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High-performance liquid chromatography for the quantitative determination of the urinary metabolites of toluene, xylene, and styrene.

A new high-pressure liquid chromatography (HPLC) method for simultaneous quantitative determination of the urinary metabolites of toluene, m-xylene, and styrene (hippuric acid, m-methylhippuric acid, phenylglyoxylic acid, mandelic acid) is described. The extraction procedure was performed on acidified urines, after addition of 4-hydroxybenzoic acid as internal standard, using a butylchloride/isopropanol mixture and drying 0.5 ml of the organic layer under nitrogen flow. The residue obtained was dissolved in 0.1 ml water/acetonitrile and 5 microliters were injected into an HPLC apparatus equipped with a 0.26 X 25 cm HC ODS SIL X column. Absorbance measures were performed at 225 nm throughout the investigation. All metabolites were clearly separated in a short time (12 min) and the amounts of other urinary compounds affecting the analysis were so small that the measurement of low concentrations of the urinary metabolites could be easily performed. Linear calibration curves were obtained from 0.1 to 3 mg/ml and a correlation coefficient greater than 0.99 was found between concentrations of the standards and areas of the peaks. Statistical analysis confirms that this method, which has a high reproducibility, is simple, reliable, and useful for the biologic monitoring of industrial exposure to aromatic compounds.

Chromatography, High Pressure Liquid↗

Non-linear kinetics of microsomal styrene monooxygenase after phenobarbital pre-treatment.

Pretreatment of rats with phenobarbital, but not with 3-methylcholanthrene, induces liver microsomal styrene monooxygenase. Under these conditions the kinetic profile is not linear and can be divided into 2 distinct curves. Evidence is presented indicating that the combined treatment with phenobarbital and CoCl2 destroy the affinity enzyme, suggesting that the native cytochrome is less sensitive to the action of CoCl2.

Animals↗

[Monitoring of persons exposed to perchloroethylene or styrene].

Forty-nine employees exposed to perchloroethylene (dry cleaning) and 41 employees exposed to styrene (fiber reinforced polyester) are compared to a control group of 68 persons. Symptoms such as dizziness, mouth dryness, fatigue, mucous membranes and skin irritation appear more frequently among the exposed groups, while liver and kidney functions remain unchanged. This study demonstrates that medical examination, industrial hygiene survey and biological monitoring can complement each other in the evaluation of work conditions.

Adult↗

Evaluation of half-mask respirator protection in styrene-exposed workers.

OBJECTIVE: The protection afforded by respirators to styrene (St)-exposed workers varies considerably. Our objective was to study the effective 'in the field' reduction in St exposure obtained by negative-pressure half-mask respirators worn by a group of fiberglass-reinforced plastics (FRP) workers. Protection was evaluated by measuring the reduction in urinary St (StU) excretion. METHODS: Seven FRP workers not using respiratory protection devices were studied for a week. External exposure to St was evaluated by personal passive sampling, and the internal dose by StU measurement. Then workers were asked to use a half-mask respirator for a week for the entire morning half-shift, and St exposure and internal dose were re-assessed. RESULTS: Mean environmental levels of St during the morning half-shift were 230-280 mg/m3, i.e., about three times the current limit proposed by ACGIH; the difference among days was not significant. Using respirators was accompanied by a large inter-individual and also intra-individual variability: the estimated reduction of StU values ranged from 30% to 90% (mean 60%). Mean StU values increased by 50% from Monday to Friday, while environmental St concentrations remained steady. Furthermore, the proportion of workers exceeding the biological equivalent exposure limit (BEEL) was 14% on Monday, double (33%) on Thursday, and triple (43%) on Friday. These data suggest a decrease of protection during the week. CONCLUSIONS: The protection afforded by negative-pressure half-mask respirators varies widely, which stresses the need to assess the effective reduction of exposure whenever these devices are introduced for St-exposed workers. If respirators are to be re-used for several days, their performance must be evaluated during the last shift of use. Measurement of urinary excretion of unmodified St proved a useful tool for the evaluation of respirator effectiveness in exposed workers.

Air Pollutants, Occupational↗

Human urine certified reference material CZ 6009: creatinine, styrene metabolites (mandelic acid and phenylglyoxylic acid).

The reference material was prepared by freeze-drying pooled urine samples obtained from healthy persons occupationally exposed to styrene. The concentrations of mandelic acid (MA), phenylglyoxylic acid (PGA), and hippuric acid (HA) in urine were determined by three modes of high-performance liquid chromatography (HPLC). For isochronous stability testing the urinary mandelic acid and phenylglyoxylic acid concentrations were followed over a 24-month period for a preliminary batch by use of HPLC. No changes of the concentration values were found. The creatinine concentration was stable for more than five years. Standard Reference Material NIST 914a Creatinine was used for traceability purposes for creatinine. Pure chemicals MA and PGA were used for traceability purposes. Control material ClinChek-Urine Control (Recipe) was analyzed simultaneously. The mean values of MA and PGA compare well with the means and fall within the control range of control samples. Results from homogeneity, stability, and traceability testing were evaluated using the statistical program ANOVA. The certified values and their uncertainties were evaluated from the results of interlaboratory comparisons, and homogeneity and stability tests. The values are unweighed arithmetical averages of accepted results and their uncertainties are combined uncertainties (coverage factor=1).

Adult↗

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding phenylacetaldehyde reductase from styrene-assimilating Corynebacterium sp. strain ST-10.

The gene encoding phenylacetaldehyde reductase (PAR), a useful biocatalyst for producing chiral alcohols, was cloned from the genomic DNA of the styrene-assimilating Corynebacterium sp. strain ST-10. The gene contained an opening reading frame consisting of 1,158 nucleotides corresponding to 385 amino acid residues. The subunit molecular weight was calculated to be 40,299, which was in agreement with that determined by polyacrylamide gel electrophoresis. The enzyme was sufficiently expressed in recombinant Escherichia coli cells for practical use and purified to homogeneity by three-column chromatography steps. The predicted amino acid sequence displayed only 20-29% identity with zinc-containing, NAD(+)-dependent, long-chain alcohol dehydrogenases. Nevertheless, the probable NAD(+)- and zinc-binding sites are conserved although one of the three catalytic zinc-binding residues of the zinc-containing, long-chain alcohol dehydrogenases was substituted by Asp in PAR. The protein contains 7.6 mol zinc/mol tetramer. Therefore, the enzyme was considered as a new member of zinc-containing, long-chain alcohol dehydrogenases with a particular and broad substrate specificity.

Alcohol Oxidoreductases↗

Synthesis of 4-O- and 6-O-monoacryloyl derivatives of sucrose by selective hydrolysis of 4,6-O-(1-ethoxy-2-propenylidene) sucrose. Polymerization and copolymerization with styrene.

The synthesis of an ethylenic orthoester of sucrose by transorthoesterification of an acrylic reagent with sucrose is described. Mild hydrolysis of this orthoester gave sucrose selectively monosubstituted by an acryloyl group at either the 4-O- or the 6-O-position. These acrylates were homopolymerized and copolymerized with styrene, and the corresponding polymers were characterized.

Acrylates↗

Chromosomal aberrations in bone marrow of Chinese hamsters exposed to styrene and ethanol.

No significant increase was detected in the number of chromosomally damaged cells in bone marrow of Chinese hamsters after either inhalation exposure to 300 ppm styrene or oral intake of 15% ethanol in drinking water (exposure time 4 days or 3 weeks). In combined exposure, a slight increase of chromosomal aberrations was observed in the group exposed for 4 days (2.0% aberrant cells vs. 0.0% in controls). The group exposed for 3 weeks to combined treatment showed no response (0.3% aberrant cells vs. 0.7% in controls).

Animals↗

Preclinical evaluation for noninvasive reversal following long-term vas occlusion with styrene maleic anhydride in langur monkeys.

A preclinical evaluation for reversal through a noninvasive approach following long-term vas occlusion with styrene maleic anhydride (SMA) has been attempted in langur monkeys at the level of semen parameters, sperm functional tests, semen biochemistry, histology and ultrastructure of reproductive organs, hematology and serum clinical biochemistry including antisperm antibodies (ASA), prostate-specific antigen (PSA) and testosterone. Noninvasive reversal through palpation, percutaneous squeezing and electrical stimulation, forced vibratory movements and suprapubic percussion in the inguinal segments and per-rectal digital massage was attempted in seven langur monkeys after 540 days following vas occlusion. The results revealed instant azoospermia reversal on the same day of reversal with impaired sperm quality, which showed gradual improvement and normospermia with normal motility and viability after 60-90 days of reversal. Sperm functional tests, including ultrastructure of spermatozoa, indicative of sterility in the initial ejaculations, reached normalcy after 90-120 days of reversal. The seminal plasma biochemistry indicative of obstructive azoospermia regained a normal pattern after 90-120 days of reversal. The morphology of testes that showed focal degeneration during 540 days of vas occlusion and that of vasa deferentia that showed exfoliation of epithelial cells resumed to normal morphology comparable with control animals after 150 days of reversal. The morphology of the epididymis, seminal vesicle and prostate did not show appreciable changes following vas occlusion and after noninvasive reversal compared with those of control animals. Hematology, serum clinical chemistry, ASA, PSA and testosterone fluctuated within control limits, indicating safety of the procedure at the level of accessory reproductive organs. The results suggest that noninvasive reversal is feasible even after long-term vas occlusion with SMA and is safe without adverse side effects.

Animals↗

DNA repair and cyclin D1 polymorphisms and styrene-induced genotoxicity and immunotoxicity.

1-SO-adenine DNA adducts, DNA single-strand breaks (SBs), chromosomal aberrations (CAs), mutant frequency (MF) at the HPRT gene, and immune parameters (hematological and of humoral immunity) were studied in styrene-exposed human subjects and controls. Results were correlated with genetic polymorphisms in DNA repair genes (XPD, exon 23, XPG, exon 15, XPC, exon 15, XRCC1, exon 10, XRCC3, exon 7) and cell cycle gene cyclin D1. Results for biomarkers of genotoxicity after stratification for the different DNA repair genetic polymorphisms showed that the polymorphism in exon 23 of the XPD gene modulates levels of chromosomal and DNA damage, HPRT MF, and moderately affects DNA adduct levels. The highest levels of biomarkers were associated with the wild-type homozygous AA genotype. The exposed individuals with the wild-type GG genotype for XRCC1 gene exhibited the lowest CA frequencies, compared to those with an A allele (P < 0.05). Cyclin D1 polymorphism seems to modulate the number of leukocytes and lymphocytes in the analyzed subjects. The number of eosinophiles was positively associated with XPD variant C allele and negatively with XRCC1 variant A allele (P < 0.05) and XPC variant C allele (P < 0.05). Immunoglobulin IgA was positively associated with an XRCC3 variant T allele (P < 0.01) and negatively with XPC variant C allele (P < 0.05). Both C3- and C4-complement components were lower in individuals with XRCC3 CT (P < 0.05) and TT genotypes (P < 0.01). Adhesion molecules sL-selectin and sICAM-1 were associated with XPC genotype (P < 0.05). Individual susceptibility may be reflected in genotoxic and immunotoxic responses to environmental and occupational exposures to xenobiotics.

Adult↗

Microemulsion polymerization of styrene in the presence of a cationic emulsifier.

The principal subject discussed in the current paper is the radical polymerization of styrene in the three- and four component microemulsions stabilized by a cationic emulsifier. Polymerization in the o/w microemulsion is a new polymerization technique which allows to prepare the polymer latexes with the very high particle interface area and narrow particle size distribution. Polymers formed are very large with a very broad molecular weight distribution. In emulsion and microemulsion polymerizations, the reaction takes place in a large number of isolated loci dispersed in the continuous aqueous phase. However, in spite of the similarities between emulsion and microemulsion polymerization, there are large differences caused by the much larger amount of emulsifier in the latter process. In the emulsion polymerization there are three rate intervals. In the microemulsion polymerization only two reaction rate intervals are commonly detected: first, the polymerization rate increases rapidly with the reaction time and then decreases steadily. Essential features of microemulsion polymerization are as follows: (1) polymerization proceeds under non-stationary state conditions; (2) size and particle concentration increases throughout the course of polymerization; (3) chain-transfer to monomer/exit of transferred monomeric radical/radical re-entry events are operative; and (4) molecular weight is independent of conversion and distribution of resulting polymer is very broad. The number of microdroplets or monomer-starved micelles at higher conversion is high and they persist throughout the reaction. The high emulsifier/water ratio ensures that the emulsifier is undissociated and can penetrate into the microdroplets. The presence of a large amount of emulsifier strongly influences the reaction kinetics and the particle nucleation. The mixed mode particle nucleation is assumed to govern the polymerization process. At low emulsifier concentration the micellar nucleation is dominant while at a high emulsifier concentration the interaction-like homogeneous nucleation is operative. Furthermore, the paper is focused on the initiation and nucleation mechanisms, location of initiation locus, and growth and deactivation of latex particles. Furthermore, the relationship between kinetic and molecular weight parameters of the microemulsion polymerization process and colloidal (water/particle interface) parameters is discussed. In particular, we follow the effect of initiator and emulsifier type and concentration on the polymerization process. Besides, the effects of monomer concentration and additives are also evaluated.

Cations↗

Affinity chromatography of DNA on nonporous copolymerized particles of styrene and glycidyl methacrylate with immobilized polynucleotide.

Nonporous particles of microsize were prepared by the dispersion polymerization of styrene and glycidyl methacrylate and chemically modified to introduce amino groups on the surface by grafting with either hexamethylenediamine or N-methyl-1,3-propanediamine. Aminated particles were then coupled with phosphorylated single-stranded polynucleotides at the 5'-end through covalent linkages. The affinity columns packed with these prepared polynucleotide-immobilized particles effectively retained single-stranded DNA, which could base-pair with the immobilized sequence. Bound DNAs could be eluted to yield a sharp peak by using an aqueous solution of 0.4M NaOH. The nonspecific adsorption due to the electrostatic interaction between the polynucleotide and the residual amino groups on the particle surface via the amination with hexamethylenediamine was significant and could only be reduced by using a high salt (NaCl) concentration. A higher salt concentration in the elution solution could result in a portion of complementary polynucleotide eluted in the nonretained fraction. However, the nonspecific adsorption of polynucleotides was insignificant in the column packed with DNA-immobilized particles prepared via amination using N-methyl-1,3-propanediamine. The column was effective for microanalysis of sequence-specific DNA.

Base Sequence↗

Leukemia and exposure to 1,3-butadiene, styrene and dimethyldithiocarbamate among workers in the synthetic rubber industry.

This study evaluated relations between exposure to 1,3-butadiene (BD), styrene (STY) and dimethyldithiocarbamate (DMDTC) and mortality from leukemia among synthetic rubber industry workers. Subjects were 13130 men employed for at least 1 year during 1943-1991 at any of six plants that manufactured synthetic rubber. Death certificates and medical records identified workers with leukemia. Cumulative exposure estimates were based on plant- and time period-specific process and task characteristics, linked to subjects' work histories. Poisson regression estimated relative rates (RRs) for workers exposed to each agent compared to unexposed workers. Leukemia (N=59) was positively associated with BD ppm-years (RRs of 1.0, 1.2, 2.0 and 3.8, for exposures of 0, >0-<86.3, 86.3-<362.2 and 362.2+ ppm-years; only the RR for the highest exposure category was statistically significant), STY ppm-years (RRs of 1.0, 1.2, 2.3 and 3.2, for exposures of 0, >0-<20.6, 20.6-<60.4 and 60.4+ ppm-years; only the RR for the highest exposure category was statistically significant) and DMDTC mg-years/cm (RRs of 1.0, 2.3, 4.9 and 2.9, for 0, >0-<566.6, 566.6-<1395.1 and 1395.1+ mg-years/cm; the RR for each non-zero exposure category was statistically significant) after adjusting for age and years since hire. After further adjusting each agent-specific set of RRs for the other two agents, a positive but imprecise relation remained for BD and DMDTC but not for STY. The association with BD was stronger for ppm-years due to exposure intensities >100 ppm than for ppm-years due to lower concentrations. BD and DMDTC, but not STY, were positively associated with leukemia in multivariable analyses. The independent effect of each agent was difficult to evaluate because of correlations with other agents and imprecision.

Butadienes↗

Isotachophoretic analysis of mandelic acid, phenylglyoxylic acid, hippuric acid and methylhippuric acid in urine after occupational exposure to styrene, toluene and/or xylene.

A simple, rapid and sensitive analytical method has been developed for the determination of phenylglyoxylic acid, mandelic acid, hippuric acid and methylhippuric acid; 0-, m- and p-methylhippuric acids are partly separated. These compounds are found as metabolites after occupational exposure to styrene, toluene and xylene. The method has been applied successfully to samples extracted from human urine by diethyl ether. The method can be used to accurately and simultaneously determine as little as 0.5 nmole of all of these acids in less than 20 min.

Chromatography, Gas↗

Gas chromatographic head-space determination of residual acrylonitrile in acrylonitrile-butadiene-styrene resins and migration into a simulated fatty foodstuffs liquid.

Head-space methods are described for the determination of residual acrylonitrile in acrylonitrile-butadiene-styrene resins and in olive oil, which simulates fatty foodstuffs. Dimethylformamide was used as solvent for the resin, with flame ionization detection. The injection of water into the resin dispersion prior to head-space analysis greatly enhances the detection capabilities. The use of a nitrogen-selective detector required dimethyl sulphoxide as the solvent. The determination of acrylonitrile in olive oil was carried out employing both types of detector. The detection sensitivity was much greater with the nitrogen-selective detector.

Acrylonitrile↗

Affinity chromatography of proteins on non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate.

Non-porous particles having an average diameter of 2.1 microm were prepared by co-polymerization of styrene, methyl methacrylate and glycidyl methacrylate, which was abbreviated as P(S-MMA-GMA). The particles were mechanically stable due to the presence of benzene rings in the backbone of polymer chains, and could withstand high pressures when a column packed with these particles was operated in the HPLC mode. The polymer particles were advantaged by immobilization of ligands via the epoxy groups on the particle surface that were introduced by one of the monomers, glycidyl methacrylate. As a model system, Cibacron Blue 3G-A was covalently immobilized onto the non-porous copolymer beads. The dye-immobilized P(S-MMA-GMA) particles were slurry packed into a 1.0 cm x 0.46 cm I.D. column. This affinity column was effective for the separation of turkey egg white lysozyme from a protein mixture. The bound lysozyme could be eluted to yield a sharp peak by using a phosphate buffer containing 1 M NaCl. For a sample containing up to 8 microg of lysozyme, the retained portion of proteins could be completely eluted without any slit peak. Due to the use of a shorter column, the analysis time was shorter in comparison with other affinity systems reported in the literature. The retention time could be reduced significantly by increasing the flow-rate, while the capacity factor remained at the same level.

Chromatography, Affinity↗

Isotope dilution gas chromatography-mass spectrometry in the determination of benzene, toluene, styrene and acrylonitrile in mainstream cigarette smoke.

A cryogenic trapping method with isotope dilution gas chromatography-mass spectrometry analysis has been developed for the determination of benzene, toluene, styrene and acrylonitrile in mainstream vapor phase cigarette smoke. The method is simple, direct, and quantitative. Vapor phase samples are collected cryogenically in a series of four traps following removal of the particulate phase with a Cambridge filter pad. For all four analytes, 75-85% of the total amounts recovered were found in the initial trap and less than 1% in the final trap. Assessment of instrumental precision by multiple injections of a sample gave relative standard deviations of less than 2%. Linear calibration for all analytes over the analysis range gave an r2 value greater than 0.99 with average relative standard deviations at the mean ranging from 1.4 to 8.2%. The cigarettes analyzed include a reference cigarette (Kentucky 1R4F), a commercial ultra-low "tar" mentholated cigarette, and two cigarettes that heat but do not burn tobacco. The values determined for the four analytes in the 1R4F samples are comparable to reported values of similar cigarettes. The cigarettes which heat rather than burn tobacco yield less of all four analytes compared to the other cigarettes in the study.

Acrylonitrile↗

Chemical compositional separation of styrene-methyl methacrylate copolymers using high-performance liquid chromatography with liquefied carbon dioxide as eluent.

Chromatographic separation of styrene-methyl methacrylate (MMA) copolymers depending on the chemical composition was studied using liquefied carbon dioxide as an adsorption promoting solvent, and tetrahydrofuran, chloroform containing ethanol as a desorption promoting solvent in the mobile phase and the column packed non-bonded silica gel by a solvent gradient method. With the increase of MMA content, the elution was retarded indicating that the typical normal-phase type of adsorption occurred. The effects of type of desorption solvents, molecular mass of sample, and column temperature on the elution were investigated.

Carbon Dioxide↗