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Objective: To develop a method for the analysis of phenylglyoxylic acid (PGA) and mandelic acid (MA) in urine by ultra-high performance liquid chromatography tandem mass spectrometry. Methods: The study was conducted in April 2022. Urine samples were directly diluted with the initial mobile phase, separated by Waters HSS T3 column after passing through the membrane, and analyzed under negative ionization mode (ESI(-)) and multiple reaction monitoring (MRM) conditions, the contents of PGA and MA in human urine were quantitatively determined by external standard method. Results: The determination of PGA and MA showed a good linear relationship within the range of 10-1000 ng/ml, with a correlation coefficient of 0.9999. The linear regression equation of PGA was y =1141.4 x +2157.3, the detection limit and lower limit of quantification of the method were 0.081 ng/ml and 0.269 ng/ml, and the recovery rate was 90.47%-99.83%. The linear regression equation of MA was y =62.8 x +140.3, the detection limit and lower limit of quantification of the method were 0.551 ng/ml and 1.836 ng/ml, and the recovery rate was 92.75%-101.09%. The intra and inter batch precision of PGA and MA were both<5%. Conclusion: An ultra-high performance liquid chromatography tandem mass spectrometry method for the analysis of PGA and MA in urine was established.The sample pretreatment operation is simple, and the accuracy and precision of the method meet the standard requirements. It can be used for monitoring and evaluating PGA and MA in urine of the general population and occupational contact population.

Industrial pollution and harmful chemicals seriously affect environment and human health. Styrene is a common air toxicant with widespread exposure sources, including smoking, automobile exhaust, and plastic pollutants. Phenylglyoxylic acid (PGA) is a typical biomarker for exposed styrene. Therefore, it is crucial to quickly identify and quantitatively detect PGA. Herein, an ultrastable terbium metal-organic framework (Tb-MOF 1 ) was developed, and the luminescence film ( 1 /PLA) consisting of polylactic acid (PLA) and 1 was fabricated as a sensor for rapid detection of PGA. The sensor possesses the advantages of efficient detection [limit of detection (LOD) is 1.05 × 10 -4 mg/mL] and rapid response speed (less than 10 s) for PGA in urine. Furthermore, this sensor exhibits high stability, outstanding anti-interference ability, and excellent recyclability. Based on this film technology, a paper-based probe was then developed for portable and convenient detection. The probe could easily distinguish different concentrations of PGA under the naked eye toward practical sensing applications. Meanwhile, photoinduced electron transfer was demonstrated to be responsible for the luminescence sensing. Hence, this study indicates that Tb-MOF is a promising material to detect PGA for evaluating the effect of styrene on the body.

Background and Objectives: Exposure to environmental chemicals has been recognized as one of the possible contributors to CKD. We aimed to identify environmental chemicals that are associated with CKD., Design, Setting, Participants, & Measurements: We analyzed the data obtained from a total of 46,748 adults who participated in the National Health and Nutrition Examination Survey (1999-2016). Associations of chemicals measured in urine or blood ( n =262) with albuminuria (urine albumin-to-creatinine ratio ≥30 mg/g), reduced eGFR (<60 ml/min per 1.73 m 2 ), and a composite of albuminuria or reduced eGFR were tested and validated using the environment-wide association study approach., Results: Among 262 environmental chemicals, seven (3%) chemicals showed significant associations with increased risk of albuminuria, reduced eGFR, or the composite outcome. These chemicals included metals and other chemicals that have not previously been associated with CKD. Serum and urine cotinines, blood 2,5-dimethylfuran (a volatile organic compound), and blood cadmium were associated with albuminuria. Blood lead and cadmium were associated with reduced eGFR. Blood cadmium and lead and three volatile compounds (blood 2,5-dimethylfuran, blood furan, and urinary phenylglyoxylic acid) were associated with the composite outcome. A total of 23 chemicals, including serum perfluorooctanoic acid, seven urinary metals, three urinary arsenics, urinary nitrate and thiocyanate, three urinary polycyclic aromatic hydrocarbons, and seven volatile organic compounds, were associated with lower risks of one or more manifestations of CKD., Conclusions: A number of chemicals were identified as potential risk factors for CKD among the general population., (Copyright © 2020 by the American Society of Nephrology.)

According to class M2.1 of the World Anti-Doping Agency (WADA) Prohibited List, the manipulation of doping control urine samples to alter their integrity and validity is prohibited both in- and out-of-competition. However, some paraplegic athletes with an overactive bladder need to be regularly treated with anti-cholinergic and anti-spasmodic drugs such as oxybutynin, which are often administered intravesically to reduce the substantial side effects observed after oral application. So far, it remains unclear whether such bladder instillations have a negative impact on analytical procedures and thus represent an anti-doping rule violation. Within this pilot study, urine samples were collected from five paraplegic athletes before and after an intravesical oxybutynin hydrochloride instillation. The samples were routinely tested for the presence of performance-enhancing drugs and afterwards fortified with 25 model compounds representing different classes of doping agents (anabolic agents, cannabinoids, diuretics, glucocorticoids, hormone and metabolic modulators, and stimulants) at low and medium concentrations. Additionally, the pH value and specific gravity were measured and the presence of oxybutynin was qualitatively determined by gas chromatography-mass spectrometry (GC-MS). In initial testing procedures, all samples were tested negative. Oxybutynin was present in most of the samples but found to have no significant effect on the detectability of the 25 model compounds subsequently added to each urine specimen. Therefore, it can be concluded that intravesical instillations with oxybutynin hydrochloride do not alter the integrity and validity of doping control urine samples., (© 2019 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

In this study, two hybrid metal organic frameworks including MOF-5@ Fe 3 O 4 -NH 2 and MOF-5@ SBA-15 for the first time were synthetized and combined with microextraction by packed sorbent (MEPS) to extract of mandelic acid (MA) from urine samples. The synthetized sorbents were characterized using FE-SEM, XRD and FT-IR techniques. The important parameters in MEPS procedure including sample volume, extraction draw&#95;discard cycles, elution solvent volume and desorption draw-eject cycles were optimized using response surface methodology (RSM) with central composite design (CCD). The results indicated that the volume of elution solvent was the most important parameter in the recovery of MA by MOF-MEPS procedure. The optimized MOF-MEPS method offered an acceptable efficiency for the recovery of MA from urine samples (MOF-5@Fe 3 O 4 -NH 2 and MOF-5@SBA-15: 94.5% and 90.3%, respectively, RSD < 3.54%). The limit of detection (LOD) of MA calculated by MOF-MEPS procedure combined with high performance liquid chromatography for MOF-5@ Fe 3 O 4 -NH 2 and MOF-5@ SBA-15 were calculated to be 0.10 and 0.13 μg mL -1 , respectively. The linearity dynamic ranges (LDRs) determination of urinary MA by MOF-5@ Fe 3 O 4 -NH 2 and MOF-5@ SBA-15 were 0.2-100 and 0.2-90 μg mL -1 , respectively. The results of the present study implied that the proposed technique is a fast and sensitive procedure for extraction and determination of MA from urine samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ethylbenzene and styrene are air toxicants with widespread nonoccupational exposure sources, including tobacco smoke and diet. Ethylbenzene and styrene (EB/S) exposure was quantified from their common metabolites measured in spot urine samples obtained from participants (≥6 years old) in the 2005-2006 and 2011-2012 cycles of the National Health and Nutrition Examination Survey (NHANES; N = 4690). EB/S metabolites mandelic acid (MA) and phenylglyoxylic acid (PGA) were measured using ultra-high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). MA and PGA were detected in 98.9% and 90.6% of tested urine specimens, respectively. Exclusive smokers had 2-fold and 1.6-fold higher median urinary MA and PGA, respectively, compared with non-users. Sampleweighted regression analysis among exclusive smokers showed that smoking 0.5 pack cigarettes per day significantly increased MA (+97.9 μg/L) and PGA (+69.3 μg/L), controlling for potential confounders. In comparison, exposure from the median daily dietary intake of grain products increased MA by 1.95 μg/L and was not associated with statistically significant changes in urinary PGA levels. Conversely, consuming vegetables and fruit was associated with decreased MA and PGA. These results confirm tobacco smoke as a major source of ethylbenzene and styrene exposure for the general U.S. population., (Published by Elsevier Inc.)

Plastic injection industry workers are exposed to toxic gases and vapors, including styrene. This study aimed to measure exposure to styrene and its relation with urine mandelic acid among plastics injection workers of the electrical parts industry. This descriptive and analytical cross-sectional study was carried out in the plastic injection halls of the electronics industry, in winter 2017 and spring 2018. Styrene gas in the workers' respiratory region was sampled by the NIOSH 1501 method and was analyzed by gas chromatography-mass spectrometry (GC/MAS). Mandelic acid concentration was determined by high-performance liquid chromatography (HPLC). Statistical data analysis was performed with STATA11. The mean of age and working experience in the population under study were 32.4 ± 8.1 and 6.4 ± 5 years, respectively. The average exposure to styrene was 83.2 ± 32.4 mg·m -3 and the mean of urine mandelic acid was 1570.1 ± 720.6 mg·g ceratinine -1 . There were 24 workers (45.3%) exposed to levels above permissible limits recommended by national and international organizations. There was a positive and significant correlation between exposure to styrene and urine mandelic acid (P = 0.006, r = 0.4). In multivariate regression, occupational exposure to styrene (P = 0.002, β = 0.5) was the strongest variable, predicting the amount of urine mandelic acid. Increased occupational exposure to styrene increases mandelic acid in the urine, and applying control measures to reduce exposure to styrene vapor is recommended in high exposure situations.

Introduction: High styrene exposures are still experienced in various occupational settings, requesting regular exposure assessments. The aims of this study were to study occupational exposures in various industrial sectors and to determine factors influencing styrene urinary metabolites levels., Methods: Biomonitoring was conducted in 141 workers from fiberglass-reinforced plastic (FRP) manufacture, thermoplastic polymers production, vehicle repair shops and cured-in-place pipe lining (CIPP). Urinary styrene (StyU) as well as Mandelic (MA) / Phenyglyoxylic Acids (PGA) were quantified at the beginning and at the end of week, and multivariate linear regression models were used., Results: StyU levels revealed very low, rarely exceeding 3 μg.L -1 . Highest concentrations of MA + PGA were observed in FRP sector, with levels reaching up to 1100 mg.g -1 of creatinine. Factors influencing end-of-week MA + PGA concentrations were levels at the beginning of week, open molding processes, proximity to the emission source, respiratory protection, styrene content in raw materials. Elevated levels were also observed during CIPP process, whereas thermoplastic injection and vehicle repair shop workers exhibited much lower exposures., Conclusions: Intervention on process (decreasing styrene proportion, using closed molding), protective equipment (local exhaust ventilation, respiratory protection) and individual practices (stringent safety rules) are expected to decrease occupational exposures. Urinary MA + PGA remain the most appropriate biomarkers for occupational biomonitoring., (Copyright © 2018 Elsevier B.V. All rights reserved.)

1. Oxybutynin hydrochloride is an antimuscarinic agent prescribed to patients with an overactive bladder (OAB) and symptoms of urinary urge incontinence. Oxybutynin undergoes pre-systemic metabolism, and the N-desethyloxybutynin (Oxy-DE), is reported to have similar anticholinergic effects. 2. We revisited the oxidative metabolic fate of oxybutynin by liquid chromatography-tandem mass spectrometry analysis of incubations with rat and human liver fractions, and by measuring plasma and urine samples collected after oral administration of oxybutynin in rats. This investigation highlighted that not only N-deethylation but also N-oxidation participates in the clearance of oxybutynin after oral administration. 3. A new metabolic scheme for oxybutynin was delineated, identifying three distinct oxidative metabolic pathways: N-deethylation (Oxy-DE) followed by the oxidation of the secondary amine function to form the hydroxylamine (Oxy-HA), N-oxidation (Oxy-NO) followed by rearrangement of the tertiary propargylamine N-oxide moiety (Oxy-EK), and hydroxylation on the cyclohexyl ring. 4. The functional activity of Oxy-EK was investigated on the muscarinic receptors (M 1-3 ) demonstrating its lack of antimuscarinic activity. 5. Despite the presence of the α,β-unsaturated function, Oxy-EK does not react with glutathione indicating that in the clearance of oxybutynin no reactive and potentially toxic metabolites were formed.

Mandelic acid (MA) is a major metabolite of ethylbenzene and styrene. For the first time, a selective, fast, and easy-to-use procedure was developed for the determination of MA in urine samples. The new procedure is based on MIMEPS, the combination of a molecularly imprinted polymer (MIP) and microextraction by packed sorbent (MEPS). High-performance liquid chromatography with ultraviolet detection (HPLC-UV) was used for the separation and determination of MA. The bulk polymerization method was used to synthesize the MIP, and the MIP and non-imprinted polymer (NIP) were characterized by Fourier transform infrared spectroscopy. The selectivity of the MIP was investigated in the presence of interferents. In addition, we investigated the various parameters that affect the performance of the MEPS, including the pH of the sample, the number of extraction cycles, sample volume, and the types and volumes of the washing and elution solvents. A six-point calibration curve was obtained in the range of 0.2-20 µg/mL (R 2  = 0.9994). The extraction recovery was more than 88.8%. The limit of detection and the limit of quantitation were 0.06 and 0.2 µg/mL, respectively. The intra- and inter-day precisions were in the range of 3.6-4.7% and 3.8-5.1%, respectively. The accuracy was -8.4 to -11.1%. The optimized procedure was selective, sensitive, and rapid, and it was both user friendly and environmentally friendly. The sample preparation process took only about 5 min, so the MIMEPS-HPLC-UV procedure is recommended as an alternative for the biomonitoring of workers exposed to ethylbenzene and/or styrene.

The harm of plastic pollutants for human and environment is being paid more and more attention. Polystyrene (PS) and styrene are toxic compounds used in large quantities in the production of fiberglass reinforced polyesters. In this work, a simple method was designed for independent detecting polystyrene and styrene biomarker (phenylglyoxylic acid, PGA) in serum and urine. We prepared Eu3+ functionalized Sc-based metal-organic frameworks as turn-on fluorescent switch for PGA. The distinct enhanced luminescence is observed from the Eu@MOFs with addition of PGA. The fabricated fluorescent switch has several appealing features including high sensitivity (LOD = 4.16 ppb), quick response time (less than 5s) and broad linear range (0.02mg/mL to 0.5mg/mL). Furthermore, Eu@MOFs exhibits excellent selectivity that it is not affected by congeneric biomarkers. More interestingly, a paper-based probe has been devised. The paper-based fluorescence probe would perform an obvious fluorescence change from navy to red with the variety of PGA content. The practicability of the on-site detection platform for quantitative analysis using a colour scanning APP in smartphone has been also demonstrated by coupled with our proposed paper based fluorescence probe. This work first provides a fast, accurate and sensitive method for independent monitoring PS biomarker PGA, and the paper-based probe exhibit a new idea for design portable and easy to operate sensing devices combine with smartphone., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: To revise the standard method for the determination of phenylglyoxylic acid(PGA)and mandelic acid(MA) in urine by ultra-performance liquid chromatography. Methods: The original standard method was evaluated by experiment, and the chromatographic column, the detection limit,quantitation limit and stabilityof the method were studied. Results: The samples were separated by BEH Phenyl(50mm×2.1mm×1.7μm)column and the internal standard working curve method was used. The regression equations were y =3.660 7 x +0.066 3 and y =5.161 2 x -0.007 3 for MA and PGA respectively. Linear correlation coefficients were 0.999 3 and 0.999 1. Linearity ranges were 0.10-1.00 mg/ml,0.04-0.40 mg/ml. The recoveries of PGA and MA were 91.6%-97.1% and 84.3%-99.0%,the precision were 0.9%-4.6% and 0.5%-1.9%. The detection limit and quantitation limit of the method were 1.1 mg/L and 3.7 mg/L for PGA, 5.4 mg/L and 17.9 mg/L for MA. Conclusion: The method uses the phenyh modified chromatographic column, determines the detection limit. The method can improve quantitation limit, the detection accuracy and meet the detection of occupational population samples.

This paper reports the development of a fast separation method employing capillary zone electrophoresis for the simultaneous determination of hippuric acid, mandelic acid, and creatinine in samples of urine using a coated capillary. The background electrolyte was composed of 10 mmol L -1 tris(hydroxymethyl)aminomethane and 30 mmol L -1 2-hydroxy-isobutyric acid at pH 3.6. The internal standard was 3,5-dihydroxybenzoic acid. Separations were performed in a fused silica capillary (32 cm total length, 8.5 cm effective length, and 50 μm internal diameter) coated with crosslinked hydroxypropyltrimethyl ammonium chloride chitosan and κ-carrageenan. Direct UV detection was performed at a wavelength of 200 nm. Samples and standards were injected hydrodynamically (-50 mbar, 3 s) using the short-end injection procedure. The electrophoretic system was operated under constant voltage of 30 kV with positive polarity on the injection side. The separation time for hippuric acid, mandelic acid, and creatinine was less than 70 s. The evaluation of some analytical parameters of the method for the three analytes showed good linearity (R 2  > 0.99), limit of detections of 0.21 to 0.63 mg L -1 , inter-day precision better than 3.0% (peak area), and recovery in the range of 98 to 106%. The method developed was applied in the analysis of the three analytes in urine samples. Graphical Abstract New method using capillary zone electrophoresis for analysis of creatinine, hippuric acid and mandelic acid in urine.

P-Synephrine is a protoalkaloid widely used as an ergogenic aid in sports. This substance has been included in the World Anti-Doping Agency monitoring program, although scientific information about its effects on performance and athletes' well-being is scarce. The purpose of this investigation was to determine the effectiveness of p-synephrine to increase performance in sprint athletes. In a randomized and counterbalanced order, 13 experienced sprinters performed 2 acute experimental trials after the ingestion of p-synephrine (3 mg·kg(-1)) or after the ingestion of a placebo (control trial). Forty-five minutes after the ingestion of the substances, the sprinters performed a squat jump, a countermovement jump, a 15-s repeated jump test, and subsequently performed 60-m and 100-m simulated sprint competitions. Self-reported questionnaires were used to assess side-effect prevalence. In comparison with the control trial, the ingestion of p-synephrine did not change countermovement jump height (37.4 ± 4.2 vs 36.7 ± 3.3 cm, respectively; P = 0.52), squat jump height (34.4 ± 3.6 vs 33.9 ± 3.7 cm; P = 0.34), or average 15-s repeated jumps height (31.8 ± 4.1 vs 32.2 ± 3.6 cm; P = 0.18). P-Synephrine did not modify maximal running speed during the 60-m (9.0 ± 0.5 vs 9.0 ± 0.4 m·s(-1), respectively; P = 0.55) and 100-m sprint competitions (8.8 ± 0.5 vs 8.8 ± 0.5 m·s(-1), respectively; P = 0.92). The ingestion of p-synephrine did not alter the prevalence of headache, gastrointestinal discomforts, muscle pain, or insomnia during the hours following the tests. Acute consumption of 3 mg·kg(-1) of p-synephrine was ineffective to increase performance in competitive sprint athletes. Moreover, p-synephrine did not increase the occurrence of side effects after the competition.

Context: Urinary biomarkers are widely used among biomonitoring studies because of their ease of collection and nonintrusiveness. Chloroform and TEX (i.e., toluene, ethylbenzene, and m-xylene) are chemicals that are often found together because of common use. Although interactions occurring among TEX are well-known, no information exists on possible kinetic interactions between these chemicals and chloroform at the level of parent compound or urinary biomarkers., Objective: The objective of this study was therefore to study the possible interactions between these compounds in human volunteers with special emphasis on the potential impact on urinary biomarkers., Materials and Methods: Five male volunteers were exposed by inhalation for 6 h to single, binary, and quaternary mixtures that included chloroform. Exhaled air and blood samples were collected and analyzed for parent compound concentrations. Urinary biomarkers (o-cresol, mandelic, and m-methylhippuric acids) were quantified in urine samples. Published PBPK model for chloroform was used, and a Vmax of 3.4 mg/h/kg was optimized to provide a better fit with blood data. Adapted PBPK models from our previous study were used for parent compounds and urinary biomarkers for TEX., Results: Binary exposures with chloroform resulted in no significant interactions. Experimental data for quaternary mixture exposures were well predicted by PBPK models using published description of competitive inhibition among TEX components. However, no significant interactions were observed at levels used in this study., Conclusion: PBPK models for urinary biomarkers proved to be a good tool in quantifying exposure to VOC.

Background: Little is known regarding the effects of environmental exposure of chemicals on androgenic system in the general population. We studied 5,107 subjects included in the National Health and Nutrition Examination Survey (2011-2012)., Methods: Urinary, serum, and blood levels of 15 subclasses comprising 110 individual chemicals were analyzed for their association with serum testosterone levels. The subjects were divided into high and low testosterone groups according to the median testosterone concentration (374.51 ng/dL). Odds ratios (ORs) of individual chemicals in association with testosterone were estimated using logistic regression after adjusting for age, ethnicity, cotinine, body mass index, creatinine, alcohol, and the poverty income ratio., Results: Adjusted ORs for the highest versus lowest quartiles of exposure were 2.12 (95% CI: 1.07, 4.21; Ptrend = 0.044), 1.84 (95% CI: 1.02, 3.34; Ptrend = 0.018) for the association between urinary mandelic acid, and strontium quartiles with low testosterone concentrations in adult men, respectively. However, no association was observed for the remaining chemicals with testosterone., Conclusions: The National Health and Nutrition Examination Survey data suggest that elevations in urinary mandelic acid, and strontium levels are negatively related to low serum testosterone levels in adult men.

Objective: To develop a method for determination of mandelic acid (MA) and phenylglyoxylic acid (PGA) in urine by reagent-free ion chromatography., Methods: Ion chromatography was performed on an AS19 column with a gradient elution solution containing 10-35 mmoL/L KOH at a flow rate of 1.00 ml/min, and MA and PGA were detected at ultraviolet wavelengths of 225 nm and 254 nm, respectively. The samples were diluted 10 times with purified water, then purified on a silver column to remove high concentrations of chloride ion, and injected after being filtered through a 0.2-µm m filter membrane., Results: The recoveries of standard addition of MA and PGA were 96.5% and 99.3%, respectively, with both relative standard deviations less than 5.0%. Good linear relationships were noted in the range of 1.0-100.0 mg/L for both MA and PGA (r >0.9995). The detection limits of MA and PGA were 0.02 mg/L and 0.05 mg/L, respectively; the minimum detectable concentrations of MA and PGA were 0.2 mg/L and 0.5 mg/L (when the sampling amount was 5.0 ml and diluted to 50.0 ml with water, and the injection volume was 300 µL)., Conclusions: This method is fast, convenient, and highly sensitive and selective. It can be used for the analysis of MA and PGA in the urine of styrene-exposed workers.

Styrene is a basic building block for manufacturing thousands of products throughout the world. The present study aimed to (1) detect the presence of styrene and/or its metabolites in the workers in one of the Egyptian plastic factories; (2) demonstrate some common health effects of styrene exposure among the same group by some laboratory investigations and compare them with the unexposed healthy individuals; and (3) correlate the duration of styrene exposure and its level in the blood with the severity of the demonstrated health effects. This study was conducted in one of Egyptian plastic factories. The exposed group was 40 male workers, ranging in age from 18 to 33 years (23.20 ± 4.09), working 12 h/day with 1 day off, and working without any protective equipment. A control group of 50 unexposed healthy males matched with the exposed group for age (21-35 yrs (23.40 ± 4.05)), sex, socioeconomic status, and smoking habit is selected. Written individual consent is obtained from all participants followed by (a) a full medical and occupational history and full clinical examination; (b) ventilatory function tests: forced vital capacity (FVC), slow vital capacity, forced expiratory volume in the 1st second (FEV₁)%, FEV₁/FVC%, peak expiratory flow, and mid-expiratory flow 25-75%; (c) analyses of β₂ microglobulin; blood styrene level; and urinary mandelic acid; and (d) cytogenetic study. The study results showed a statistically significant difference between the exposed and the control groups as regard the blood styrene level, urinary mandelic acid level, β₂ microgloblin in urine, and chromosomal study. The study also showed a statistically significant correlation between the duration of styrene exposure and ventilatory function parameters, also between the duration of styrene exposure and some detectable chromosomal aberrations. Our study recommends the implementation of preemployment and periodic medical examinations and health education programs using personal protective equipments and following the recommended allowable concentrations of styrene exposure.

Objective: To investigate the role of genetic polymorphisms of epoxide hydrolase 1 (EPHX1) in the metabolism of styrene in vivo., Methods: Fifty-six styrene-exposed workers, who worked in the painting workshop of an enterprise for manufacturing glass fiber-reinforced plastic yachts in Shandong Province, China for over one year and were protected in approximately the same way, were selected as study subjects. The 8-hour time-weighted average concentration (8 h-TWA) of styrene and the concentrations of mandelic acid (MA) and phenyl glyoxylic acid (PGA) as urinary metabolites were measured. The genetic polymorphisms of EPHX1 were detected by polymerase chain reaction-restriction fragment length polymorphism analysis., Results: The urinary concentrations of MA and PGA were 177.25±82.36 mg/g Cr and 145.91±69.73 mg/g Cr, respectively, and the 8 h-TWA of styrene was 133.28±95.81 mg/m3. Urinary concentrations of MA and PGA were positively correlated with 8 h-TWA of styrene (R=0.861, P < 0.05; R=0.868, P < 0.05). The subjects were divided into high-exposure group (8 h-TWA >50 mg/m(3)) and low-exposure group (8 h-TWA ≤ 50 mg/m(3), and in the two groups, the urinary concentrations of MA and PGA were significantly higher in the individuals carrying high-activity genotypes of EPHX1 than in those carrying low-activity genotypes of EPHX1 (P < 0.05)., Conclusion: Genetic polymorphisms of EPHX1 play an important role in the metabolic process of styrene in vivo.

Background: Styrene is used in manufacturing fiberglass reinforced plastics: and occupational exposure was related to neurotoxicology and genotoxicity. The sum of the metabolites mandelic and phenylglyoxylic acids is the ACGIH biomarker for occupational exposure with a BEI of 400 mg/g of creatinine in end shift urine corresponding to a airborne styrene concentration of 85 mg/m3. There are two main molding processes, open and closed, the last more effective at controlling worker's styrene exposure., Objectives: To compare the open molding process to the compression of fiber reinforced resin foils, a kind of closed molding, monitoring the styrene exposure of workers in two production sites (A and B)., Methods: Environmental Monitoring was carried out by Radiello samplers and Biological Monitoring by means of the determination of MA and PGA with HPLC/MS/MS in 10 workers at Site A and 14 at Site B., Results: The median values for styrene exposure resulted 31.1 mg/m3 for Site A and 24.4 mg/m for Site B, while the medians for the sum of the two metabolites in the end shift urine were 86.7 e 33.8 mg/g creatinine respectively. There is a significant linear correlation between personal styrene exposure and the excretion of styrene metabolites (R = 0.74)., Conclusions: As expected the exposure markers of the workers of the two production sites resulted higher in the open process. The analytical results of both environmental and biological monitoring were all below the occupational exposure limits, confirming the efficacy of the protective devices.

Mandelic acid (MA) is an important metabolite of styrene. In humans, measurement of its concentration in urine provides an important assessment of the overall level of styrene exposure in workers of the reinforced plastic manufacturing industry. The aim of our study was to investigate in these workers the relationship between MA concentration and styrene exposure time and intensity as well as its dependence on work occupation. The concentration of MA in the urine samples of 35 employees was analyzed with HPLC (high performance liquid chromatography). Out of 35 workers, 11 performed laminating, 11 milling and finalizing, 6 laying-up and spraying-up, and 7 worked in background support. Urinal samples were obtained twice a day over the course of three weeks, at the beginning and the end of the work shift. We found a significant increase in MA concentrations during a work shift in all tested days (Wilcoxon test p < 0.05). Employees working in elevated atmospheric concentrations of styrene (93.77-159.88 mg/m3) had significantly higher MA concentrations in urine compared to other groups at both the beginning and the end of the shift (Kruskal Wallis test p < 0.001) (p < 0.001). Only samples from laminating workers exceeded the biological limit of MA concentration (640 mg/L) at the end of the shift. Normalisation of MA concentration to body mass index (BMI, normal range: 21.7 +/- 3.2 kg/m2) refined differences within groups (Kruskal-Wallis analysis p < 0.001). The accumulation of MA at the end of the work shift for measured time period was not significant for the measured time period (Friedman analysis p > 0.11). Our results confirmed that MA is a sensitive metabolic marker of styrene exposure without cumulative effect. However, normalization of MA concentrations to BMI can improve the accuracy of styrene exposure estimates in certain groups of employees.

A convenient and reliable gas chromatographic method was developed for the simultaneous determination of six aromatic acid metabolites of styrene and styrene-oxide in rat urine; i.e., benzoic (BA), phenylacetic (PAA), mandelic (MA), phenylglyoxylic (PGA), hippuric (HA) and phenylaceturic (PAUA) acids. The method involves a one-pot esterification-extraction procedure, performed directly on urine without prior treatment. Analyses were performed on a RTX-1701 capillary column and the recovered isopropyl esters derivatives were detected by flame ionization detection. The analytical method was validated for selectivity, linearity, detection and quantification limits, recovery and intra-day and inter-day precisions. Calibration curves showed linearity in the range of 8-800 mg/L, except for HA and PAUA (40-800 mg/L). Limits of detection were between 0.2 (PPA) and 7.0 (PAUA) mg/L. The intra-day precisions determined at three concentrations levels were less than 5% for BA, PAA, MA and PGA and 9% for HA and PAUA, respectively. The corresponding mean inter-day precisions for these two groups were 8 and 16%, respectively. The method was successfully applied to quantitatively analyze styrene, styrene-oxide, ethylbenzene and toluene metabolites in urine samples from rats exposed by inhalation to these compounds at levels close to the occupational threshold limit values. Provided that this method can be transposed to human urine, it could have applications as part of biological monitoring for workers exposed to styrene or related compounds.

Ultra-performance liquid chromatography coupled with tandem mass spectrometry was used for the biological monitoring of workers occupationally exposed to solvents. The method was developed using a triple quadrupole to investigate the relevant urinary metabolites of styrene, namely mandelic acid and phenylglyoxylic acid. The method provides quantitative and qualitative data to give additional assurance about the nature of the contaminant analyzed in workers' urine. A full scan and a product ion scan were acquired within the chromatographic peak acquired in MRM. For the two metabolites, the repeatability was 96%, the precision ≥97%, and the accuracy ≥93 ± 3%. The quantitative performances were not influenced by the inclusion of simultaneous full scan acquisition as compared to a usual quantitative approach. Footprints of each substance of interest were obtained at each injection, and full scan data can be interrogated for the presence of interferences and other contaminants. The method developed has been submitted to random real samples from both non-occupationally and occupationally exposed workers. The urines of non-occupationally exposed workers were all free of mandelic acid, phenylglyoxylic acid and putative interferences showing the high selectivity of the method. However, the urines of occupationally exposed workers were robustly quantified. The levels of mandelic acid and phenylglyoxylic acid ranged between 0.2 and 9 mM, and the footprints of each metabolite and structural information were acquired in parallel with the quantitative results, thus providing unquestionable data about the nature of the contaminant and the levels reported. The combination of qualitative information acquired simultaneously with quantitative results provides the structural information needed in case of questions, without any harmful effect on the robustness and throughput of the quantitative analysis., (Copyright © 2012 John Wiley & Sons, Ltd.)

Styrene is a widely used chemical in the manufacture of synthetic rubber, resins, polyesters, and plastics. The highest levels of human exposure to styrene occur during the production of reinforced plastic products. The objective of this study was to examine occupational exposure to styrene in a multistage approach, in order to integrate the following endpoints: styrene in workplace air, mandelic and phenylglyoxylic acids (MA + PGA) in urine, sister chromatid exchanges (SCE), micronuclei (MN), DNA damage (comet assay), and genetic polymorphisms of metabolizing enzymes (CYP2E1, EPHX1, GSTM1, GSTT1, and GSTP1). Seventy-five workers from a fiberglass-reinforced plastics factory and 77 unexposed controls took part in the study. The mean air concentration of styrene in the breathing zone of workers (30.4 ppm) and the mean concentration of urinary metabolites (MA + PGA = 443 ± 44 mg/g creatinine) exceeded the threshold limit value (TLV) and the biological exposure index (BEI). Significantly higher SCE frequency rate and DNA damage were observed in exposed workers, but MN frequency was not markedly modified by exposure. With respect to the effect of genetic polymorphisms on different exposure and effect biomarkers studied, an increase in SCE levels with elevated microsomal epoxide hydrolase activity was noted in exposed workers, suggesting a possible exposure-genotype interaction.

This study assessed the relationships between ethylbenzene exposure and levels of 8-hydroxydeoxyguanosine (8-OHdG) among spray painters. Sixty-four male workers employed at a large shipyard were recruited for this investigation. Fifteen spray painters exposed to paint, together with two non-exposed groups, namely 19 sandblasting workers and 30 office staffs were selected as the subjects. Personal exposure to xylene and ethylbenzene in air were collected using diffusive samplers. Urine samples of the spray painters were collected after a month-long holiday leave and during the pre- and post-workshifts. Urine samples of sandblasting workers and office staffs were gathered after their shift. Urinary mandelic acid and methyl hippuric acid were used as biological indices of dose of ethylbenzene and xylene, respectively. Urinary 8-OHdG was used as biomarker of oxidative DNA damage. The post-workshift concentration of urinary 8-OHdG for 10 spray painters (30.3 ± 9.28 μg g(-1) creatinine) significantly exceeded that of holiday leave (7.20 ± 1.08 μg g(-1) creatinine; P = 0.001). The post-workshift concentration of urinary 8-OHdG was higher among 15 spray painters (29.0 ± 6.52 μg g(-1) creatinine) than sandblasting workers (9.14 ± 2.05 μg g(-1) creatinine; P = 0.01) and office staffs (8.35 ± 0.84 μg g(-1) creatinine; P = 0.007). A stepwise regression model revealed an 8.11 μg g(-1) creatinine increase per 1 p.p.m. increase in ethylbenzene [95% confidence interval (CI) 4.13-12.1]. A stepwise regression model revealed an increase of 6.04 μg g(-1) creatinine (95% CI 2.23-9.84) per 1 p.p.m. in ethylbenzene after adjustment of age (95% CI 2.23-9.84). This pilot study suggests that occupational exposure to paint increases oxidative DNA injury. Moreover, urinary 8-OHdG levels displayed greater DNA damage in spray painters compared to other unexposed groups and their holiday leave samples. A significant correlation was found between urinary 8-OHdG and the exposure to ethylbenzene. The ethylbenzene exposure could not explain all urinary 8-OHdG measured. Other components of paint deserve further investigation.

The present study aimed to evaluate the effects of styrene exposure at levels below the recommended standards of the Threshold Limit Value (TLV-TWA(8)) of 20 ppm (ACGIH, 2004) in reinforced-fiberglass plastics workers. Study subjects comprised 50 exposed workers and 40 control subjects. The exposed workers were stratified by styrene exposure levels, i.e. group I (<10 ppm, <42.20 mg/m(3)), group II (10-20 ppm, 42.20-84.40 mg/m(3)), and group III (>20 ppm, >84.40 mg/m(3)). The mean styrene exposure levels of exposed workers were significantly higher than those of the control workers. Biomarkers of exposure to styrene, including blood styrene and the urinary metabolites, mandelic acid (MA) and phenylglyoxylic acid (PGA), were significantly increased with increasing levels of styrene exposure, but were not detected in the control group. DNA damage, such as DNA strand breaks, 8-hydroxydeoxyguanosine (8-OHdG), and DNA repair capacity, were used as biomarkers of early biological effects. DNA strand breaks and 8-OHdG/10(5)dG levels in peripheral leukocytes of exposed groups were significantly higher compared to the control group (P<0.05). In addition, DNA repair capacity, determined by the cytogenetic challenge assay, was lower in all exposed groups when compared to the control group (P<0.05). The expression of CYP2E1, which is involved in styrene metabolism, in all styrene exposed groups, was higher than that of the control group at a statistically significant level (P<0.05). Levels of expression of the DNA repair genes hOGG1 and XRCC1 were significantly higher in all exposed groups than in the control group (P<0.05). In addition to styrene contamination in ambient air, a trace amount of benzene was also found but, the correlation between benzene exposure and DNA damage or DNA repair capacity was not statistically significant. The results obtained from this study indicate an increase in genotoxic effects and thus health risk from occupational styrene exposure, even at levels below the recommended TLV-TWA(8) of 20 ppm., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Exposure to volatile organic compounds (VOCs) was known to result in immunologic, respiratory, carcinogenic, reproductive, neurologic, and cardiovascular effects. However, the mechanisms by which VOCs induce these adverse health effects are not well understood. To evaluate the change of oxidative stress biomarker and gene expression levels in workers exposed to VOCs, we obtained urine and blood samples from 21 subjects before and after occupational exposure to VOCs. We measured levels of muconic acid (MuA), hippuric acid (HA), mandelic acid (MaA), and methyl hippuric acid (MHA) as urinary exposure biomarkers for benzene, toluene, ethylbenzene, and xylene (collectively BTEX), and malondialdehyde (MDA) and 8-hydroxydeoxyguanine (8-OHdG) as oxidative stress biomarkers in all subjects. We also evaluated BTEX-mediated RNA expression using cDNA microarray in 14 subjects. HA and MHA levels were higher following occupational exposure to VOCs (p < 0.01). In the linear regression analysis, HA ratios of after- and before-exposure were found to be significantly associated with increase of MDA ratios of after- and before-exposure after controlling for age, body mass index, and smoking (β = 0.06, p = 0.031). Evaluation of the gene expressions by HA showed that 23 gene expressions were found to be significantly associated with HA levels after adjusting for age, body mass index, and smoking (p < 0.001). In particular, expressions of ENO3 and CDNA FLJ39461 fis among the 23 genes were significantly associated with the change in MDA level (p < 0.05). Our study results suggest that exposure to VOCs, specifically toluene, induces oxidative stress and various gene expression change of which some may be responsible for oxidative stress.

The metabolic impact of polyphenol-rich red wine and grape juice consumption in humans was studied using a metabolomics approach. Fifty-eight men and women participated in a placebo-controlled, double-crossover study in which they consumed during a period of 4 wk, either a polyphenol-rich 2:1 dry mix of red wine and red grape juice extracts (MIX) or only a grape juice extract (GJX). Twenty-four-hour urine samples were collected after each intervention. (1)H NMR spectroscopy was applied for global metabolite profiling, while GC-MS was used for focused profiling of urinary phenolic acids. Urine metabolic profiles after intake of both polyphenol-rich extracts were significantly differentiated from placebo using multilevel partial least squares discriminant analysis. A significant 35% increase in hippuric acid excretion (p<0.001) in urine was measured after the MIX consumption as) or only a red grape juice dry extract (GJX). 24-h urine samples were collected after each intervention. 1H-NMR spectroscopy was applied for global metabolite profiling, while gas chromatography-mass spectrometry (GC-MS) was used for focused profiling of urinary phenolic acids. Urine metabolic profiles after intake of both polyphenol-rich extracts were significantly differentiated from placebo using multilevel partial least squares discriminant analysis (ML-PLS-DA). A significant 35% increase in hippuric acid excretion (p<0.001) in urine was measured after the MIX consumption compared with placebo, whereas no change was found after GJX consumption. GC-MS-based metabolomics of urine allowed identification of 18 different phenolic acids, which were significantly elevated following intake of either extract. Syringic acid, 3- and 4-hydroxyhippuric acid and 4-hydroxymandelic acid were the strongest urinary markers for both extracts. MIX and GJX consumption had a slightly different effect on the excreted phenolic acid profile and on endogenous metabolite excretion, possibly reflecting their different polyphenol composition.

Ethylbenzene is an important industrial chemical that has recently been classified as a possible human carcinogen (International Agency of Research on Cancer class 2B), but the available data do not support the genotoxic mechanism of ethylbenzene-induced tumors in kidney. We investigated the effects of ethylbenzene on renal ultrastructure and explored the nongenotoxic mechanism of mitochondria-mediated apoptosis pathway. Forty male Sprague-Dawley rats were used as a vivo model with ethylbenzene inhalation for 13 weeks, and the metabolites of ethylbenzene, mandelic acid (MA), and phenylglyoxylic acid (PGA) in urine were examined by high-performance liquid chromatography. Meanwhile, the ultrastructure of renal tubular epithelial cells was observed, and cell apoptosis was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Furthermore, we investigated the expression levels of messenger RNA (mRNA) and protein of bax, bcl-2, cytochrome c, caspase-9, and caspase-3 in rat kidney. With respect to levels of MA, PGA, and MA + PGA, a significant dose-dependent increase was observed in 4335 and 6500 mg/m(3) ethylbenzene-treated groups against the control group. The mitochondria of renal tubular epithelial cells became a compact and vacuolar structure in 6500 mg/m(3) ethylbenzene-treated group, and ethylbenzene induced a significant increase in the number of apoptotic cells as compared to the control group. In addition, enhanced mRNA and protein expression levels of all measured genes were observed in various ethylbenzene-treated groups except the decreased bcl-2 expression levels. Our results indicated that ethylbenzene may induce apoptosis of renal tubular epithelial cells via mitochondria-mediated apoptotic pathways. MA and PGA in urine might be a parameter of biological dose in vivo after ethylbenzene inhalation.

The CYP2E1 has been identified as the main cytochrome P450 isoform involved in human styrene metabolism. CYP2E1 presents polymorphism in humans and the different genotypes may, at least partly, be related to the different levels of individual expression of enzyme activity. We studied whether the genetic polymorphisms and phenotype of CYP2E1 modulate the level of urinary styrene metabolites and if they can be used for assessing risks of occupational exposure to styrene. A population of 49 male workers exposed to styrene (average level 362.7mg/m(3)) and a control group were selected. Samples of urine, blood and buccal swab were taken to determine the urinary biological indicators (phenylglyoxylic acid and mandelic acid), to quantify mRNA of CYP2E1 in blood using RT-PCR and to analyse different polymorphisms of enzyme CYP2E1 from buccal swab. We found decreased expression of mRNA of the enzyme, as well as decreased excretion of the styrene metabolites in individuals carrying the CYP2E1*5B heterozygote allele (cl/c2) with respect to the wild-type homozygote (c1/c1), which indicates a reduction in the inducibility of the enzyme in the presence of this polymorphism. The results show that the combined effect of both the CYP2E1 phenotype, measured by the expression of the specific mRNA in blood samples, and the CYP2E1*5B allele genotype, may explain the variability of urinary excretion of the styrene metabolites., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

The aim of this work is to compare several short-term biomarkers of styrene exposure, namely urinary styrene (StyU), mercapturic acids (M1+M2), mandelic acid (MA), phenylglyoxylic acid (PGA), phenylglycine (PHG), and 4-vinylphenol conjugates (VP), for use as biomarkers of exposure in epidemiologic studies. A repeated measurements protocol (typically 4 measurements per worker over 6 weeks) was applied to measure airborne styrene (StyA) and urinary biomarkers in 10 varnish and 8 fiberglass reinforced plastic workers. Estimated geometric mean personal exposures to StyA were 2.96mg/m(3) in varnish workers and 15.7mg/m(3) in plastic workers. The corresponding levels of StyU, M1+M2, MA, PGA, MA+PGA, PHG and VP were 5.13microg/L, 0.111, 38.2, 22.7, 62.6, 0.978, and 3.97mg/g creatinine in varnish workers and 8.38microg/L, 0.303, 146, 83.4, 232, 2.85 and 3.97mg/g creatinine in plastic workers. Within-worker (sigma(wY)(2)) and between-worker (sigma(bY)(2)) variance components were estimated from the log-transformed data as were the corresponding fold ranges containing 95% of the respective lognormal distributions of daily levels ((w)R(0.95)) and subject-specific mean levels ((b)R(0.95)). Estimates of (w)R(0.95) (range: 4-26) were generally smaller than those of (b)R(0.95) (range: 5-790) for both environmental and biological markers; this indicates that exposures varied much more between workers than within workers in these groups. Since attenuation bias in an estimated exposure-response relationship increases with the variance ratio lambda=sigma(wY)(2)/sigma(bY)(2), we estimated values of lambda for all exposure measures in our study. Values of lambda were typically much less than one (median=0.220) and ranged from 0.089 for M1+M2 in plastic workers to 1.38 for PHG in varnish workers. Since values of lambda were 0.147 and 0.271 for StyA in varnish workers and plastic workers, respectively, compared to 0.178 and 0.210 for MA in the same groups, our results suggest that either air measurements or conventional biomarker measurements (urinary MA) would be comparable surrogates for individual exposures in epidemiologic studies., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

Objective: To investigate the influence of ethylbenzene on the levels of mandelic acid (MA) and phenylglyoxylic acid (PGA) in urine, the ultrastructure and the expressions of mitochondrial apoptotic-related genes in the rat nephridial tissues., Methods: Four groups of 10 males of Sprague-Dawley rats were allocated randomly into four groups: control (C) group, low (L) group, moderate (M) group and high (H) group, and inhaled daily with different doses of ethylbenzene: 0, 433.5 mg/m(3), 4335 mg/m(3), and 6500 mg/m(3) 6 h per day, 5 days per week for 13 weeks. The mandelic acid and phenylglyoxylic acid in the urine was assayed by high performance liquid chromatography. The ultrastructure of nephridial tissue was observed via electron microscope. The protein expression levels of Bax, Bcl-2, cytochrome C, Caspase-9 and Caspase-3 in nephridial tissues were measured by Western blot, respectively., Results: The levels of MA [(0.303 +/- 0.148) mg/L, (0.404 +/- 0.154) mg/L] and PGA [(0.168 +/- 0.104) mg/L, (0.174 +/- 0.092) mg/L] in the urine of M and H groups were significantly higher than that in the control and L group [(0.084 +/- 0.070) mg/L, (0.041 +/- 0.029) mg/L] (P < 0.05, respectively). It has been shown a dose-effect relationship between the contents of MA, PGA and MA + PGA and inhaled ethylbenzene, respectively. The mitochondria of rat nephridial tissue of H group became a compact and vacuolar structure with disorder and loss of cristae. The expression levels of Bax in mitochondria of nephridial tissues of M and H groups were significantly lower than that in the control group (P < 0.05). Caspase-3 expression level in H group was remarkably higher than that in the control group (P < 0.05). Compared with the control group, the expression levels of cytochrome C and Caspase-9 were enhanced, while the expression levels of Bcl-2 were restrained in all ethylbenzene-treated groups (P < 0.05, P < 0.05, respectively). The expression levels of Caspase-3 in M and H groups were significantly higher than that in the control group and L group (P < 0.05)., Conclusion: Ethylbenzene can induce apoptosis in the cells of nephridial tissues. The apoptotic mechanism might be involved with up-regulation of Bax, cytochrome C, Caspase-9 and Caspase-3, as well as restraint of Bcl-2. The level of MA and PGA in the rat urine could be a parameter of biological dose in vivo after ethylbenzene inhalation.

Objective: To establish a GC/MS method for analysis of cotinine (COT), phenylglyoxylic acid (PA) and mandelic acid (MA) in human urine., Methods: Human urine samples were extracted by CCl(3) and derivatized with MSTFA after dried completely. The contents of COT, PA and MA were measured by GC/MS method with DB-5MS capillary column and EI ion-source., Result: The calibration curves for COT in urine samples were linear over the concentration ranges of 0.0002 approximately 3.5 microg ml(-1), while PA and MA were both of 1.25 approximately 160 microg ml(-1). The limits of quantification were 0.0002 microg ml(-1), 1.25 microg ml(-1) and 1.25 microg ml(-1) for COT, PA and MA, respectively. The assay recoveries for COT, PA and MA ranged from 89.53% approximately 102.4%, 84.88% approximately 91.46% and 83.46% approximately 13.6%, respectively., Conclusion: The established method can detect cotinine, phenylglyoxylic acid and mandelic acid simultaneously, which would be used in routine assessment and monitoring of the internal exposure to nicotine and styrene in human body.

Objective: Associations between occupational styrene exposures and impairment of visual functions were investigated with a view to answering three questions: (1) are the published findings for colour vision deficiencies and impaired contrast sensitivity to reproduce in a new study approach, (2) if such effects exist, are they related to current or chronic exposures and (3) if effects exist, are there reductions in the effects during an exposure-free period?, Methods: Workers from a boat building plant were examined in groups of current low [n = 97, mean mandelic acid (MA) + phenylglyoxylic acid (PGA) = 51 mg/g creatinine], medium (n = 115, mean = 229 mg/g creatinine) and high (n = 30, mean = 977 mg/g creatinine) level exposure to styrene. Job tenure was about 6 years. In addition, subgroups chronically exposed to low-short (n = 34, lifetime weighted mean 200 mg/g creatinine for 6 years) and high-long (n = 17, mean = 660 mg/g creatinine, 15 years) styrene levels were analysed. The examinations were carried out during normal working days and during the company holidays. Colour vision was investigated with the Lanthony desaturated panel D-15d using the colour confusion index (CCI) as a relevant variable. Contrast sensitivity was investigated with the Vistech charts VCTS 6500 using frequency-related results as well as total scores as variables. Co-variance analyses with repeated measurements and multiple linear regressions were used for statistical analysis., Results: There was no evidence of significant associations between exposure parameters and CCI. This is true for the analyses with all participants as well as for those with the subgroups with high-long versus low-short exposure. Thus, no exposure related changes in the relevant variables were found during the exposure-free period. The analyses for contrast sensitivity show similar results. The largest portions of the variances in both tests were explained by age. German as mother tongue covered a considerable portion of the CCI variances. Education, long-term alcohol use and job tenure explain only partly significant portions of the test variances exhibited., Conclusion: Both acute styrene exposure levels of 40 ppm (range of standard deviation up to 54 ppm) and long term exposures to 27 ppm (range of standard deviation up to 44 ppm with higher exposure levels in the past) for a period of about 15 years were not identified as causing elevated risks for the investigated parameters of colour vision and contrast sensitivity. This statement contradicts the published results for styrene-related colour vision deficiencies but it seems to be compatible with published results for contrast sensitivity due to styrene exposure.

Purpose of the Study: The purpose of this study is to examine the effectiveness of several types of personal respiratory protection equipment at styrene exposed laminators under real work place conditions., Subjects and Method: 99 male styrene exposed workers were examined. During their lamination activities the average styrene concentrations in air ranged between 30 to 60 ppm (maximum: 205 ppm). The laminators were followed during an usual workweek from Monday to Thursday. The external styrene exposure was measured by means of passive active carbon badges. The excretion of mandelic acid (MA) and phenyl glyoxylic acid (PGA) in end-of-shift urine samples was used to quantify the internal styrene load. During the work shift some laminators did not use respiratory protection masks. The majority used either a half face mask with active carbon filter or an air purifying respirator., Results: The respiratory masks were worn during an average between 31% and 72% of the work time. The styrene concentrations of the ambient air were -depending on the activity- in the range of 30 to 60 ppm. The end-of-shift concentrations of MA and PGA in urine samples varied considerably, their means range from 153 to 606 mg/g creatinine. The comparison shows that workers with air purifying respirators experience the lowest internal styrene body burden in spite of high external exposures. Their effectiveness during usual working condition was around 83% whereas the use of half face masks with active carbon filters reduce styrene exposure only of 26% as an average., Conclusions: The use of styrene-containing resins in boatbuilding can be associated with increased external styrene exposure of the laminators. During the use of different types of respiratory protection masks it is shown that only the application of air purifying respirators leads to a significant reduction of the internal styrene body burden of 83% when worn during 72% of the total work time. In this way it is possible to comply with or to stay clearly below the biological limit value of 600 mg MA + PGA/g creatinine (BAT-value).

Objective: Associations between occupational styrene exposure and impairment of hearing function were investigated, guided by three questions: are there hearing losses concerning high frequency and standard audiometric test? Are there dose-response relationships and measurable thresholds of effects? Are there signs of reversibility of possible effects if the workers are examined during times of improvement from their work?, Methods: A group of workers from a boat building plant, some of whom were laminators, were examined in subgroups of current low (n = 99, mean mandelic acid MA + phenylglyoxylic acid PGA = 51 mg/g creatinine), medium (n = 118, mean 229 mg/g creat.) and high (n = 31, mean 970 mg/g creat.) exposure to styrene. In addition, subgroups chronically exposed to high-long (n = 17) and low-short (n = 34) styrene levels were analysed. The examinations were carried out during normal work days and during the company holidays. Hearing thresholds and transient evoked otoacoustic emissions (TEOAE) were measured. Statistics included multiple co-variance analyses with repeated measures, linear regressions, and logistic regressions., Results: The analyses of all participants demonstrated no clear exposure effects. Particularly no sufficient proof of dose-response relationship measured against parameters of current exposure (MA + PGA, styrene/blood) and of chronic exposure (cumulative and average life time exposure resp.) was found. The analyses of groups exposed to high levels show elevated thresholds at frequencies up to 1,500 Hz among the subgroup exposed to high styrene levels (e.g. 40-50 ppm as average) for a longer period of time (e.g. more than 10 years). These participants also demonstrated signs of "improvement" at frequencies above 2,000 Hz during work holidays, when they were not exposed to styrene. A significantly elevated odds ratio for cases of hearing loss (more than 25 dB (A) in one ear, 3,000-6,000 Hz) was found among the group exposed to high levels (above 30 ppm as average) for a longer period of time (more than 10-26 years). The measurements of TEOAE did not exhibit significant results related to exposure., Conclusion: This study found, that chronic and intensive styrene exposure increases the hearing thresholds. At levels of about 30-50 ppm as an average inhaled styrene per work day over a period of about 15 years with higher exposure levels above 50 ppm in the past, an elevated risk for impaired hearing thresholds can be expected. The formerly published results on ototoxic effects below 20 ppm could not be confirmed. With few exceptions (at frequencies of 1,000 and 1,500 Hz) no dose-response relationship between threshold and exposure data was found. Improvements of hearing thresholds during work- and exposure-free period are possible.

Objectives: We aimed to investigate whether styrene can affect the vibration perception threshold (VPT) and to examine the dose-effect relationship at current and past styrene exposure levels., Methods: VPT was examined using a Vibrometer (TM-31A) for 67 subjects exposed to styrene in a fiberglass reinforced plastic boat plant and 151 non-exposed subjects. We selected 67 age-matched controls out of the non-exposed subjects for the analyses. End shift urinary metabolites of styrene were measured for evaluation of the dose-effect relationship for the past eight years and at the time of VPT measurement. The current exposure level was expressed by the end shift urinary mandelic acid (MA) and phenylglyoxylic acid (PGA) levels. Cumulative exposure index (CEI) were calculated based on the exposure frequency and urinary MA concentrations measured for the past eight years., Results: The VPT of the exposed group was higher than that of non-exposed group. Multiple regression analysis revealed that past maximum exposure level and age were significant factors explaining the variation of VPT. Dose-effect relationship was recognized in upper limbs but not in lower limbs among exposure groups. When the exposed group was divided into high- and low-level groups for the past maximum exposure level by the cutoff point of MA 0.83 g/g cr (equivalent to 50 ppm in air) and compared to the control group, we found significant differences in the VPT in upper limbs, between the high-level exposed group and control group, and in lower limbs, between both of the high- and low-level exposed groups and the control group., Conclusion: If the maximum concentration of styrene exposure exceeded 50ppm in the past, effects of exposure to styrene on the VPT are likely to persist.

Background: Many natural substances and drugs have long been known to cause goiter or thyroid dysfunction. More recently, several environmental pollutants, such as pesticides and industrial compounds, have been investigated for their thyroid-disrupting activity and related adverse effects on human health. The aim of this study was to evaluate the effects of styrene on the thyroid axis in occupationally exposed workers., Methods: Thirty-eight exposed (E) and 123 nonexposed (NE) male workers (controls) were assessed. Serum concentrations of thyrotropin (TSH; basal and after thyrotropin-releasing hormone [TRH] administration.), free thyroxine (FT(4)), free triiodothyronine (FT(3)), anti-thyroglobulin, thyroid peroxidase antibody, and calcitonin were measured. Thyroid ultrasound examination was also performed. In E workers, urinary creatinine, mandelic acid (MA), and phenylglyoxylic acid (PGA) were also measured., Results: No significant differences between E and NE workers were demonstrated, as far as thyroid volume, nodularity, serum thyroid antibodies, and calcitonin were analyzed. However, in the E group a positive correlation between duration of exposure and thyroid volume was detected. After exclusion of subjects with nodular or autoimmune thyroid diseases, serum concentrations of FT(4), FT(3), and TSH did not differ between the two groups. In E workers there was a positive correlation between the urinary concentrations of styrene metabolites (MA plus PGA) and FT(4) or FT(4)/FT(3) ratio (p < 0.05; r = 0.45 and p < 0.005; r = 0.61, respectively), while no correlation was observed between urinary concentrations of MA plus PGA and serum TSH (either basal and stimulated)., Conclusions: Chronic exposure to styrene is not associated with an increase in nodular or autoimmune thyroid diseases. However, styrene could interfere with peripheral metabolism of thyroid hormones by inhibiting T(4) to T(3) conversion. Whether this is a direct effect on iodothyronine deiodinases or a consequence of a general distress, such as in nonthyroidal illnesses, remains to be established. Further studies in a larger population of exposed workers are needed to confirm these preliminary observations.

Occupational exposure biological monitoring techniques were applied for the diagnosis of inhalation abuse and for the evaluation of the levels of exposure to benzene, toluene, ethylbenzene, xylenes, and n-hexane, in 44 Tunisian adolescents and children suspected for volatile substance addiction. Urinary trans,trans-muconic acid, hippuric acid (HA), mandelic acid, and methylhippuric acids determinations were performed by high performance liquid chromatography with a photodiode array detector, and urinary o-cresol (o-Cr) and 2,5-hexanedione (HD) were extracted simultaneously and measured using gas chromatography with a flame ionization detector. Given the high linearity ranges, HD and o-Cr occupational exposure monitoring techniques could be applied without modification. However, urinary sample dilution was necessary before HA analysis. Concentrations were compared with the maxima of normal values (MNVs) in the general population and to the biological exposure indices (BEIs) used in occupational toxicology. Values as high as 6610-fold the MNV and 68 times the BEI were registered. The subjects showed high exposure to toluene and hexane. Measured metabolites HA and/or o-Cr and HD enabled the easy detection and evaluation of exposure levels. The problem of inhalant abuse should be given more attention and treated through an effective prevention strategy.

Phenylglyoxylic acid (PhGA) and mandelic acid (MA) are two popular urinary markers of occupational exposure to styrene, but PhGA has been considered to be relatively unstable when urine samples are stored. This study was initiated to examine the stability of PhGA in urine under two storage conditions, i.e., at room temperature (at 25 degrees C) and in refrigeration (at 4 degrees C) for up to 14 days. The experiments showed that no substantial decrease was observed in either PhGA or MA even at room temperature within one day, but, depending on urine samples, a gradual decrease in PhGA took place both at 4 degrees C and more markedly at 25 degrees C when kept for a week. Further reduction was observed in two weeks even at 4 degrees C. No reduction was observed in MA up to two weeks both at 4 degrees C and at 25 degrees C. The observation on stability of MA and limited stability of PhGA (i.e., no significant decrease for 4 days when stored as refrigerated) was confirmed by a repeated experiment. Further analyses disclosed that PhGA when stored at 4 degrees C tended to be more unstable when urine samples were alkaline (e.g., at pH 8) rather than acidic (e.g., at pH 6 or below), but the trend varied subject to individual samples. Thus, the practical recommendation is that urine samples should be analyzed on the day of collection if all possible, or kept at 4 degrees C, or more preferably at -20 degrees C. Refrigeration will allow storage of up to 4 days without substantial decrease in either PhGA or MA.

Objectives: To determine the effectiveness of protective suits and gloves by biomonitoring., Methods: Fifteen male spray painters at a ship coating factory were studied for two weeks. Workers wore no protective clothing during the first week and wore protective suits and gloves during the second week. Sampling was conducted on four consecutive working days each week. Ethyl benzene and xylene in the air were collected by using 3M 3500 organic vapour monitors. Urine was collected before and after each work shift., Results: Urinary mandelic acid (MA) and methyl hippuric acid (MHA) levels were divided by the personal exposure concentrations of ethyl benzene and xylene, respectively. Mean (SE) corrected MA and MHA concentrations in the first week were 1.07 (0.18) and 2.66 (0.68) (mg/g creatinine)/(mg/m3), and concentrations in the second week were 0.50 (0.12) and 1.76 (0.35) (mg/g creatinine)/(mg/m3) in the second week, respectively. Both MA and MHA concentrations in the second week (when spray painters wore protective suits and gloves) were lower than in the first week, respectively (p<0.001, p = 0.011). Mean decrease in MA and MHA biomarkers were 69% and 49%, respectively., Conclusion: This study successfully evaluated the effectiveness of chemical protective suits and gloves by using biomarkers as urinary MA and MHA. This method is feasible for determining the performance of workers wearing personal protective equipment. Moreover, the experimental results suggest that dermal exposure may be the major contributor to total body burden of solvents in spray painters without protective suits and gloves.

Styrene is one of the most important organic chemicals used worldwide. In humans, styrene metabolism involves oxidation by cytochrome P450 monooxygenases (CYPs) to styrene-7,8-oxide, an epoxide thought to be responsible for the genotoxic effects of styrene exposure, and detoxification by means of epoxide hydrolase (mEH) and glutathione S-transferases (GSTs). The objective of this study was to investigate if genetic polymorphisms of metabolic enzymes modulate the level of urinary styrene metabolites and styrene oxide adducts with N-terminal valine of human globin (SO-Hb) in 75 workers occupationally exposed to styrene and 77 unexposed controls. The mean air concentration of styrene in the breathing zone of workers (30.4ppm) was higher than the threshold limit value of 20ppm recommended by the American Conference of Governmental Industrial Hygienists (ACGIH), and the biological exposure index adopted by the ACGIH for exposure to styrene prior to the next shift (MA+PGA=400mg/g creatinine) was exceeded, indicating that styrene exposure for this group of workers was higher than recommended. A highly significant correlation was observed between styrene concentration in the breathing zone and the MA+PGA in urine of workers (r=0.85, P<0.001). The levels of SO-Hb adducts in exposed workers were significantly increased as compared with controls, although no difference was observed between subjects stratified as high and medium exposure categories based on MA+PGA excretion. Regarding the effect of the genetic polymorphisms we found that the level of SO-Hb adducts might be modulated by the predicted mEH enzymatic activity in the exposed workers. From our data we conclude that SO-Hb adduct measurement is a complementary method to MA+PG measurement for assessing exposure to styrene at occupational and environmental levels, which reflects a more extensive exposure period.

A reversed-phase HPLC method for the simultaneous quantitative determination of mandelic acid enantiomers (MA) and phenylglyoxylic acid (PGA) in urine is described. MA and PGA were extracted with ethyl acetate from urine at acidic pH and derivatized with S-(-)-1-(1-naphthyl) ethylamine. A ZORBAX SB-C(18) column (250 mm x 4.6mm i.d., 5 microm, Agilent, USA) was used with a mobile phase composed of methanol-10 mmol/L phosphate buffer [pH 2.5 (65:35, v/v)] at a flow-rate of 0.8 ml/min. Detection was set at UV wavelength of 254 nm. The mean absolute recoveries were 94.2%, 91.9%, 92.5% and 86.3% for S-MA, R-MA, PGA and salicylic acid (I.S.), respectively. The intra- and inter-day precisions determined at three different concentrations ranged from 2.8% to 4.8%, 0.7% to 7.7% and 1.3% to 6.8%, respectively. The lower limits of detection for MA enantiomers and PGA in urine were 1 microg/ml and the lower limits of quantification were 5 microg/ml (R.S.D.<10%, n=5). The method has been applied to determine the urinary excretion of MA enantiomers and PGA from Sprague-Dawley rats after orally administered with styrene.

Pregnant Sprague-Dawley rats were exposed to ethylbenzene (EB; 0, 250, or 1000 ppm) and methylethylketone (MEK; 0, 1000, or 3000 ppm), alone and in combination, by inhalation, for 6h/day, during days 6-20 of gestation. Maternal toxicity, evidenced by decreased in body weight gain and food consumption, tended to be greater after simultaneous exposures to the high concentrations of 1000 ppm EB and 3000 ppm MEK, when compared to the treatments with individual compounds. No significant increase in embryo/fetal lethality or incidence of malformations and variations was observed in any of the treatment groups. Fetal body weight was significantly reduced after individual treatment with 1000 ppm EB or 3000 ppm MEK, and in the combined groups. There was no evidence of interaction between EB and MEK in causing developmental toxicity.

The use of a supercritical carbon dioxide reaction medium for the determination of the (R)- and (S)-enantiomers of mandelic acid (MA) is proposed. The process involves a previous derivatization step under supercritical conditions by which the carboxyl group is esterified with methanol, then followed by acylation of the hydroxyl group in methyl MA with pentafluoropropionic anhydride in the absence of a catalyst. These derivatization steps cause no enantiomeric inversion. The derivatized enantiomers are extracted and quantified by gas chromatography. A BETA DEX 225 capillary column allows the separation of (R)-MA and (S)-MA as pentafluoropropionyl methyl esters with good resolution and precision. The overall method was used to determine both enantiomers in urine samples.

Objective: To study the effects of antimuscarinics excreted into human urine on normal bladder in a rat model of detrusor overactivity., Materials and Methods: Two 'normal' adult volunteers collected voided urine after taking trospium (20 mg, twice daily), tolterodine LA (4 mg, four times daily), or oxybutynin XL (10 mg, four times daily). The drugs were taken in a random order for 5 days with a 7-day washout period between the drugs. The urine collected from the two volunteers was mixed together and then blindly labelled and used for testing. Control human urine (no oral antimuscarinics) was also used. The effect of intravesical administration of human urine on carbachol-induced bladder overactivity was studied in female Sprague-Dawley rats anaesthetised with urethane. Cystometric variables during continuous infusion (0.04 mL/min) for >1 h each of saline, human urine, then a mixture of carbachol (30 microm) and human urine were compared in the four groups (control and the three different antimuscarinics tested; six rats per group)., Results: Human urine, with or with no intake of antimuscarinic agents, had no effect on normal bladder function. Bladder capacity and intercontraction intervals were significantly decreased after adding carbachol to urine containing vehicle, tolterodine or oxybutynin. However, urine collected from the humans who had taken trospium prevented the carbachol-induced reduction in bladder capacity and intercontraction intervals. Maximum voiding pressure and pressure threshold were not changed in any case., Conclusion: This is the first report that the urine excreted after oral ingestion of trospium (20 mg, twice daily) has a significant inhibitory effect in a rat model of detrusor overactivity. This suggests that antimuscarinic agents have a local bladder effect during the bladder-storage phase in addition to the smooth muscle-mediated voiding phase.

Here we evaluate the influence of individual genetic polymorphisms of drug-metabolizing enzymes as well as body mass index (BMI) and lifestyle (smoking, alcohol consumption) on urinary metabolites after occupational exposure to styrene. Seventy-three workers exposed to styrene in a reinforced-plastics workplace were studied. The personal styrene exposure in the air and the urinary styrene metabolites mandelic acid and phenylglyoxylic acid were measured. The subjects' genetic polymorphisms in the genes that encode the styrene-metabolizing enzymes CYP2E1, CYP2B6, EPHX1, GSTM1, GSTT1 and GSTP1 were determined. Neither genotype nor lifestyle significantly affected urinary metabolites. There was, however, an interaction between the CYP2E1 genotype and smoking. Among non-smokers, urinary styrene metabolites were significantly decreased in subjects with c1/c1 alleles of CYP2E1 as compared with those with the c1/c2 genotype. There was no significant difference in urinary metabolites among smokers. When the combined influence of the CYP2B6 genotype and the predicted activity of EPHX1 were examined, urinary metabolites in subjects with low enzyme activity were lower than in those with medium or high activity after high styrene exposure (>or=50 ppm). The results suggest that genetic susceptibility and lifestyle should be considered in biological monitoring of exposure to styrene.

Objective: To apply mass spectrometry-based techniques for the determination of background levels of metabolites of widespread organic solvents, such as styrene, in unexposed subjects from the general population., Methods: The study population consisted of 129 healthy Italian subjects, not occupationally exposed to styrene (67 men; 37% smokers). Urinary levels of mandelic acid (MA) and phenylglyoxylic acid (PGA) were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS)., Results: Using LC-MS/MS, we were able to detect the peaks of MA and PGA in 100% of samples. The distribution of styrene metabolites was log-normal, the geometric means of MA and PGA concentrations being 0.443 mg/g creatinine [geometric standard deviation (GSD) 2.34] and 0.107 mg/g creatinine (GSD 3.49), respectively. The reference intervals estimated for MA and PGA were 0.084-2.339 and 0.009-1.238 mg/g creatinine, respectively., Conclusion: The application of highly sensitive and selective mass spectrometry-based analytical techniques could be useful in the definition of reference values for metabolites of organic pollutants and for better assessment of low-level occupational exposure to organic solvents.