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The reactive metabolite of styrene, styrene-7,8-oxide (SO), reacts with a variety of nucleophilic sites in hemoglobin (Hb) to form SO-Hb adducts. Following the in vitro incubation of SO with blood from humans, NMRI mice and Sprague-Dawley rats, the second-order reaction rate constants were determined for the reaction of SO with cysteine (through both the alpha- and beta-carbons of SO), N-terminal valine (through the beta-carbon of SO), and carboxylic acid (presumably through both the alpha- and beta-carbons of SO) residues in Hb. The rate constants for cysteine adducts vary dramatically between species [2.04, 10.7, 133 L (mol Hb)-1 h-1 (alpha binding) for humans, mice and rats, respectively] and [0.078, 2.16, 20.4 L (mol Hb)-1 h-1 (beta binding), respectively]. The considerably higher rate of reaction with cysteine in rat Hb probably reflects the presence of an additional cysteine residue at position beta 125. Although the rate constants for valine adducts (1.82, 0.80, 0.29 L (mol Hb)-1 h-1, respectively) and COOH adducts (3.55, 1.94, 2.37 L (mol Hb)-1 h-1, respectively) are much more consistent, the inter-species differences are statistically significant for the reaction of SO with the N-terminal valine of Hb. Following the i.p. administration of styrene to mice and styrene and SO to rats, the levels of adducts at each of these sites were used in conjunction with the calculated rate constants to predict the integrated blood doses of SO. While the SO doses predicted from cysteine and valine adducts were very similar, that based upon COOH-binding was significantly different, presumably due to the instability of SO-COOH adducts. This research affirms the use of both cysteine and valine adducts, but not carboxylic acid adducts, as biomarkers of exposure to styrene and SO.

Objectives: To study the load of selected organochlorine compounds in the blood of aluminium foundry workers who use hexachloroethane as a degassing agent for aluminium and to measure some possible effects on internal organs., Methods: Plasma from nine male aluminium foundry workers with past experience of use of hexachloroethane and 18 controls (two controls per exposed case) matched for residence, sex, age, and socioeconomic status was analysed for hexachlorobenzene (HCB), (P-HCB), and octachlorostyrene (P-OCS) with low resolution gas chromatography-mass spectrometry. Serum samples from the same subjects were analysed for standard kidney, pancreas, and liver function variables. Analysis of variance (ANOVA) with the triplets retained, a non-parametric test, and linear regression were used for the analysis., Results: A fourfold increase of mean P-HCB was found among the exposed subjects compared with the controls (313.1 v 66.9 ng/g lipid; P < 0.01; (ANOVA model)). For P-OCS this difference was even larger (54.6 v 0.7 ng/g lipid; P < 0.01). Results were still significant (P < 0.05) with non-parametric testing. Within the exposed group there was a good correlation between the ln P-HCB (r = 0.80) and ln P-OCS (r = 0.91), respectively, with the cumulative number of years of exposure to hexachloroethane. No significant difference in kidney, pancreas, or liver function was found between the two groups., Conclusions: Aluminium degassing with hexachloroethane may increase the body burden of selected organochlorine compounds as reflected by HCB and OCS measurements. With the inherent limitations of this investigation no signs of subclinical organ toxicity were found.

Objective: To understand whether previous styrene exposure increases the human liver's ability to convert styrene into styrene oxide., Methods: The hypothesis was tested that the average linear metabolic rate constant kappa was the same in both exposed and unexposed groups, when the exposed group comprised people with a history of styrene exposure and the unexposed group had no exposure. In an experimental chamber, these two groups of subjects were exposed to a concentration of 80 ppm styrene for two hours. A three compartment pharmacokinetic model was used to define kappa. Based on large sample theory, the comparison of estimated mean values of kappa in the exposed and unexposed groups was shown to be equivalent to a comparison of the estimated mean values of the hepatic clearance X in the two groups. A method was developed to estimate X for each subject in both groups from the subject's height, weight, and estimated asymptotic styrene decay constant alpha. Here, alpha was estimated individually from observed blood concentrations over time when sufficient time had elapsed after the controlled exposure., Results: The proposed methodology of comparing the estimated mean values of kappa in exposed and unexposed groups reduced the number of specific physiological variables involved to three, all of which were estimable from data based on simple direct measurements. In contrast, other methods based on pharmacokinetic models usually involved many variables that were non-estimable on an individual basis. Consequently, statistical comparisons were impossible. These methods were applied to analyse previously published data on the time course of styrene concentrations in arterial blood of subjects in both exposed and unexposed groups. A Wilcoxon non-parametric rank sum test with the individually estimated X values was used, and no significant difference in the means of X in the two groups was found., Conclusion: The linear metabolic rate constant kappa for humans is probably not altered by previous exposure to styrene. This result is in agreement with some experimental studies on animals. However, in the data analysis, it was noted that the number of subjects in each group was small (6-7) and that the styrene concentration data did not exactly reflect true behaviour of asymptotic decay. Further studies are still needed to draw more definitive conclusions.

Rats (Sprague Dawley) and mice (NMRI) were administered nonlabelled or labelled styrene and styrene oxide by i.p. injection. Blood samples were collected 6 and 24 h after treatment for studies of dose-response and 6 h to 32 days after treatment for studies of adduct stability. Haemoglobin (Hb) and plasma protein adduct levels were determined by radioactivity measurements or, in the case of adducts to N-terminal valine in Hb, by the so-called N-alkyl Edman procedure. Adducts to N-terminal valine were found to be chemically stable during the life-span of the erythrocytes, whereas adducts to carboxylic acid residues showed a reduced stability. The Hb-adduct levels found after styrene oxide treatment were compatible with a linear dose-response at low doses (< or = 0.4 mmol/kg body weight). At higher doses the detoxification of styrene oxide was overloaded resulting in a higher than proportional increase in adduct levels. Saturation of detoxification of styrene oxide could also explain the non-linear dose-response relationship observed in the mouse following treatment with styrene. Styrene oxide gave 4-7 times higher adduct levels than styrene when administered to the animals at equimolar low concentration. For both compounds, the levels of adducts to N-terminal valine were 2-3 times higher in the mouse than in the rat. A comparison of Hb-adduct levels in the styrene-exposed animals with adduct levels in styrene-exposed reinforced plastics workers (Christakopoulos et al., Scand. J. Work Environ. Health, 19(4) (1993) 255-263) suggests that styrene is less effective in humans than in mice and rats.

The urinary excretion of styrene represents a promising indicator of exposure to this solvent. Nevertheless extensive research under field conditions is scant. In this investigation 214 styrene-exposed workers from 10 fiberglass-reinforced plastics factories were studied. Environmental monitoring was performed by personal passive sampling. Blood styrene and the urinary excretion of styrene and its main metabolites, mandelic acid (MA) and phenylglyoxylic acid (PGA), were measured. The correlation coefficient between the time-weighted average of environmental styrene and the mean urinary excretion of styrene was 0.88 (0.91 after logarithmic transformation), compared with the 0.82 and 0.78 of the end-of-shift MA and PGA values, respectively. A high correlation (0.86) was also found between styrene in the blood and urine. The results, obtained under field conditions with a large group of exposed workers, confirm the usefulness of the urinary excretion of styrene as an exposure index for the biological monitoring of styrene exposure.

Prevention of environmentally related cancer will be enhanced by the availability of sensitive early warning systems and by improvements in quantitative assessment of human risks. Accordingly, we have carried out a series of molecular epidemiologic studies aimed at validating a panel of biologic markers, including carcinogen-DNA and -protein adducts, sister chromatid exchange, micronucleus formation, DNA strand breaks, and DNA repair capacity. Results from three such studies illustrate the usefulness of these biomarkers in elucidating low-dose-response relationships, correlations between biomarkers, and the range of variation in biomarkers between individuals exposed to similar concentrations of carcinogens. Low-level workplace or ambient exposures to styrene, ethylene oxide, and polycyclic aromatic hydrocarbons (PAH) were associated with significant increases in both molecular dose of carcinogens (adducts) and various markers of preclinical effects. Correlations between biomarkers varied by exposure. For example, in the styrene study, sister chromatid exchange frequency was not correlated with any of the markers, in contrast to the studies of ethylene oxide and PAH. Significant molecular effects were observed not only in occupationally exposed people but also in residents of an area in Poland characterized by high levels of air pollution. For example, the mean PAH-DNA level in exposed residents (winter sample) was 30.4 adducts per 10(8) nucleotides. This level was significantly higher than that of adducts seen in summer samples from the same area (4.2/10(8), or in winter samples from residents of a rural area (11.01/10(8). Significant seasonal variation in PAH-DNA adduct formation in this group was consistent with recorded fluctuations in air pollution levels. Striking interindividual variation was observed in all three exposed populations.

A cross sectional study of biological markers of neurochemical function in peripheral blood cells, and self reported nervous system symptoms, was conducted among 60 workers exposed to styrene in three reinforced plastics plants and 18 reference workers not exposed to styrene or other solvents. Concentrations of styrene in the air at the plants ranged from less than 1 to 160 ppm. Biomarkers of neurochemical function measured were: sigma receptor binding in lymphocytes, monoamine oxidase type B (MAO-B) activity in platelets, and serotonin uptake by platelets. Blood styrene concentration was used as the exposure index to take account of the use of protective equipment and dermal uptake. Four blood styrene exposure groups were defined as: non-exposed (reference) and exposed to less than 0.05, 0.05-0.19, and greater than or equal to 0.20 micrograms/ml. The prevalences of headache, dizziness, light headedness, fatigue, irritability, memory loss, and feeling "drunk" at work increased with increasing blood styrene concentration. No effect on sigma receptor binding was seen. A slight positive correlation was found for uptake of serotonin, which has been used as an exposure related effect indicator in previous studies of workers exposed to solvents. The MAO-B activity decreased with increasing blood styrene concentration; the mean (SE) MAO-B values for the four groups were 34.2 (3.0), 28.1 (5.3), 20.1 (4.8), and 16.9 (7.7) pmol/10(7) cells/min. The MAO-B activity also correlated negatively with the number of reported nervous system symptoms, whereas no associations were seen between prevalence of symptoms and either serotonin uptake or sigma receptor binding. The findings for MAO-B activity are consistent with previously reported experimental data, and suggest that MAO-B may be a useful marker of styrene neurotoxicity.

Given a styrene tissue/blood partition coefficient of ca. 39 and a relatively low perfusion rate of ca. 0.03 ml/min-g tissue, adipose tissue provides a useful physiologically damped integrative measure of environmental exposure. Styrene in the adipose tissue of nonoccupationally exposed individuals was measured for the first time. Tissue samples obtained from elective surgery patients and postmortem donors were analyzed by capillary gas chromatography and found to contain 1.12 +/- 1.06 (mean +/- SD) ppm styrene. Using these measured tissue levels and an apparent clearance of styrene (defined as the ratio of blood clearance to adipose tissue/blood partition coefficient), environmental intake of styrene was estimated to be 2.23 mg/hr, corresponding to an inhaled concentration of 1.96 mg/m3 (476 ppb). This value is two to three orders of magnitude higher than typical breathing zone air measurements, indicating additional undiscovered sources of styrene exposure.

The pharmacokinetic profile of inhaled styrene was examined in rats exposed to levels of 80, 200, 600 or 1,200 ppm for periods of up to 24 h. At levels up to 200 ppm for 6 h, styrene was cleared from the blood according to a two-compartment linear pharmacokinetic model, but at levels of 600 ppm and above the clearance was saturated. In going from 80 to 1,200 ppm (a 15-fold increase), the area under the blood concentration/time curves (AUC) increased by a factor or 112. Fat tissue was shown to comprise the second compartment of the two-compartment pharmacokinetic model. It is suggested that saturation of styrene clearance is due mostly to saturation of the metabolic capacity for styrene. In humans exposed to 80 ppm of styrene for 6 h, styrene was cleared from the blood according to a two-compartment linear pharmacokinetic model similar to that for rats. A maximum blood concentration of 0.9 microgram/ml was reached at the end of the exposure. Most of the inhaled styrene was excreted in the urine as phenylglyoxylic and mandelic acids, and only a small amount as styrene in the expired air. Simulation of the pharmacokinetic model showed that no continued accumulation of styrene would occur during repeated, daily 8-h exposures to 80 ppm. These data reveal that the rat is a reasonable pharmacokinetic model for styrene in humans. At levels of exposure up to 200 ppm, styrene is cleared from the body very efficiently and will not continue to accumulate upon repeated exposure. But at levels of styrene sufficiently high to saturate the metabolic clearance capacity, the integrated dose (measured by the AUC) will be much greater than expected based on exposure levels alone. Therefore, the extrapolation of toxicity observed at high levels of styrene exposure to that expected at low levels may not be justified.

Objective methods for critically evaluating the toxic effect of industrial solvents are highly desirable. As many of these solvents are suspected to cause vertigo, an animal experimental model was set up for studying the effects of solvents on the vestibular systems. The vestibular function was studied by registration of involuntary eye movements--nystagmus--which are elicited via central vestibulo-oculomotor connections. During exposure to styrene a so-called positional nystagmus was demonstrated that indicated vestibular disturbances. Nystagmus is normally elicited by rotatory acceleration. During exposure to styrene the direction of this rotatory nystagmus was reversed. The incidence of the positional nystagmus correlated well with the blood level of the solvent, measured by gas chromatography. Kinetic studies also demonstrated a rapid equilibration between the level of the solvent in arterial blood and cerebrospinal fluid, and therefore suggested that estimation of the arterial level reliably indicates the level in the central nervous system.

Styrene monomer is a greatly used chemical, chiefly in the production of polystyrene. A cross-sectional health survey of 493 production workers was undertaken at the oldest and largest monomer production, polymerization, and extrusion facility in the United States. Relative exposure durations and levels were obtained from occupational histories and corroborated by spot air sampling, blood and fat styrene concentrations and levels of urinary mandelic and phenylglyoxylic acids. Statistically significant differences between the prevalence of abnormalities in high and low exposure groups were found for the following: history of acute prenarcotic symptoms, history of acute lower respiratory symptoms, peroneal nerve conduction velocities, relative lymphocytosis, and elevated gamma glutamyl transpetsidase. The following showed no distinct pattern in prevalence when analyzed by exposure group: chest radiographic changes; indices or restrictive, obstructive and small airway dysfunction; other hepatic and hematological parameters; carcinoembryonic antigen level; sputum cytopathology; radial nerve conduction velocities; and ophthalmological findings. Clinically significant abnormalities were rare.

A group of men exposed to styrene in a factory building glass-fibre boats performed a series of behavioural tests at the beginning and end of their shift, and the results were compared with those of a referent group from the same factory. Changes in mood were noted in both groups of workers but were greater in the exposed men; moreover, the change in mood was correlated with blood styrene concentration. In the styrene workers the morning reaction time was slower than that for the referents. During the day the reaction time of the men with low blood styrene concentration (less than or equal to 5.4 mumol/l) speeded up and in the afternoon was similar to that of the referents; the reaction time for the men with high blood styrene concentration (greater than or equal to 5.5 mumol/l) was unchanged. Data taken from a questionnaire indicated that the men exposed were much more likely than was the referent group to report feeling unduly tired. They also reported feeling more tired on Friday night than Monday night, suggesting that the styrene might have a cumulative effect through the week.

The concentrations of mandelic and phenylglyoxylic acids, urinary metabolites of styrene, and styrene in blood were determined for 491 styrene polymerization workers. Styrene in subcutaneous fat was determined for 25 workers. The levels of styrene exposure were estimated to be less than 10 ppm, and urinary metabolite and blood styrene concentrations indicated that significant recent exposure (within 4 h) had occurred among workers in areas of styrene polymerization and styrene monomer production. Styrene analysis of subcutaneous fat suggested that the monomer may have been present for as long as 3 d after exposure, a time when urinary metabolites and blood styrene were almost invariably undetectable. All three biological parameters were correlated with recency of exposure and estimated intensity of exposure within job categories.

A variety of food containers, drinking cups and cutlery, fabricated from polystyrene (PS) or polystyrene-related plastic, were analyzed for their styrene monomer content. Samples of yogurt, packaged in PS cups, were similarly analyzed and the leaching of styrene monomer from PS containers by some food simulants was also determined. Blood level studies with rats, dosed with styrene monomer by various routes, illustrated uptake phenomena that were dependent on the dose and route of administration and were also affected by the vehicle used to convey the styrene monomer.

Eight male workers from a glass reinforced plastics industry were experimentally exposed for 2 hours to 2.84 mmol/m3 (296 mg/m3) styrene during light physical exercise (50 W). About 63% of the amount supplied (4.6 mmol styrene) was taken up in the body. The arterial blood concentration of styrene reached a relatively stable level of 15 mumol/l at the end of exposure which was about 70% of the blood concentration in a group of volunteers with no previous exposure to solvents. The apparent blood clearance was significantly higher in the occupationally exposed subjects 2.01/h X kg compared with 1.51/h X kg. Contrary to the relatively stable level of styrene at the end of exposure the concentration of non-conjugated styrene glycol increased throughout the exposure and reached about 3 mumol/l in both groups. Like styrene, the non-conjugated styrene glycol seemed to be eliminated faster from the occupationally exposed workers. The blood concentration of styrene-7,8-oxide was low and seldom exceeded the detection limit of 0.02 mumol/l. The results show that long term exposure in a glass reinforced plastics industry may facilitate the metabolism of styrene.

Styrene induces sister chromatid exchanges (SCEs) in human whole-blood lymphocyte cultures without exogenous metabolizing systems, which indicates that styrene is metabolically activated in this in vitro system. Whole-blood lymphocyte cultures from 11 male donors showed a clear increase in SCEs after a 48-hr treatment with styrene (2 mM) or with the reactive metabolite styrene 7,8-oxide (0.15 mM). Styrene (0.5 to 4 mM) induced a distinct dose-dependent increase of SCEs in whole-blood cultures (with 200 to 400 million red blood cells/ml) but only a slight effect in purified lymphocyte cultures (with 20,000 red blood cells/ml). SCE induction by styrene (2 mM) depended on the amount of red blood cells (0.02 to 2000 million/ml) added to the purified lymphocyte cultures. Cyclophosphamide, studied for comparison, clearly increased SCEs irrespective of the presence of erythrocytes. The results show that erythrocytes are essential for the activation of styrene in the lymphocyte test system. This activation probably results from the conversion of styrene into styrene 7,8-oxide by oxyhemoglobin.

Six male subjects were exposed for two hours during light physical exercise to 2.81 mmol/m3 (293 mg/m3) of styrene on one occasion and to a mixture of 2.89 mmol/m3 (301 mg/m3) of styrene and 21.3 mmol/m3 (1240 mg/m3) of acetone on another (combination study). About 68% of the dose (somewhat more than 4 mmol) of styrene was taken up. The arterial blood concentration of styrene reached a relatively stable level after about 75 minutes of exposure of about 18 and 20 mumol/l after the single and combined exposure, respectively. Calculated values of mean blood clearance were 1.9 l/min in the styrene study and 1.6 l/min in the combination study; the half life of styrene in blood was about 40 minutes in both studies. The concentration of non-conjugated styrene glycol increased linearly during exposure and reached about 3 mumol/l at the end of exposure and was eliminated with a half life of about 70 minutes. Styrene-7,8-oxide was detected and quantified in the blood in a complementary study. The half lives for the excretion of mandelic and phenylglyoxylic acid in the urine were about four and nine hours, respectively, in both studies.