Biomarkers of Exposure to Pyrimethanil After Controlled Human Experiments.

Pyrimethanil (PYM) is a fungicide used pre- and post-harvest on many crops. It has a low acute toxicity but is of toxicological concern because of its antiandrogenic properties. The aim of the current work was to investigate some metabolism and estimate elimination kinetics of PYM in humans after experimental oral and dermal exposure. A liquid chromatography triple quadrupole mass spectrometry (LC-MS-MS) method was developed and validated for the analysis of PYM and its metabolite 4-hydroxypyrimethanil (OH-PYM) in human urine. The method was applied to analyze urine obtained from two volunteers experimentally exposed to PYM. The elimination of OH-PYM seemed to follow first-order kinetics and a two-phase excretion. After the oral exposure, the elimination half-life of OH-PYM in the rapid phase was 5 and 3 h for the female and male volunteer, respectively. In the slower phase, it was 15 h in both volunteers. After the dermal exposure, the half-life in the rapid phase was 8 h in both volunteers. In the slower phase, it was 30 and 20 h, respectively. About 80% of the oral dose was recovered as urinary OH-PYM in both volunteers. The dermal dose recovered as urinary OH-PYM was 9.4% and 19%, in the female and male volunteer, respectively. OH-PYM was mainly found as a conjugate of sulfonate and glucuronic acid. No free PYM was found. The analytical method showed good within-run, between-run and between-batch precision with a coefficient of variation between 6% and 12%. A limit of detection of 0.1 ng/mL and a limit of quantification of 0.4 ng/mL were achieved for both the analytes. The method was applied to biomonitor PYM exposure in populations in Sweden. OH-PYM was detected in nearly 50% and 96% of samples from the environmentally and occupationally exposed populations, respectively.

Unexpected, ubiquitous exposure of pregnant Brazilian women to diisopentyl phthalate, one of the most potent antiandrogenic phthalates.

BACKGROUND: Human exposure to phthalates and other non-persistent chemicals in developing countries is largely unknown. A preliminary analysis of urinary samples from pregnant Brazilian women revealed the presence of metabolites of Diisopentyl phthalate (DiPeP). OBJECTIVES: Reliably quantify DiPeP metabolites in human urine and investigate the potential antiandrogenic activity of this phthalate in rats. METHODS: We initiated a pilot pregnancy cohort in Curitiba, Brazil, to examine phthalate exposure in urine samples collected in early pregnancy (n = 50) or pooled samples from early, mid and late pregnancy (n = 44). Our well established phthalate method was modified to include the primary DiPeP metabolite, monoisopentyl phthalate (MiPeP), and two additional secondary oxidized metabolites, 3OH-MiPeP and 4OH-MiPeP. In a parallel approach, we orally exposed pregnant rats to DiPeP or Di-n-butyl phthalate (DnBP; reference phthalate) at 0, 125, 250, and 500 mg/kg/day from gestation day 14 to 18 and measured ex vivo fetal testis testosterone production. RESULTS: We were able to detect and quantify specific DiPeP metabolites in nearly all (98%) of the early pregnancy urine samples and in all gestational pool samples with a median concentration for MiPeP of 3.65 and 3.15 µg/L, respectively, and for the two oxidized metabolites between 1.00 and 1.70 µg/L. All three urinary DiPeP metabolites were strongly correlated (r = 0.89 to 0.99). In the rat model, the effective dose (mg/kg/day) inhibiting fetal testosterone production by 50% (ED50 [95% confidence interval]) was 93.6 [62.9-139.3] for DiPeP which was significantly lower than for DnBP (220.3 [172.9-280.7]), highlighting the strong antiandrogenic potency of DiPeP within the spectrum of the phthalates. CONCLUSIONS: We unveiled and confirmed the exposure of pregnant Brazilian women to DiPeP via specific urinary metabolites. This unexpected and ubiquitous DiPeP exposure indicates to unique DiPeP exposure sources in Brazil. These exposures spark considerable concern because DiPeP is one of the most potent antiandrogenic phthalates.

Analysis of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutylanilide (DTMBA) as a new potential biomarker of exposure to vinclozolin in urine.

Vinclozolin (V) is a fungicide with anti-androgenic properties whose metabolism is not fully understood, and data on urinary elimination of either V or its metabolites are limited. Therefore the kinetics of urinary elimination of V and its metabolites, after an oral dose in adult male rats were investigated. A single oral dose of V (100 mg/kg) suspended in corn oil was administered to male adult Wistar rats, and urine was collected at different times after dosing. V and its metabolites were extracted from urine, then enzymatically hydrolyzed using ß-glucuronidase/sulfatase of H. pomatia, and analyzed by HPLC/DAD. Urinary pharmacokinetic parameters were calculated using the analyte concentrations adjusted by creatinine levels. V and its metabolites 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutylanilide (DTMBA, formerly denoted as M5), 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1), 3,5-dichloroaniline (M3), and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) were efficiently detected. The mean urine concentrations of V and M1 metabolite were fitted to a two-compartmental model for pharmacokinetic analysis. DTMBA approximately represented 88% of the total excreted metabolites, it was easily detected up to 168 h after dosing and its half-lives were 21.5 and 74.1 h, respectively. M1 was the second most abundant metabolite and was detected up to 144 h after being void. V and M3 were detected before 48 h, and M2 exhibited the lowest levels during the first 8 h after dosing. DTMBA, the most abundant V metabolite is quickly eliminated by urine, it is chemically stable, specific and could represent a useful alternative to be used as a biomarker of exposure to V.

Selective androgen receptor modulators: comparative excretion study of bicalutamide in bovine urine and faeces.

Besides their development for therapeutic purposes, non-steroidal selective androgen receptor modulators (non-steroidal SARMs) are also known to impact growth-associated pathways as ligands of androgenic receptors (AR). They present a potential for abuse in sports and food-producing animals as an interesting alternative to anabolic androgenic steroids (AAS). These compounds are easily available and could therefore be (mis)used in livestock production as growth promoters. To prevent such practices, dedicated analytical strategies should be developed for specific and sensitive detection of these compounds in biological matrices. The present study focused on Bicalutamide, a non-steroidal SARM used in human treatment of non-metastatic prostate cancer because of its anti-androgenic activity exhibiting no anti-anabolic effects. To select the most appropriate matrix to be used for control purposes, different animal matrices (urine and faeces) have been investigated and SARM metabolism studied to highlight relevant metabolites of such treatments and establish associated detection time windows. The aim of this work was thus to compare the urinary and faecal eliminations of bicalutamide in a calf, and investigate phase I and II metabolites. The results in both matrices showed that bicalutamide was very rapidly and mainly excreted under its free form. The concentration levels were observed as higher in faeces (ppm) than urine (ppb); although both matrices were assessed as suitable for residue control. The metabolites found were consistent with hydroxylation (phase I reaction) combined or not with glucuronidation and sulfation (phase II reactions). Copyright © 2016 John Wiley & Sons, Ltd.

Development and assessment of a novel Arxula adeninivorans androgen screen (A-YAS) assay and its application in analysis of cattle urine.

The novel A-YAS assay for the detection of androgenic activity in liquid samples such as urine has been developed and assessed. The assay is based on transgenic Arxula adeninivorans yeast cells as the bio-component. The cells were engineered to co-express the human androgen receptor (hAR) gene and the inducible phytase reporter gene (phyK, derived from Klebsiella sp. ASR1), under the control of an Arxula derived glucoamylase (GAA) promoter, which had been modified by the insertion of hormone-responsive elements (HREs). The Arxula transformation/expression platform Xplor®2 was used to select stable mitotic resistance marker free transformants and the most suitable cells were characterized for performance as a sensor bio-component. The assay is easy-to-use, fast (6-25 h) and is currently the most sensitive yeast-based androgen screen with an EC50, limit of detection and of quantification values for 5α-dihydrotestosterone (DHT) of 277.1±53.0, 56.5±4.1 and 76.5±6.7 ng L(-1), respectively. Furthermore, the assay allows the determination of androgenic and anti-androgenic activity of various compounds such as naturally occurring androgens and estrogens, pharmaceuticals and biocides. The robustness of the A-YAS assay enables it to be used for analysis of complex samples such as urine. The results of the analysis of a number of cattle urine samples achieved by the A-YAS assay correlate well with GC-MS analysis of the same samples.

Dual-color bioluminescent bioreporter for forensic analysis: evidence of androgenic and anti-androgenic activity of illicit drugs.

Bioassays represent promising complementary techniques to conventional analytical approaches used in doping analysis to detect illicit drugs like anabolic-androgenic steroids (AAS). The fact that all AAS share a common mechanism of action via the human androgen receptor (hAR) enables the use of bioassays, relying on the activation of hAR as antidoping screening tools. Previously, we developed a dual-color bioreporter based on yeast cells engineered to express hAR and androgen response elements driving the expression of the bioluminescent (BL) reporter protein Photinus pyralis luciferase. A second reporter protein, the red-emitting luciferase PpyRE8, was introduced in the bioreporter as internal viability control. Here, we report the first forensic application of a straightforward, accurate, and cost-effective bioassay, relying on spectral resolution of the two BL signals, in 96-microwell format. The bioreporter responds to dihydrotestosterone as reference androgen in a concentration-dependent manner from 0.08 to 1,000 nM with intra- and inter-assay variation coefficients of 11.4 % and 13.1 %, respectively. We also demonstrated the suitability of this dual-color bioreporter to assess (anti)-androgenic activity of pure AAS, mixtures of AAS, and other illicit drugs provided by the Scientific Police. Significant anti-androgenic activity was observed in samples labeled as marijuana and hashish, containing Δ(9)-tetrahydrocannabinol as major constituent.

Screening for two selective androgen receptor modulators using gas chromatography-mass spectrometry in doping control analysis.

Selective androgen receptor modulators (SARMs) have become a major field of clinical research enabling the tissue-selective stimulation of androgen receptors. The treatment of debilitating diseases, osteoporosis and frailty are primary goals and promising results have been obtained from clinical trials. However, the potential for misuse of SARMs in sport is great and drug testing methods based on liquid chromatography were established for different classes including arylpropionamide-, 2-quinolinone- and bicyclic hydantoin-derived compounds. As gas chromatography and mass spectrometry (GC-MS) are still important analytical tools in sports drug testing, a method to determine 2-quinolinone- and bicyclic hydantoin-derived SARMs established. Spiked urine samples were subjected to routine doping control protocols including enzymatic hydrolysis, liquid-liquid extraction, concentration and derivatisation to trimethylsilylated analogues followed by GC-MS analysis. The method was validated for the items specificity, lower limit of detection (0.2-10 ng mL(-1)), recovery (83-85%), intraday and interday precision (9-15% and 13-18%, respectively), which demonstrates the suitability of conventional GC-MS systems to determine representatives of an emerging class of compounds in doping control specimens.

Doping control analysis of tricyclic tetrahydroquinoline-derived selective androgen receptor modulators using liquid chromatography/electrospray ionization tandem mass spectrometry.

Selective androgen receptor modulators represent an emerging class of therapeutics to counteract various diseases such as osteoporosis and muscle wasting. Numerous drug candidates have been developed and investigated including a group that comprises a tricyclic tetrahydroquinoline nucleus such as 2-methyl-2-(8-nitro-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]chinolin-4-yl)propan-1-ol. Due to their novelty and medicinal purpose, these compounds also possess great potential for misuse in sports, and studies on the mass spectrometric behavior of three synthesized model substances and drug candidates were conducted to provide information on typical dissociation pathways following electrospray ionization and collision-induced dissociation. Product ion mass spectra derived from protonated molecules were studied using high resolution/high accuracy orbitrap mass spectrometry, and characteristic fragmentation routes and product ions were elucidated. Major and general findings include the elimination of a hydroxyl radical from [M+H](+), the elimination of the 2-substituted side chain, and the gas-phase rearrangement of the investigated tricyclic tetrahydroquinolines to 6-nitroquinoline yielding a common product ion at m/z 175. Knowledge of these dissociation pathways supports the identification of related substances as well as metabolic products, which is of utmost importance to drug testing laboratories. The compounds were implemented into existing screening procedures, and detection limits (0.2-0.6 ng/mL), recoveries (92-97%), and intraday and interday precision (<22%) were evaluated.

Mass spectrometry of selective androgen receptor modulators.

Nonsteroidal selective androgen receptor modulators (SARMs) are an emerging class of drugs for treatment of various diseases including osteoporosis and muscle wasting as well as the correction of age-related functional decline such as muscle strength and power. Several SARMs, which have advanced to preclinical and clinical trials, are composed of diverse chemical structures including arylpropionamide-, bicyclic hydantoin-, quinoline-, and tetrahydroquinoline-derived nuclei. Since January 2008, SARMs have been categorized as anabolic agents and prohibited by the World Anti-Doping Agency (WADA). Suitable detection methods for these low-molecular weight drugs were based on mass spectrometric approaches, which necessitated the elucidation of dissociation pathways in order to characterize and identify the target analytes in doping control samples as well as potential metabolic products and synthetic analogs. Fragmentation patterns of representatives of each category of SARMs after electrospray ionization (ESI) and collision-induced dissociation (CID) as well as electron ionization (EI) are summarized. The complexity and structural heterogeneity of these drugs is a daunting challenge for detection methods.

Determination of benzimidazole- and bicyclic hydantoin-derived selective androgen receptor antagonists and agonists in human urine using LC-MS/MS.

Selective androgen receptor modulators (SARMs) represent a novel class of drugs with tissue-specific agonistic and antagonistic properties, which are prohibited in sports from January 2008 according to the World Anti-Doping Agency. Preventive approaches to restrict the use of SARMs include early implementation of target analytes into doping control screening assays. Five model SARMs were synthesized, four of which are analogs to prostate-specific androgen receptor antagonists with a 5,6-dichloro-benzimidazole nucleus. The fifth SARM is a muscle-tissue specific agonist with a bicyclic hydantoin structure (BMS-564929). Dissociation pathways after negative electrospray ionization were studied using an LTQ-Orbitrap mass analyzer, and diagnostic product ions and common fragmentation patterns were employed to establish a screening procedure that target the intact SARMs as well as putative metabolic products. Sample preparation based on solid-phase extraction and subsequent LC-MS/MS measurement allowed for detection limits of 1-20 ng/mL, intra- and interday precisions of between 2.4 and 13.2% and between 6.5 and 24.2%, respectively. Recoveries varied from 89 to 106%, and tests for ion suppression or enhancement effects were negative for all analytes. [figure: see text]

Screening for 2-quinolinone-derived selective androgen receptor agonists in doping control analysis.

Selective androgen receptor modulators (SARMs) represent a class of emerging drugs with high potential for misuse in sports, and therefore members of this group are banned as anabolic agents by the World Anti-Doping Agency. Preventive approaches to restrict their use include early implementation of target analytes into doping control screening assays and evaluation of the mass spectrometric behavior of these drugs to allow their unequivocal identification as well as the characterization of structurally related compounds and metabolic products. Four model SARMs with the 6-alkylamino-2-quinolinone structure, including the advanced drug candidate LGD-2226, were synthesized. Fragmentation pathways after positive electrospray ionization and collision-induced dissociation were studied using an LTQ Orbitrap mass analyzer, and diagnostic product ions and common dissociation pathways were employed to establish a screening procedure targeting intact quinolinone-based SARMs as well as putative metabolic products such as dealkylated analogues. Therefore, features of a triple quadrupole mass analyzer such as multiple reaction monitoring and precursor ion scanning were utilized. Sample preparation based on commonly employed liquid-liquid extraction and subsequent liquid chromatographic/tandem mass spectrometric measurement allowed for detection limits of 0.01-0.2 ng/mL, and intra- and interday precisions between 3.2 and 8.5% and between 6.3 and 16.6%, respectively. Recoveries varied from 81 to 98%, and tests for ion suppression or enhancement effects were negative for all analytes.

NMR analysis of male fathead minnow urinary metabolites: a potential approach for studying impacts of chemical exposures.

The potential for profiling metabolites in urine from male fathead minnows (Pimephales promelas) to assess chemical exposures was explored using nuclear magnetic resonance (NMR) spectroscopy. Both one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy was used for the assignment of metabolites in urine from unexposed fish. Because fathead minnow urine is dilute, we lyophilized these samples prior to analysis. Furthermore, 1D 1H NMR spectra of unlyophilized urine from unexposed male fathead minnow and Sprague-Dawley rat were acquired to qualitatively compare rat and fish metabolite profiles and to provide an estimate of the total urinary metabolite pool concentration difference. As a small proof-of-concept study, lyophilized urine samples from male fathead minnows exposed to three different concentrations of the antiandrogen vinclozolin were analyzed by 1D 1H NMR to assess exposure-induced changes. Through a combination of principal components analysis (PCA) and measurements of 1H NMR peak intensities, several metabolites were identified as changing with statistical significance in response to exposure. Among those changes occurring in response to exposure to the highest concentration (450 microg/L) of vinclozolin were large increases in taurine, lactate, acetate, and formate. These increases coincided with a marked decrease in hippurate, a combination potentially indicative of hepatotoxicity. The results of these investigations clearly demonstrate the potential utility of an NMR-based approach for assessing chemical exposures in male fathead minnow, using urine collected from individual fish.

Flutamide metabolism in four different species in vitro and identification of flutamide metabolites in human patient urine by high performance liquid chromatography/tandem mass spectrometry.

A new metabolic scheme of flutamide is proposed in this article. Some patients treated with flutamide, a nonsteroidal antiandrogen, have developed severe hepatic dysfunction. Toxic metabolites have been proposed to be responsible for these negative effects. In this study, the qualitative aspects of the in vitro metabolism of flutamide in liver microsomes from human, dog, pig, and rat were evaluated. A direct comparison of the flutamide metabolism in liver and prostate microsomes from pig was made, and the in vivo metabolism of flutamide was investigated in urine from orally treated prostate cancer patients. Liquid chromatography/tandem mass spectrometry was used for analysis. The mass spectrometer was equipped with an electrospray interface and operated in the negative ion mode. In liver microsomes from pig, dog, and rat, extensive hydroxylation of flutamide occurred. One, two, or three hydroxy groups were attached, and isomeric forms were detected for both monohydroxylated and trihydroxylated drug. In pig liver microsomes, isomers of a third metabolite, hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, were also found after incubation with either flutamide or 2-hydroxyflutamide. In human liver microsomes, the pharmacologically active 2-hydroxyflutamide was the only metabolite detected. Several phase I metabolites as well as four intact phase II metabolites could be recovered from the urine samples. For the first time in humans, glucuronic acid conjugates of hydroxylated 4-nitro-3-(trifluoromethyl)-aniline, and mono- and dihydroxylated flutamide were identified, together with hydroxylated 4-nitro-3-(trifluoromethyl)-aniline conjugated with sulfate. In addition, one mercapturic acid conjugate of hydroxylated flutamide, probably formed from flutamide via a reactive intermediate, was detected.

Detection of a new N-oxidized metabolite of flutamide, N-[4-nitro-3-(trifluoromethyl)phenyl]hydroxylamine, in human liver microsomes and urine of prostate cancer patients.

Flutamide (2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]-propanamide), a nonsteroidal antiandrogen, is used in the treatment of prostate cancer but is occasionally associated with hepatic dysfunction. In the present study, the metabolism of flutamide including the formation of the possible reactive toxic metabolites was investigated using human liver microsomes and 10 isoforms of recombinant human cytochrome P450 (P450). 2-Hydroxyflutamide (OH-flutamide) and 4-nitro-3-(trifluoromethyl)phenylamine (FLU-1) were the main products of flutamide metabolism in human liver microsomes. The formation of OH-flutamide was markedly inhibited by ellipticine, an inhibitor of CYP1A1/1A2, and was mainly catalyzed by the recombinant CYP1A2. FLU-1 was also produced from OH-flutamide, but its metabolic rate was much less than that from flutamide. An inhibitor of carboxylesterase, bis-(p-nitrophenyl)phosphoric acid, completely inhibited the formation of FLU-1 from flutamide in human liver microsomes. A new metabolite, N-[4-nitro-3-(trifluoromethyl)phenyl]hydroxylamine (FLU-1-N-OH), was detected as a product of the reaction of FLU-1 with human liver microsomes and identified by comparison with the synthetic standard. The formation of FLU-1-N-OH was markedly inhibited by the addition of miconazole, an inhibitor of CYP3A4, and was mediated by recombinant CYP3A4. Furthermore, FLU-1-N-OH was detected mostly as the conjugates (glucuronide/sulfate) in the urine of prostate cancer patients collected for 3 h after treatment with flutamide. The formation of FLU-1-N-OH, however, did not differ between patients with and without abnormalities of hepatic functions among a total of 29 patients. The lack of an apparent association of the urinary excretion of FLU-1-N-OH and hepatic disorder may suggest the involvement of an additional unknown factor in the mechanisms of flutamide hepatotoxicity.

Metabolism of osaterone acetate in dogs and humans.

Osaterone acetate (17 alpha-acetoxy-6-chloro-2-oxa-4,6-pregnadiene-3,20-dione; OA) is a steroidal antiandrogen. In order to clarify the species differences, metabolites of OA were examined in plasma, urine, and feces of dogs and humans after oral administration of OA. Eleven metabolites in plasma, urine, and feces were identified by their spectral properties and comparison to appropriate standards. The primary routes of OA metabolism involve 11 beta-, 15 beta- and 21-hydroxylation, 17 alpha-deacetylation, and dechlorination. Other metabolites arise from combinations of these pathways to form multiple oxidized metabolites. All metabolites observed in humans occurred in dogs. 11 beta-Hydroxylated metabolites (11 beta-OH OA and 11-oxo OA) were found in the plasma and urine of dogs, but there was no evidence of their presence in humans. 11 beta-Hydroxylation of exogenous steroids represents a distinctive biotransformation pathway.