quantitative differences in the metabolism of biphenyl exist between the guinea pig and the rabbit even though 4-hydroxybiphenyl was the most prominent metabolite of biphenyl in both species.

2-phenylphenol may be applied as active agent in hand disinfectants which have a simultaneous cleaning effect. Such a preparation (Primasept M, Schülke & Mayr GmbH, Norderstedt) which is also suited for the inactivation of Hepatitis B viruses was applied according to prescription and the intake of the active agent by the skin was determined indirectly by the quantity separated in the urine. After 10 minutes successive application of the preparation a mean value of 6.2 mg was observed. Compared with the ADI-value, admitting significantly higher values, no particular risk has to be expected by application of the preparation.

Several mono- and dihydroxy metabolites of di-, tri, and tetrachlorobiphenyl have been identified in the urine of rats fed prolonged diets of Aroclor 1016 or Aroclor 1242. Combined gas chromatography-mass spectrometry was used for characterization of the metabolic products.

The mass spectra and gas chromatographic properties of 17 synthetic fluoro-, chloro- and bromomethoxy-biphenyls and 12 dichlorodimethoxybiphenyls have been examined. From this representative series it appears that the position of the methoxy group (ortho, meta and para to the biphenyl bond) in all monomethoxy compounds examined, and the positions of the two methoxy groups in most of the dimethoxy compounds, can be assigned unambiguously by their difference in fragmentation pattern. The value of this method was shown by metabolism experiments in which 4,4'-difluoro- and 4,4'-dibromobiphenyl were fed to rats and 4,4'-dichlorobiphenyl was administered to plants. All hydroxylated metabolites found were identified by gas chromatography mass spectrometry. Relationships between structure and gas chromatographic retention time of these compounds are discussed.

The identification of the hydroxylated rat urinary metabolites of the 2-, 3- and 4-chlorobiphenyls and 2-, 3- and 4-bromobiphenyls has been determined by gas chromatographic mass spectrometric analysis of their corresponding methyl ether derivatives. The electron impact fragmentation patterns of the bromotheoxybiphenyls and chloromethoxybiphenyls were used to assign the position of the methyoxyl group (ortho, meta or para to the biphenyl bond); the mass spectra of the corresponding [2H5]halobiphenyls confirmed the sites of the hydroxylation by distinguishing between the halophenyl and phenyl rings. The results illustrated that ring hydroxylation occurs predominantly at the para positions of the biphenyl nucleus and at sites which are ortho and para to the halogen substituents. 4,4'-Dimethoxyhalobiphenyls are major urinary metabolites of the 2- and 3-halobiphenyls and the rapid formation of these metabolites is illustrated in a time course study which monitors the urinary metabolites formed after the separate coadministration of the isomeric chlorobiphenyl and bromobiphenyl substrates to rats.

4'Chloro-4-biphenylol, the major metabolite of 4-chlorobiphenyl in the rat, was given intraperitoneally to rats, and the urine and feces were examined for possible metabolic degradation products. The structure of the major urinary metabolite was elucidated by mass and nuclear magnetic resonance spectroscopy and shown to be 4'-chloro-3,4-biphenyldiol. Two chloromethoxbiphenylols (m-4) were also identified in the urine extracts but they could not be separated by chromatographic procedures. Demethylation of the mixture gave 4'-chloro-3,4-biphenyldiol as the sole product, thus indicating that the two components of the mixture were 4'-chloro-3-methoxy-4-biphenylol and 4'-chloro-4-methoxy-3-biphenyldiol. No 4'-chloro-4-biphenylol metabolites were isolated in the fecal extracts, and mass spectrometric analysis of the crude urine and feces extracts did not reveal any chlorine-containing degradation products that could be derived by oxidative fission of the biphenyl nucleus.

Groups of rats were fed biphenyl at various dose levels in a semisynthetic diet and in a commercial chow. The effect levels for induction of polycystic kidney lesions were established by means of urinalysis, organ weight changes, light and electron microscopy, and enzyme histochemistry. The no-effect level, was less than 50 mg/kg bw./day and 300 mg/kg bw./day, when feeding the semisynthetic diet and the commercial chow respectively. This difference in effect level due to the diet is an indication that the diet is of great influence on the results of toxicological experiments.

Brequinar sodium is a 4-quinolinecarboxylic acid analogue that inhibits dihydroorotate dehydrogenase and subsequent de novo pyrimidine biosynthesis. It has shown dose-dependent antineoplastic activity against several mouse and human tumor models. This trial evaluated Brequinar given as a single daily i.v. bolus over a 5-day period repeated every 28 days. One hundred seven courses of treatment at dosages ranging from 36 to 300 mg/m2/day x 5 were administered to 45 patients (31 male and 14 female) with refractory solid tumors; median age was 58 years (range 30-74); median Southwest Oncology Group performance status was 1 (range, 0-3). Thirty patients had prior cytotoxic chemotherapy. Dose-limiting toxicities were thrombocytopenia and a severe desquamative maculopapular dermatitis. Two of 5 good risk patients at 300 mg/m2 and 3 of 6 poor risk patients at 170 mg/m2 developed a platelet count less than 25 x 10(3)/microliters. Two of 5 good risk patients at 300 mg/m2 and 1 of 6 poor risk patients at 170 mg/m2 developed a severe desquamative dermatitis. Moderate to severe mucositis was usually associated with the thrombocytopenia and/or the dermatitis. Nonhematological drug-related toxicities included nausea and vomiting, malaise, anorexia, diarrhea, phlebitis, reversible transaminase elevation, and mucositis. Other hematological toxicities were anemia, granulocytopenia, and leukopenia. There were no drug-related deaths. There were no objective tumor responses. Plasma and urine levels of Brequinar were quantified by high pressure liquid chromatography in 28 patients. Plasma levels and areas under the curve increased proportionally with increased dose. Brequinar had a harmonic mean terminal t1/2 of 8.1 +/- 3.6 h with a model-independent determined apparent volume of distribution at steady state of 9.0 +/- 2.9 liters/m2 and a total body clearance of 19.2 +/- 7.7 ml/min/m2. Renal excretion was a minor route of elimination for Brequinar. The maximally tolerated dose of Brequinar on a daily x 5 i.v. schedule was 250 mg/m2 for good risk patients. For the daily x 5 i.v. schedule, the recommended dose of Brequinar for phase II evaluation is 250 mg/m2 for good risk patients and 135 mg/m2 for poor risk patients.

Dechlorinated products were isolated from the urine of rats that were administered chlorobiphenylols, the primary hydroxylated metabolites of PCB in mammals. The mechanism of chlorine loss from chlorobiphenylols is different from the mechanism of dechlorination via arene oxides whereby concomitant hydroxylation is always observed.

Specific procedures are described for the determination of fenbufen and its metabolites in serum and urine using high-pressure liquid chromatography. Serum or urine extracts were chromatographed on a bounded reversed-phase partitioning column. The sensitivity of the assay for fenbufen was 0.5 microgram/ml in serum with 2-ml samples and 1.0 microgram/ml in urine with 1-ml samples. The procedures are suitable for bioavailability and pharmacokinetic studies.

Urinary N-hydroxy metabolites of the bladder carcinogens, 2-aminofluorene and 4-aminobiphenyl, were examined for the induction of unscheduled DNA synthesis (UDS) in urothelial cells of several susceptible species. N-Hydroxy-2-aminofluorene, N-hydroxy-2-acetylaminofluorene (N-OH-AAF), N-hydroxy-4-aminobiphenyl, N-hydroxy-4-acetylaminobiphenyl, and the N-glucuronides of these two hydroxylamines induced UDS in the urothelial cells of dogs, rats, and rabbits. N-Hydroxy-2-aminonaphthalene, N-hydroxy-2-acetylaminonaphthalene, and the N-glucuronide of the hydroxylamine were not active. The induction of UDS in dog cells by N-OH-AAF or N-acetoxy-2-acetylaminofluorene, but not by N-hydroxy-2-aminofluorene, was inhibited by paraoxon. The microsomal fraction of dog urothelial cells catalyzed the binding of N-OH-AAF to transfer ribonucleic acid; the enzyme activity was completely inhibited by paraoxon, suggesting that N-deacetylase, but not N-,O-acetyltransferase, was responsible for the binding. The O-glucuronide of N-OH-AAF did not induce UDS in the urothelial cells of dogs, rats, or rabbits, nor did it bind to tRNA in the presence of dog urothelial enzymes, which suggest that N-OH-AAF is detoxified by O-glucuronidation. These results are consistent with the hypothesis that nonacetylated, N-hydroxylated metabolites play a major role in arylamine-induced bladder carcinogenesis. The importance of arylacethydroxamic acid metabolites in bladder carcinogenesis for various species may be inversely related to the rate of hepatic O-glucuronidation.