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Introduction: Exposures to volatile organic compounds and metals have previously been associated with liver diseases including steatohepatitis, although more data are needed. Benzene, toluene, ethylbenzene, xylenes, styrene (BTEXS) and metals were measured in blood samples collected between May 2012-July 2013 from volunteers participating in home visits for the Gulf Long-term Follow-up (GuLF) Study. This cross-sectional analysis evaluates associations of exposure biomarkers with serum liver injury and adipocytokine biomarkers in a sample of 214 men., Methods: Adult nonsmoking men without a history of liver disease or heavy alcohol consumption were included. The serologic disease biomarkers evaluated were the hepatocellular injury biomarker, cytokeratin 18 [whole (CK18 M65) and caspase-cleaved fragment (CK18 M30)]; and adipocytokines. Confounder-adjusted beta coefficients were determined using linear regression models for the overall sample (primary endpoints) and for obesity-classified sub-groups (secondary endpoints). A product interaction term between the exposure of interest and a dichotomized indicator of obesity was included to determine the disease modifying effects of obesity on the biomarker associations., Results: The study sample was 57% white and 51% obese. In the overall sample, lead was positively associated with CK18 M30 (β = 21.7 ± 6.0 (SE), p = 0.0004); IL-1β (β = 32.8 ± 5.2, p < 0.0001); IL-6 (β = 72.8 ± 18.3, p = 0.0001); and IL-8 (β = 140.8 ± 42.2, p = 0.001). Cadmium exposures were associated with increased IL-1β (β = 77.8 ± 26.3, p = 0.003) and IL-8 (β = 419.5 ± 201.2, p = 0.04). There were multiple significant interactions between obesity and exposure to lead, cadmium, benzene and toluene in relation to outcome biomarkers. Among obese participants (n = 108), benzene, lead, and cadmium were each positively associated with CK18 M30, IL-1β, IL-6, and IL-8. In obese subjects, lead was also inversely associated with leptin, and toluene was positively associated with IL-1β., Conclusion: For the overall sample, heavy metal exposures were associated with liver injury (lead only) and/or systemic inflammation (lead and cadmium). Obesity modified the associations between BTEXS and heavy metal exposures on several of the outcome variables. In the obesity subgroup, liver injury was positively associated with lead, cadmium and benzene exposures; systemic inflammation was increased with lead, cadmium, benzene, and toluene exposures; and leptin was inversely associated with lead exposures. The cross-sectional design of this study makes it difficult to determine causality, and all results should be interpreted cautiously. Nonetheless, the potential impact of exposures to lead, cadmium, benzene and toluene in steatohepatitis, an obesity-associated inflammatory liver disease, warrants further investigation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Cave previously received a contract from Lakeside Life Sciences related to the clinical development of the cytokeratin 18 biomarker for alcoholic hepatitis. That contract is not directly related to this work., (Published by Elsevier Ltd.)

Amphotericin B (AmB), which plays a central role in the treatment of systemic fungal infections, is difficult to formulate because it's sparingly soluble in water and organic solvents. We previously prepared AmB-loaded micelles using styrene-maleic acid copolymer (SMA). Although solubilization was achieved by this formulation, stability in the blood circulation was as insufficient as that of Fungizone®, which is a conventional formulation of AmB. Meanwhile, it is well known that polymer-drug conjugates are more stable in circulation than drug-loaded micelles. Therefore, in this study, we developed covalently conjugated SMA-AmB (SMA-AmB conjugate). The SMA-AmB conjugate was found to be soluble and present as micelles in aqueous solution. Furthermore, it was revealed that this micelle behaves as a larger molecule by forming a complex with albumin. The circulation in the blood increased significantly compared to that of Fungizone®, which was suggested to be due to this complex-forming ability. Although in vitro and in vivo antifungal activity of the SMA-AmB conjugate against Saccharomyces cerevisiae was reduced by 1/3 compared to that of Fungizone®, hemolysis decreased to 1/40 or less, and the LD 50 decreased to 1/10. In conclusion, it is expected that the SMA-AmB conjugate can be a polymer-therapeutic agent with high antifungal selectivity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Long-term exposure to certain volatile organic compounds is a significant public health concern. A variety of food containers and drinking cups prepared from polystyrene or polystyrene-related plastics could contain styrene monomer. In the current study, the concentrations of styrene in plasma and liver were surveyed and determined after oral doses of 25 mg/kg to rats and 200 mg/kg to control and humanized-liver mice. Plasma concentrations of styrene in rats were still detected 2 hr after 10-25 mg/kg oral doses. In contrast, after an order of magnitude higher oral dose of styrene (200 mg/kg) to mice, styrene in mouse plasma was rapidly cleared within 15 min to the limit-of-detection level. However, unmetabolized styrene was detected in mouse liver 24 hr after oral treatment. A simple physiologically based pharmacokinetic (PBPK) model capable of estimating blood and liver concentrations of styrene was established for rats. A human PBPK model was then set up for styrene by using the same intrinsic hepatic clearances in rats and humans and by applying allometric scaling to rat parameters obtained from the plasma concentrations of styrene in rats. By reverse dosimetry analysis (from concentrations to doses), we found that the 95th percentile values of styrene concentrations (0.132 ng/mL) reported in United States biomonitoring data of more than 1000 human blood samples may imply exposure to repeated oral doses of styrene of 2.89 µg/kg/day. These results suggest that styrene biomonitoring data in human blood samples imply exposures roughly similar to or lower than the established tolerable daily intake level of 7.7 μg/kg/day.

Data from National Health and Nutrition Examination Survey were analyzed to evaluate detection rates, trend in and factors affecting the observed levels of 1,4-dichlorobenzene, benzene, ethylbenzene, o-xylene, styrene, toluene, and m/p-xylene among US adolescents and adults over 2005-2012. Over 2005-20102, among adolescents, detection rates declined by more than 50% for benzene, ethylbenzene, and o-xylene, and among adults, detection rates declined by more than 50% for ethylbenzene and o-xylene and by a little less than 50% for benzene. Among adults, adjusted levels of 1, 4-dichlorobenzene, benzene, ethylbenzene, o-xylene, toluene, and m/p-xylene decreased by 13.7%, 17.1%, 20%, 17.7%, 23.2%, and 18.7% respectively for every two-year survey cycle. Among adolescents, percentage decline in the levels of 1, 4-dichlorobenzene, benzene, ethylbenzene, o-xylene, styrene, toluene, and m/p-xylene was 15.2%, 21.4%, 19.3%, 16.1%, 47.8%, and 17.7% respectively for every two year survey period. The ratio of adjusted geometric means for adult smokers as compared to adult nonsmokers was 10.7 for benzene, 3.5 for ethylbenzene, 2.0 for o-xylene, 3.4 for styrene, 3.5 for toluene, and 2.2 for m/p-xylene. Among adolescents, gender did not affect the adjusted levels of any of the seven VOCs, and the order in which adjusted levels for 1, 4-dichlorobenzene by race/ethnicity was observed was: non-Hispanic white (0.038ng/mL)

There is potential for oral exposure of humans to styrene (ST) such as from migration of residual levels in polystyrene food containers. After absorption, ST is metabolised to styrene-7,8-oxide (SO), an alkylating epoxide. Hence, a comparison of blood burdens of SO resulting from oral exposures to ST was made with SO burdens possibly warranting genotoxic concern. A validated physiological toxicokinetic model was used for the assessment. Model calculations predicted for exposures to ST that maximum concentrations of SO in venous blood of rats and humans should not exceed 0.33 μg/ml and 0.036 μg/ml, respectively, because of saturation of the SO formation from ST. The daily area under the concentration-time curve of SO in venous blood (AUCSO) was directly proportional to the dose of ST (mg/kg body weight; BW), independent of the exposure route (inhalation or oral exposure). In resting humans, the daily AUCSO was about half that in rats at the same amount of ST/kg BW (calculated up to 100mg ST/kg BW in humans). Taking into account the results of cytogenetic studies in ST-exposed rats, it was deduced that no genotoxic effects of SO are to be expected in ST-exposed humans, at least up to a daily amount of 100mg ST/kg BW, which is equivalent to 100 times the amount originating from the Overall Migration Limit in the EU for ST migrating from food contact plastics. Therefore, no potential genotoxic concern is predicted for ST uptake from food packaging, based on the reported combined measured and modelled data., (Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Introduction: The comet assay is a fluorescent microscopic method that is able to detect DNA strand-breaks even in non-proliferative cells in samples with low cell counts., Aim: The aim of the authors was to measure genotoxic DNA damage and assess oxidative DNA damage caused by occupational exposure in groups exposed to benzene, polycyclic aromatic carbohydrates and styrene at the workplace in order to clarify whether the comet assay can be used as an effect marker tool in genotoxicology monitoring., Method: In addition to the basic steps of the comet assay, one sample was treated with formamido-pirimidine-DNA-glycolase restriction-enzyme that measures oxidative DNA damage., Results: An increase was observed in tail moments in each group of untreated and Fpg-treated samples compared to the control., Conclusions: It can be concluded that occupational exposure can be detected with the method. The comet assay may prove to be an excellent effect marker and a supplementary technique for monitoring the presence or absence of genotoxic effects.

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: The aim of this study was to evaluate the expression of a panel of genes involved in toxicology in response to styrene exposure at levels below the occupational standard setting., Methods: Workers in a fiber glass boat industry were evaluated for a panel of stress- and toxicity-related genes and associated with biochemical parameters related to hepatic injury. Urinary styrene metabolites (MA+PGA) of subjects and environmental sampling data collected for air at workplace were used to estimate styrene exposure., Results: Expression array analysis revealed massive upregulation of genes encoding stress-responsive proteins (HSPA1L, EGR1, IL-6, IL-1β, TNSF10 and TNFα) in the styrene-exposed group; the levels of cytokines released were further confirmed in serum. The exposed workers were then stratified by styrene exposure levels. EGR1 gene upregulation paralleled the expression and transcriptional protein levels of IL-6, TNSF10 and TNFα in styrene exposed workers, even at low level. The activation of the EGR1 pathway observed at low-styrene exposure was associated with a slight increase of hepatic markers found in highly exposed subjects, even though they were within normal range. The ALT and AST levels were not affected by alcohol consumption, and positively correlated with urinary styrene metabolites as evaluated by multiple regression analysis., Conclusion: The pro-inflammatory cytokines IL-6 and TNFα are the primary mediators of processes involved in the hepatic injury response and regeneration. Here, we show that styrene induced stress responsive genes involved in cytoprotection and cytotoxicity at low-exposure, that proceed to a mild subclinical hepatic toxicity at high-styrene exposure.

Background: Biomonitoring of chemicals in the workplace provides an integrated characterization of exposure that accounts for uptake through multiple pathways and physiological parameters influencing the toxicokinetics., Objectives: We used the case of styrene to (i) determine the best times to sample venous blood and end-exhaled air, (ii) characterize the inter-individual variability in biological levels following occupational exposure and (iii) propose biological limit values using a population physiologically based pharmacokinetic (PBPK) model., Methods: We performed Monte Carlo simulations with various physiological, exposure and workload scenarios. Optimal sampling times were identified through regression analyses between levels in biological samples and 24-h area under the arterial blood concentration vs. time curve. We characterized the variability in levels of styrene in biological samples for exposures to a time weighted average (TWA) of 20ppm., Results: Simulations suggest that the best times to sample venous blood are at the end of shift in poorly ventilated workplaces and 15min after the shift in highly ventilated workplaces. Exhaled air samples are most informative 15min after the shift. For a light workload, simulated styrene levels have a median (5th-95th percentiles) of 0.4mg/l (0.2-0.6) in venous blood at the end of shift and 0.5ppm (0.3-0.8) in exhaled air 15min after the end of shift., Conclusion: This study supports the current BEI(®) of the ACGIH of 0.2mg/l of styrene in venous blood at the end of shift and indicates a biological limit value of 0.3ppm in end-exhaled air 15min after the end of shift., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The cytogenetic alterations in leukocytes and the increased risk for leukemia, lymphoma, or all lymphohematopoietic cancer observed in workers occupationally exposed to styrene have been associated with its hepatic metabolisation into styrene-7,8-oxide, an epoxide which can induce DNA damages. However, it has been observed that styrene-7,8-oxide was also found in the atmosphere of reinforced plastic industries where large amounts of styrene are used. Since the main route of exposure to these compounds is inhalation, in order to gain new insights regarding their systemic genotoxicity, Fisher 344 male rats were exposed in full-body inhalation chambers, 6 h/day, 5 days/week for 4 weeks to styrene-7,8-oxide (25, 50, and 75 ppm) or styrene (75, 300, and 1000 ppm). Then, the induction of micronuclei in circulating reticulocytes and DNA strand breaks in leukocytes using the comet assay was studied at the end of the 3rd and 20th days of exposure. Our results showed that neither styrene nor styrene-7,8-oxide induced a significant increase of the micronucleus frequency in reticulocytes or DNA strand breaks in white blood cells. However, in the presence of the formamidopyridine DNA glycosylase, an enzyme able to recognize and excise DNA at the level of some oxidized DNA bases, a significant increase of DNA damages was observed at the end of the 3rd day of treatment in leukocytes from rats exposed to styrene but not to styrene-7,8-oxide. This experimental design helped to gather new information regarding the systemic genotoxicity of these two chemicals and may be valuable for the risk assessment associated with an occupational exposure to these molecules., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The impact of cigarette smoking on volatile organic compound (VOC) blood levels is studied using 2003-2004 National Health and Nutrition Examination Survey (NHANES) data. Cigarette smoke exposure is shown to be a predominant source of benzene, toluene, ethylbenzene, xylenes and styrene (BTEXS) measured in blood as determined by (1) differences in central tendency and interquartile VOC blood levels between daily smokers [≥1 cigarette per day (CPD)] and less-than-daily smokers, (2) correlation among BTEXS and the 2,5-dimethylfuran (2,5-DMF) smoking biomarker in the blood of daily smokers, and (3) regression modeling of BTEXS blood levels versus categorized CPD. Smoking status was determined by 2,5-DMF blood level using a cutpoint of 0.014 ng/ml estimated by regression modeling of the weighted data and confirmed with receiver operator curve (ROC) analysis. The BTEXS blood levels among daily smokers were moderately-to-strongly correlated with 2,5-DMF blood levels (correlation coefficient, r, ranging from 0.46 to 0.92). Linear regression of the geometric mean BTEXS blood levels versus categorized CPD showed clear dose-response relationship (correlation of determination, R(2), ranging from 0.81 to 0.98). Furthermore, the pattern of VOCs in blood of smokers is similar to that reported in mainstream cigarette smoke. These results show that cigarette smoking is a primary source of benzene, toluene and styrene and an important source of ethylbenzene and xylene exposure for the U.S. population, as well as the necessity of determining smoking status and factors affecting dose (e.g., CPD, time since last cigarette) in assessments involving BTEXS exposure., (Published by Elsevier Ltd.)

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.)

Decreased levels of single-strand breaks in DNA (SSBs), reflecting DNA damage, have previously been observed with increased styrene exposure in contrast to a dose-dependent increase in the base-excision repair capacity. To clarify further the above aspects, we have investigated the associations between SSBs, micronuclei, DNA repair capacity and mRNA expression in XRCC1, hOGG1 and XPC genes on 71 styrene-exposed and 51 control individuals. Styrene concentrations at workplace and in blood characterized occupational exposure. The workers were divided into low (below 50 mg/m³) and high (above 50 mg/m³)) styrene exposure groups. DNA damage and DNA repair capacity were analyzed in peripheral blood lymphocytes by Comet assay. The mRNA expression levels were determined by qPCR. A significant negative correlation was observed between SSBs and styrene concentration at workplace (R=-0.38, p=0.001); SSBs were also significantly higher in men (p=0.001). The capacity to repair irradiation-induced DNA damage was the highest in the low exposure group (1.34±1.00 SSB/10⁹ Da), followed by high exposure group (0.72±0.81 SSB/10⁹ Da) and controls (0.65±0.82 SSB/10⁹ Da). The mRNA expression levels of XRCC1, hOGG1 and XPC negatively correlated with styrene concentrations in blood and at workplace (p<0.001) and positively with SSBs (p<0.001). Micronuclei were not affected by styrene exposure, but were higher in older persons and in women (p<0.001). In this study, we did not confirm previous findings on an increased DNA repair response to styrene-induced genotoxicity. However, negative correlations of SSBs and mRNA expression levels of XRCC1, hOGG1 and XPC with styrene exposure warrant further highly-targeted study., (Copyright © 2010 Elsevier Inc. All rights reserved.)

This study investigated nucleic acid oxidation associated with styrene exposure, mRNA expression levels of hOGG1 gene and the role of the genetic polymorphism Ser326Cys of human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) in 60 styrene-exposed workers and 50 unexposed clerks. Biomarkers of exposure (styrene in blood, mandelic and phenylglyoxylic acids and 4-vinylphenol in urine) and urinary biomarkers of nucleic acid oxidation, namely 8-oxo-7,8-dihydro-2'-deoxyguanosine (U-8-oxodGuo), 8-oxo-7,8-dihydroguanosine (U-8-oxoGuo) and 8-oxo-7,8-dihydroguanine (U-8-oxoGua) were determined by liquid chromatography-tandem mass spectrometry. The levels of 8-oxodGuo adduct and 2'-deoxyguanosine (dGuo) were measured by HPLC in DNA from white blood cells (WBC). Genomic DNA and RNA from blood samples were used to characterize the Ser326Cys polymorphism and the mRNA expression levels of the hOGG1 gene, respectively, by PCR-based methods. Exposed workers showed lower values of 8-oxodGuo/10(5) dGuo ratio in WBC-DNA but higher concentrations of U-8-oxoGuo compared to controls (p=0.002 and p=0.008, respectively, t-test for independent samples). In the whole group, all urinary biomarkers of nucleic acid oxidation correlated with both the sum of mandelic and phenylglyoxylic acids (rho>0.33, p<0.0001) and 4-vinylphenol (rho>0.29, p<0.001), whereas 8-oxodGuo/10(5) dGuo in WBC showed a negative correlation with exposure parameters (rho<-0.24, p<0.02). Subjects bearing the hOGG1 Ser/Ser genotype showed lower values of 8-oxodGuo/10(5) dGuo in WBC than those with at least one variant Cys allele (0.34+/-0.16 vs 0.45+/-0.21, p=0.008). In the subgroup of hOGG1 Ser/Ser subjects, laminators showed lower levels of WBC 8-oxodGuo/10(5) dGuo ratio and significantly higher concentrations of U-8-oxoGua than controls (p=0.07 and p=0.01, respectively, t-test for independent samples). Interestingly, workers showed higher levels of hOGG1 expression compared to controls (p<0.0005). Styrene exposure seems to be associated with oxidation damage to nucleic acids, particularly to RNA and with an induction of the BER system.

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.

Epidemiological and laboratory investigations have shown that toluene and styrene are toxic compounds that lead to impairment of the nervous system. To quantitate toluene and styrene in biological samples, liquid-liquid phase, headspace (HS), and solid-phase microextraction (SPME) methods are generally used. Most of these methods are not sensitive enough for applications involving small sample volumes. Here, we present a method for quantitative analysis of low concentrations of styrene and toluene in very small volumes of biological samples using HS-SPME and gas chromatography (GC) equipped with a flame-ionization detector. The method was developed by optimizing operating parameters that affect the HS-SPME-GC process [i.e., desorption time (30 s), depth of the fiber in the GC injection port (3.7 cm), adsorption time (4 min), and adsorption temperature (room temperature)]. It has a wide range of linearity (0.5-500 ng/10 microL), high precision (coefficient of variation < 5%), good accuracy (deviation < 11%), and low detection limits of 0.13 and 0.08 ng/10 microL for styrene and toluene in serum, respectively. This analytical technique can be applied to the estimation of styrene and toluene in small volumes of biological fluids (blood, serum, and perilymph) and tissues of low lipid content (cochlea).

Exposure assessment of individuals exposed to certain chemicals plays an important role in the analysis of occupational-as well as environmental-health problems. Biological monitoring, as an alternative to direct environmental measurements, may be applied to relate the exterior exposure with the amount of individual intake. In this paper, we estimate individuals' (inhalation) exposure retrospectively from their blood concentrations via a simplified one-compartment toxicokinetic model. Considering stochastic variations to the toxicokinetic model, the solution to the resultant stochastic differential equation (SDE), together with measurement error, is transformed into a dynamic linear state-space model. The unknown model parameters and the mean inhalation concentration are then estimated via Markov Chain Monte Carlo (MCMC) simulations. The proposed method is used in the analysis of the styrene data (Wang et al. in Occup Environ Med 53:601-605, 1996) to backward estimate the inhalation concentration, assuming it is unknown. The data analysis showed that the internal stochastic variations, often ignored in toxicokinetic model analysis, outweighed in standard deviation almost twice that of the measurement error. Also, the simulation results showed that the method performed relatively well to the approach considering measurement error only.

It is known that styrene is ototoxic and causes cochlear damage starting from the middle turn. However, the cellular mechanism underlying styrene ototoxicity is still unclear. In this study, rats were exposed to styrene by gavage at different doses once a day for varying periods. Styrene levels in the cochlear tissues, styrene-induced permanent hearing loss, cochlear disruptions, and cell death pathways were determined. Styrene concentration in the cochlea varied along with the basilar membrane with the lowest level in the basal turn being consistent with the lowest styrene-induced threshold shift and hair cell loss in this region. After 3 weeks of exposure (5 days per week), a dose-dependent permanent hearing loss and a hair cell loss, especially in the midfrequency region, were observed. The styrene exposure at a dose of 200 mg/kg, which induced a blood level of 6.0 +/- 1.0 microg/g, caused an average of 4.4 +/- 0.5% OHC (outer hair cell) loss and 2-5 dB threshold shift in the cochlear region of 20-70% from the apex. A significant OHC loss was not observed until 7 days of exposure at a dose of 800 mg/kg. Deiters cells appeared to be the most vulnerable target of styrene. When condensed nuclei were observed in Deiters cells after a few days of styrene exposure (800 mg/kg), other cells were still intact. Apoptotic cell death appeared to be the main cell death pathway in the cochlea after styrene exposure. In the styrene-induced apoptotic OHCs, histochemical staining detected activated caspases-9 and 8, indicating that both mitochondrial-dependent pathway and death receptor-dependent pathway were involved in the styrene-induced cell death.

Objective: To identify demographic and work related factors that predict blood levels of styrene and styrene-7,8-oxide (SO) in the fibreglass reinforced plastics (FRP) industry., Methods: Personal breathing-zone air samples and whole blood samples were collected repeatedly from 328 reinforced plastics workers in the Unuted States between 1996 and 1999. Styrene and its major metabolite SO were measured in these samples. Multivariable linear regression analyses were applied to the subject-specific levels to explain the variation in exposure and biomarker levels., Results: Exposure levels of styrene were approximately 500-fold higher than those of SO. Exposure levels of styrene and SO varied greatly among the types of products manufactured, with an 11-fold range of median air levels among categories for styrene and a 23-fold range for SO. Even after stratification by job title, median exposures of styrene and SO among laminators varied 14- and 31-fold across product categories. Furthermore, the relative proportions of exposures to styrene and SO varied among product categories. Multivariable regression analyses explained 70% and 63% of the variation in air levels of styrene and SO, respectively, and 72% and 34% of the variation in blood levels of styrene and SO, respectively. Overall, air levels of styrene and SO appear to have decreased substantially in this industry over the last 10-20 years in the US and were greatest among workers with the least seniority., Conclusions: As levels of styrene and SO in air and blood varied among product categories in the FRP industry, use of job title as a surrogate for exposure can introduce unpredictable measurement errors and can confound the relation between exposure and health outcomes in epidemiology studies. Also, inverse relations between the intensity of exposure to styrene and SO and years on the job suggest that younger workers with little seniority are typically exposed to higher levels of styrene and SO than their coworkers.

Widespread exposure to benzene, toluene, ethylbenzene, xylene, and styrene (BTEXS) and the potential for this exposure to cause health effects drives the need to develop improved methods for measuring exposure. In this work, we demonstrate our latest assay for quantifying BTEXS in blood and characterize sources of both positive and negative biases. This method involves blood sample collection using common techniques followed by static headspace sampling using solid-phase microextraction and gas chromatography/mass spectrometry analysis. We found that the greatest and unexpected source of positive bias was from contamination of butyl rubber materials used in sample preparation consumables such as Vacutainer stoppers, syringe plungers, and sample vial septa. Conversely, the primary cause of negative bias observed was from the diffusion loss of BTEXS from blood during transfer into sample vials. By minimizing or eliminating these and other sources of bias, we improved method accuracy and precision to within 10% while maintaining low-picogram per milliliter detection. Furthermore, upon comparison of these results with those from other laboratories, we observe substantially lower blood BTEXS levels reported to date for nonoccupationally exposed nonsmokers. A relatively unbiased method, as such, will help elucidate any potential associations between adverse health effects and human exposure to low levels of BTEXS.

There have been several epidemiological and experimental studies about styrene from the neuroendocrinological viewpoint. Some reported that styrene exposure affected the neuroendocrinological system and enhanced prolactin (PRL) secretion, but others have denied those effects. It was assumed that styrene exposure caused depletion of dopamine (DA), which is a PRL inhibitor, and that, in consequence, the PRL level increased. However, not only DA but also many other factors control PRL secretion. Therefore, the mechanism of hypersecretion of PRL has not yet been clearly elucidated. In addition, effects of styrene on the female reproductive system have been reported, but the susceptibility needs to be further studied. Therefore, to investigate what causes hypersecretion of PRL and how different the susceptibility is in males and females, we studied acute effects of styrene exposure on the neuroendocrinological system in male and female rats. Immediately after exposure to 150 ppm styrene vapor for 10 days (8 h/day), male and female rats were killed, and blood and brain samples were collected. The styrene concentration in blood, hormones such as PRL, growth hormone (GH) and thyroid-stimulating hormone (TSH) in plasma and neurotransmitters in various brain regions were measured. The styrene concentration in the blood of female rats was higher than that in male rats, and the PRL level was significantly increased in female exposed rats compared with controls. No significant change was observed in male rats. We did not observe any significant changes in DA, 5-hydroxytryptamine (5-HT) or their metabolites. Because neurotransmitters were not affected in either male or female rats, the mechanism enhancing PRL secretion remains unclear. These results suggest that styrene exposure may cause hypersecretion of PRL and that the sensitivity to styrene exposure of the female may be higher than that of the male.

Two experimental studies were conducted with the intention to simulate exposure characteristics of work places with styrene exposure and to investigate the risk for neurobehavioral impairments. In experiment I 16 volunteers (8 in the morning, 8 in the afternoon) were exposed to 0.5 and 20 ppm styrene on a constant level for 3h. In experiment II 24 volunteers (12 in the morning, 12 in the afternoon) were exposed for 4h to 0.5 and 20 ppm styrene on a constant level as well as to a changing exposure between 0.5 and 40 ppm with a TWA of 14 ppm. Simple reaction, choice reaction, attention, acute symptoms, and ratings for well-being were measured. Exposure related performance effects could not be detected. However, 6h time change resulted in delayed choice reactions in the morning hours. Analysing acute symptoms and the state of well-being the impact of styrene did not reach adverse extents of impaired well-being.

There is clear evidence that aromatic solvents can disrupt the auditory system in humans and animals. As far as animal models are concerned, solvent-induced hearing loss seems to be species-dependent. Indeed, most published data have been obtained with the rat, which shows mid-frequency cochlear deficits, whereas the guinea pig does not show any permanent hearing loss after solvent exposure. In the current investigation, the effects of two solvents, toluene (600 ppm) and styrene (1000 ppm), were studied in both Long-Evans rats and pigmented guinea pigs exposed 6 h/day for 5 consecutive days. Cochlear function was tested by using distortion product otoacoustic emissions (DPOAE) measured prior to the solvent exposure, 20 min after the end of the exposure and successively at 2 and 4 weeks post-exposure. In addition to cochlear testing, solvent concentrations in blood and urinary metabolites were measured. A cochlear histological analysis was performed at the end of the experiment. No decrease in DPOAE amplitude was observed in the guinea pig, even immediately following the end of exposure. The rat model showed severe disruption of auditory function and cochlear pathology, whereas the guinea pig had no disruption of DPOAE or cochlear pathological alterations. Therefore, the vulnerability of the cochlear function was strictly dependent on the species. As expected, an important difference in the styrene concentration in blood was observed: the solvent concentrations were fourfold higher in the rat than in the guinea pig. Therefore, it is clear that a pharmacokinetic or an uptake difference might explain the difference in susceptibility observed between the two species. Moreover, the metabolism pathways of the solvents were different depending on the species. Attempts to explain differences of vulnerability between the rat and guinea pig are addressed in the present paper.

A purge-and-trap gas chromatographic (PT-GC) method for determining styrene concentrations in urine and blood samples has been used in the biological monitoring of workers exposed to styrene and acetone. Blood and urine samples were collected from 34 individuals exposed to both solvents at the end of a 4-h shift and measured for styrene in urine (Su), blood (Sb), and the two major urinary metabolites, mandelic acid (MA) and phenylglyoxylic acid (PGA). A second urine sample was taken at the beginning of the next shift. Environmental exposure was measured using passive personal monitoring and GC. Urinary excretion of MA and PGA was measured by high-performance liquid chromatography. The average exposures to styrene and acetone were 70.5 mg/m3 and 370.5 mg/m3, respectively. In end-of-shift samples there was a significant correlation between concentrations of Su and Sb and the metabolites PGA, MA (r = 0.714 and 0.788, p < 0.001 for Su and r = 0.644 and 0.566, p < 0.005 for Sb). A high correlation between Sb and Su (r = 0.732, p < 0.001) also existed. Poor correlations were found between Su and metabolites in samples collected at the beginning of the next shift (r = 0.491 and 0.474 for PGA and MA, respectively, p < 0.05). There was a better correlation between the biological parameters at the end of the shift and the environmental styrene (r = 0.841 for PGA, r = 0.834 for MA, r = 0.788 for Su, and r = 0.698 for Sb; p < 0.001) compared with those at the start of the shift (r = 0.81 for PGA, 0.675 for MA, and 0.650 for Su; p < 0.001). We found that the concentration of excreted metabolites decreased significantly when environmental concentrations of acetone increased (p < 0.05), particularly at the end of the shift. Although the best correlation with environmental styrene was obtained with the sum of PGA and MA at the end of the shift (r = 0.862, p < 0.001), urine and blood styrene were shown to be more useful biological monitoring indicators because their concentrations were not affected by acetone co-exposure.

Workers in the reinforced plastics industry are exposed to large quantities of styrene and to small amounts of the carcinogen, styrene-7,8-oxide (SO), in air. Since SO is also the primary metabolite of styrene, we modified a published physiologically based pharmacokinetic (PBPK) model to investigate the relative contributions of inhaled SO and metabolically derived SO to the systemic levels of SO in humans. The model was tested against air and blood measurements of styrene and SO from 252 reinforced plastics workers. Results suggest that the highly efficient first-pass hydrolysis of SO via epoxide hydrolase in the liver greatly reduces the systemic availability of SO formed in situ from styrene. In contrast, airborne SO, absorbed via inhalation, is distributed to the systemic circulation, thereby avoiding such privileged-access metabolism. The best fit to the model was obtained when the relative systemic availability (the ratio of metabolic SO to absorbed SO per unit exposure) equaled 2.75 x 10(-4), indicating that absorbed SO contributed 3640 times more SO to the blood than an equivalent amount of inhaled styrene. Since the ratio of airborne styrene to SO rarely exceeds 1500 in the reinforced plastics industry, this indicates that inhalation of SO presents a greater hazard of cytogenetic damage than inhalation of styrene. We conclude that future studies should assess exposures to airborne SO as well as styrene.

Styrene is an extensively used industrial chemical that has been classified as a possible human carcinogen. The possible carcinogenicity may be related to the covalent DNA binding properties of styrene 7,8-oxide, a major metabolite of styrene. We have developed a sensitive and a highly specific phosphorus-32-postlabeling method for the determination of 1-styrene 7,8-oxide-adenine DNA adducts. These adducts were analyzed in white blood cells from workers exposed to styrene at mean level of 76.2 mg/m3. Three of nine exposed workers showed adducts above the detection limit; the mean was 0.79 +/- 0.14 1-styrene 7,8-oxide-adenine DNA adducts/10(9) nucleotides. None of the 11 control cells showed adducts above the detection limit, which was 0.4 adducts/10(9) nucleotides. The results show a potential of 1-adenine DNA adducts for predicting risks in the workers exposed to styrene.

Methods of isotope-dilution gas chromatography-mass spectrometry (GC-MS) are described for the determination of styrene and styrene-7,8-oxide (SO) in blood. Styrene and SO were directly measured in pentane extracts of blood from 35 reinforced plastics workers exposed to 4.7-97 ppm styrene. Using positive ion chemical ionization, styrene could be detected at levels greater than 2.5 microg/l blood and SO at levels greater than 0.05 microg/l blood. An alternative method for measurement of SO employed reaction with valine followed by derivatization with pentafluorophenyl isothiocyanate and analysis via negative ion chemical ionization GC-MS-MS (SO detection limit=0.025 microg/l blood). The detection limits for SO by these two methods were 10-20-fold lower than gas chromatographic assays reported earlier, based upon either electron impact MS or flame ionization detection. Excellent agreement between the two SO methods was observed for standard calibration curves while moderate to good agreement was observed among selected reinforced plastics workers (n = 10). Levels of styrene in blood were found to be proportional to the corresponding air exposures to styrene, in line with other published relationships. Although levels of SO in blood, measured by the direct method, were significantly correlated with air levels of either styrene or SO among the reinforced plastics workers, blood concentrations were much lower than previously reported at a given exposure to styrene. The two assays for SO in blood appear to be unbiased and to have sufficient sensitivity and specificity for applications involving workers exposed to styrene and SO during the manufacture of reinforced plastics.

Background: Styrene is a widely used industrial chemical. Immune and hematological parameters were examined in 29 hand laminators and sprayers exposed to styrene for an average of 14 years and in 19 in-factory unexposed controls. The workers performed hand lamination procedures in a production area with an average area airborne styrene level of 139.5 mg/m(3). Mean concentration of styrene in the blood of exposed workers was 945.7 microg/L and the mean styrene in exhaled air was 38.8 microg/L., Methods: Parameters of internal and external exposure, immune function assays, immunoglobulins, acute phase reactants and hematology were evaluated in exposed and non-exposed populations., Results: Using multifactorial analysis of variance we found a significant decrease in proliferation of lymphocytes stimulated by Concanavalin A but not by pokeweed mitogen (PWM) in workers occupationally exposed to styrene. Proliferative response to PWM was significantly correlated with the levels of styrene in blood. Phagocytic activity of monocytes, levels of IgG, IgA, IgM, IgE and alpha-2-macroglobulin in serum were indistinguishable in the two groups. The population exposed to styrene had increased levels of C4-component of complement. Levels of C3-component of complement were positively correlated with duration of exposure. A significant elevation in the percentage and number of monocytes and a significantly decreased number of lymphocytes were seen in exposed workers. Styrene concentrations in both blood and exhaled air were associated with decreased percentage of large granular lymphocytes., Conclusions: These results suggest immune alterations of cell-mediated immune response of T-lymphocytes and imbalance in leucocyte subsets in peripheral blood of workers exposed to styrene., (Copyright 2000 Wiley-Liss, Inc.)

A simple purge-and-trap gas chromatographic method with flame ionization detection was developed for the determination of styrene in urine and blood. Styrene present in a 5 ml sample at room temperature was swept by helium at 40 ml/min for 11 min, trapped on a Tenax trap, desorbed by heating, cryofocused, and injected by flash heating into a DB-5 capillary GC column. The oven temperature program was from 80 degrees C, held for 8 min, to 120 degrees C at 5 degrees C/min, and then held for 2 min. The detector temperature was 250 degrees C. The calibration curves were linear in the range of 2.5-15 ppb styrene in urine and 25-150 ppb in blood. The detection limits calculated were 0.4 microg/l in urine and 0.6 microg/l in blood. The coefficients of variations within the day and day-to-day were 3 and 3.1%, respectively, for 2.5 ppb of styrene in urine, and 1 and 1.6% for 25 ppb of styrene in blood. The results obtained from samples taken from workers exposed to styrene were reported.

Cysteinyl adducts of hemoglobin (Hb) and albumin (Alb) formed via reactions with reactive species were measured in 48 subjects exposed to styrene (0.24-55.2 ppm) and to styrene-7,8-oxide (SO) (2.65-107 ppb) in a factory producing boats in the USA. Hb and Alb adducts were also investigated among 88 workers exposed to benzene (0-138 ppm) in several Chinese factories. The particular adducts were S-(2-hydroxy-1-phenylethyl) cysteine, from reactions of SO with Alb (designated SO-Alb), and S-phenylcysteine, from reactions of the CYP450 benzene metabolite, benzene oxide (BO), with Hb and Alb (designated BO-Hb and BO-Alb, respectively). The relationships between adduct levels and exposures were investigated in both studies. The estimated slopes varied considerably among the particular combinations of adduct and agent to which the workers were exposed, ranging from 0.815 pmol BO-Hb/g Hb per ppm benzene to 24400 pmol SO-Alb/g Alb per ppm SO. We used these estimated slopes, along with kinetic constants, to predict the systemic doses of SO and BO in humans per mg of styrene, SO or benzene per kg body weight, under certain assumptions. Using RX to signify the particular electrophile (SO or BO) the doses of RX to the blood per unit of dose varied between 2.21 and 4110 nM RX-h/mg agent per kg b.w. The dose of RX to the blood arising from inhalation of SO was almost 2000 times that of styrene (i.e. 4110 vs. 2.21 nM RX/mg agent per kg b.w.) and 430-781 times that of benzene (i.e. 4110 vs. 5.26-9.55 nM RX/mg agent per kg b.w.), depending upon the study. Comparable estimates of the blood dose of BO were obtained from adducts of Hb and Alb and two independent studies of BO-Alb yielded similar dose estimates. These results point to the utility of protein adducts as dosimeters of reactive electrophilic species in occupational studies. Finally, significant levels of background adducts of SO and BO with Hb and Alb were observed among workers, among control subjects and in commercial human proteins. Levels of these background adducts were too great to have arisen from non-occupational exposures to styrene or benzene or from cigarette smoking.

It is well established that organic solvents such as toluene and styrene are ototoxic in the rat; however, the intoxication route used to reach the organ of Corti is still questionable. The distribution of toluene and styrene in various tissues of Long-Evans rats (n = 2 x 8) was studied after inhalation of either 1750 ppm toluene or 1750 ppm styrene for 10 h (6 consecutive h + 4 h the following day). At the end of the solvent exposures, blood, brain, auditory nerves, the organ of Corti, cerebrospinal (CSF), and inner ear fluids (IEF) were sampled or removed to measure the rates of solvent uptake in each tissue by gas chromatography. Results indicate that CSF and IEF were free from detectable solvents, whereas the organ of Corti, the nerves, and the brain were contaminated. Therefore, both toluene- and styrene-induced hearing losses are caused by tissue intoxication rather than by fluid contamination. It is proposed that the outer sulcus is used as an intoxication route to reach the organ of Corti.

Short-term inhalation exposure of B6C3F1 mice to styrene causes necrosis of centrilobular (CL) hepatocytes. However, in spite of continued exposure, the necrotic parenchyma is rapidly regenerated, indicating resistance by regenerated cells to styrene toxicity. These studies were conducted to test the hypothesis that resistance to repeated styrene exposure is due to sustained cell proliferation, with production of hepatocytes that have reduced metabolic capacity. Male mice were exposed to air or 500 ppm styrene (6 h/day); hepatotoxicity was evaluated by microscopic examination, serum liver enzyme levels, and bromodeoxyuridine (BrdU)-labeling index (LI). Metabolism was assessed by measurement of blood styrene and styrene oxide. Both single and repeated exposures to styrene resulted in mortality by Day 2; in mice that survived, there was CL necrosis with elevated BrdU LI at Day 6, and complete restoration of the necrotic parenchyma by Day 15. The BrdU LI in mice given a single exposure had returned to control levels by Day 15. Re-exposure of these mice on Day 15 resulted in additional mortality and hepatocellular necrosis, indicating that regenerated CL cells were again susceptible to the cytolethal effect of styrene following a 14-day recovery. However, in mice repeatedly exposed to styrene for 14 days, the BrdU LI remained significantly increased on Day 15, with preferential labeling of CL hepatocytes with enlarged nuclei (karyomegaly). If repeated exposures were followed by a 10-day recovery period, CL karyomegaly persisted, but the BrdU LI returned to control level and CL hepatocytes became susceptible again to styrene toxicity as demonstrated by additional mortality and acute necrosis after a challenge exposure. These findings indicated a requirement for continued styrene exposure and DNA synthesis in order to maintain this resistant phenotype. Analyses of proliferating-cell nuclear-antigen (PCNA) labeling were conducted to further characterize the cell cycle kinetics of these hepatocytes. The proportion of cells in S-phase was increased by repeated exposure. However, PCNA analysis also revealed an even larger increase in the G1 cell compartment with repeated exposures, without a concurrent increase in G2 phase or in mitotic cell numbers. These data indicate that resistance to styrene-induced necrosis under conditions of repeated exposure is not due to sustained cell turnover and production of new, metabolically inactive cells, but rather is due to some other, as yet unknown, protective phenotype of the regenerated cells.

The concentration of styrene in blood of the occupationally styrene-exposed workers was checked by gas chromatographic headspace analysis. Mandelic acd in urine, that is a major metabolite of styrene, and hippuric acid were also analyzed by high performance liquid chromatography. For the biological monitoring of styrene-exposed workers, the routine method of the quantitative determination of styrene and its metabolites in the biological samples were studied.