Styrene-7,8-oxide in blood of workers exposed to styrene

A field study was carried out on 13 workers exposed to styrene vapors at time-weighted average concentrations between 10 and 73 ppm. The reactive intermediate styrene-7,8-oxide was determined in blood samples using a direct gas chromatographic method. Styrene-7,8-oxide concentrations were in the range between 0.9 and 4.1 μg/l blood. Linear correlations were found between styrene-7,8-oxide in blood and styrene in ambient air and blood. For an exposure concentration of 20 ppm styrene (German MAK value) a steady-state level of about 1 μg styrene-7,8-oxide/l blood was calculated. Received: 3 February 1994/Accepted: 7 April 1994     

Individual sensitivity to DNA damage induced by styrene in vitro: influence of cytochrome p450, epoxide hydrolase and glutathione S-transferase genotypes

Styrene is a monomer of great commercial interest; its polymers and copolymers are used in a wide range of applications. In humans, styrene metabolism involves oxidation by cytochrome p450 monooxygenases (CYPs) to styrene-7,8-oxide, an epoxide thought to be responsible for the genotoxic effects of styrene exposure and detoxification by means of epoxide hydrolase (EH) and glutathione S-transferases (GSTs). The objective of this study was to investigate if genetic polymorphisms of metabolic enzymes modulate styrene-induced DNA damage in human leukocytes. CYP2E1, CYP1A1, EH, GSTP1, GSTM1 and GSTT1 polymorphisms were determined in 30 healthy donors and alkaline comet assay was carried out in isolated leukocytes exposed to 5 and 10 mM styrene, using 1% acetone as solvent control. The results obtained suggest that CYP1A1 m1, m2 and m4, CYP2E1 Dra I and GSTP1 (exons 5 and 6) polymorphisms may affect styrene induction of DNA damage in human leukocytes.     

Induction of single-strand breaks in DNA of mice by trichloroethylene and tetrachloroethylene

Trichloroethylene (TRI) (4-10 mmol/kg body wt) and tetrachloroethylene (PER) (4-8 mmol/kg body wt) were given to male mice by i.p. injection. The induction of single-strand breaks (SSB) in DNA of liver, kidney and lung was studied by the DNA unwinding technique. There was a linear increase of the level of SSB in kidney and liver DNA but not in lung DNA 1 h after administration. The damage was completely repaired 24 h after injection. The capability of TRI and PER to induce SSB in liver DNA is compared to that of three other substances, i.e., methyl methanesulfonate (MMS), styrene-7,8-oxide and styrene, which have been studied earlier by the same technique. The potency of the substances for induction of SSB was in the following order: MMS greater than styrene-7,8-oxide greater than styrene greater than PER greater than TRI.     

Styrene-oxide N-terminal valine haemoglobin adducts in reinforced plastic workers: possible influence of genetic polymorphism of drug-metabolising enzymes

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

Modulation of different stress pathways after styrene and styrene-7,8-oxide exposure in HepG2 cell line and normal human hepatocytes

Styrene is one of the most important monomers produced worldwide. IARC classified styrene as a possible carcinogen to humans (group 2B). Styrene-7,8-oxide (SO) is the main reactive metabolite of styrene, and it is found to be genotoxic in several in vitro test systems.Styrene and styrene-7,8-oxide (SO) toxicity to HepG2 cells was investigated by evaluating end-points such as heat shock proteins (Hsps), metallothioneins (MT), apoptosis-related proteins, accumulation of styrene within the cells and expression of two isoforms of cytochrome P450. The potential activity of styrene and styrene-7,8-oxide in modulating gene expression was also investigated. The results showed induction of Hsp70, metallothioneins, BclX(S/L) and c-myc expression and a decrease in Bax expression in HepG2 after treatments, confirming that these compounds activated protective mechanisms. Moreover, up-regulation of TGFbeta2 and TGFbetaRIII in HepG2 cells was found after exposure to styrene, while in human primary hepatocytes these genes were down-regulated after both treatments. Finally, it was found that styrene and SO treatments did not induce CYP1A2 and CYP2E1 protein expression.In conclusion, both compounds caused toxic stress in HepG2 cells, with SO being more toxic; in the meantime, a different effect of the two compounds in HepG2 cells and primary human hepatocytes was observed regarding their activity in gene modulation.     

Assessment of biotransformation of the arene moiety of styrene in volunteers and occupationally exposed workers

Styrene is a chemical widely used in the plastic industry. The main pathway of styrene metabolism in humans occurs via the oxidation to styrene-7,8-oxide (7,8-SO). The aim of this study was the investigation of a minor metabolic route, involving the oxidation of the arene moiety of styrene, by means of the characterization of the conjugated urinary metabolites of 4-vinylphenol (4-VP). 4-vinylphenol-glucuronide (4-VP-G) and -sulfate (4-VP-S), were measured by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS) from 174 workers belonging to three cohorts recruited in European countries and from 26 volunteers exposed to 50 mg/m(3) (11.8 ppm) of styrene for 8 h. The 4-VP conjugates represented about 0.5-1% of the total excretion of styrene metabolites. Both 4-VP-G and 4-VP-S are eliminated with a monophasic kinetic, the glucuronide being excreted faster (half-time, 2.2 +/- 0.2 h) than the sulfate (half-time 9.7 +/- 1.7 h). The urinary 4-VP was found to be significantly correlated both with airborne styrene (r = 0.607, p < 0.001) and the sum of MA and PGA (r = 0.903, p < 0.001 in "end-of-shift" samples). Apart from 7,8-SO, 4-VP is the only styrene metabolite not shared with ethylbenzene and therefore thought to be a highly specific marker of styrene exposure. However, a measurable background excretion of 4-VP was also found in all urine samples from controls not occupationally exposed to styrene. This background appears to be highly correlated to smoking (p < 0.001) and possibly also to the dietary intake of styrene or 4-VP. Consequently, the use of 4-VP as a biomarker of styrene exposure is recommended for exposures exceeding 1 ppm.     

Quantification of DNA adducts formed in liver, lungs, and isolated lung cells of rats and mice exposed to (14)C-styrene by nose-only inhalation.

Bronchiolo-alveolar tumors were observed in mice exposed chronically to 160 ppm styrene, whereas no tumors were seen in rats up to concentrations of 1000 ppm. Clara cells, which are predominant in the bronchiolo-alveolar region in mouse lungs but less numerous in rat and human lung, contain various cytochrome P450s, which may oxidize styrene to the rodent carcinogen styrene-7,8-oxide (SO) and other reactive metabolites. Reactive metabolites may form specific DNA adducts and induce the tumors observed in mice. To determine DNA adducts in specific tissues and cell types, rats and mice were exposed to 160 ppm [ring-U-(14)C]styrene by nose-only inhalation for 6 h in a recirculating exposure system. Liver and lungs were isolated 0 and 42 h after exposure. Fractions enriched in Type II cells and Clara cells were isolated from rat and mouse lung, respectively. DNA adduct profiles differed quantitatively and qualitatively in liver, total lung, and enriched lung cell fractions. At 0 and 42 h after exposure, the two isomeric N:7-guanine adducts of SO (measured together, HPEG) were present in liver at 3.0 +/- 0.2 and 1.9 +/- 0.3 (rat) and 1.2 +/- 0.2 and 3.2 +/- 0.5 (mouse) per 10(8) bases. Several other, unidentified adducts were present at two to three times higher concentrations in mouse, but not in rat liver. In both rat and mouse lung, HPEG was the major adduct at approximately 1 per 10(8) bases at 0 h, and these levels halved at 42 h. In both rat Type II and non-Type II cells, HPEG was the major adduct and was about three times higher in Type II cells than in total lung. For mice, DNA adduct levels in Clara cells and non-Clara cells were similar to total lung. The hepatic covalent binding index (CBI) at 0 and 42 h was 0.19 +/- 0.06 and 0.14 +/- 0.03 (rat) and 0. 25 +/- 0.11 and 0.44 +/- 0.23 (mouse), respectively. The pulmonary CBIs, based on tissues combined for 0 and 42 h, were 0.17 +/- 0.04 (rat) and 0.24 +/- 0.04 (mouse). Compared with CBIs for other genotoxicants, these values indicate that styrene has only very weak adduct-forming potency. The overall results of this study indicate that DNA adduct formation does not play an important role in styrene tumorigenicity in chronically exposed mice.     

T(H1)-directed modulation of in vitro cytokine production in human peripheral blood mononuclear cells by styrene-7.8-oxide

Styrene and its chiral main metabolite styrene-7.8-oxide are well characterized regarding their cytotoxic, genotoxic and neurotoxic properties. To our knowledge, no data exist on the influence of styrene and styrene-7.8-oxide on immune reactions. Epidemiological studies suggest that exposure to environmental pollutants, specifically volatile organic compounds (VOCs), including styrene is one factor contributing to increasing prevalence rates of allergic diseases. In this study, we investigated the modulation of the immune system by styrene-7.8-oxide in vitro. Human peripheral blood mononuclear cells were incubated with styrene-7.8-oxide, either as (S)-enantiomer, (R)-enantiomer, or racemic styrene-7.8-oxide. Subsequently, the secretion of T(H1)-cytokines IFNgamma and IL-12 as well as T(H2)-cytokines IL-4 and IL-5 were measured by ELISA. We introduced a novel mathematical approach to quantify and compare cytokine responses. The results revealed a stimulation of cytokine secretion with emphasis on T(H1)-cytokines IFNgamma and IL-12. The stimulating effects were elicited at concentrations of styrene-7.8-oxide comparable to what would be encountered at industrial workplaces where styrene is processed. Therefore, we conclude that styrene-7.8-oxide exhibits immunomodulating capacities, which can be of clinical relevance for individuals with high styrene exposure.     

Comparison of Styrene Hepatotoxicity in B6C3F1 and Swiss Mice

Inhalation exposure to styrene at concentrations that causemetabolic saturation results in significantly greater hepatotoxicityin B6C3F1 mice than in Swiss mice; females of both strains aremore susceptible than males. These studies were conducted toinvestigate the mouse strain and gender differences in susceptibilityto hepatotoxicity caused by repeated exposure to styrene atconcentrations that do not cause metabolic saturation. Maleand female B6C3F1 and Swiss mice (8 weeks old) were exposedto 0, 150, or 200 ppm styrene for 6 hr/day, 5 days/week, forup to 2 weeks. Changes in body and liver weights, serum alanineaminotransferase (ALT) and sorbitol dehydrogenase (SDH) levels,liver histopathology, and total liver glutathione (GSH) wereevaluated after 2, 3, 5, and 10 exposures (six mice/sex/strain/timepoint/concentration). Blood levels of styrene and styrene-7,8-oxide(SO) were measured in mice exposed to 200 ppm styrene for 2,3, or 5 days (six mice/sex/strain/time point/concentration).Serum ALT and SDH levels were significantly elevated only infemale B6C3F1 mice after 3 exposures to 200 ppm styrene; enzymelevels had returned to control levels when measured after 5and 10 exposures. Degeneration and coagulative necrosis of centrilobularhepatocytes were observed in female B6C3F1 mice exposed 2, 3,and 5 days to 150 or 200 ppm styrene; incidences of these lesionswere greater in the 200 ppm than in the 150 ppm dose group.After 10 days of exposure to 150 or 200 ppm styrene, hepatocellularlesions had resolved, although a residual chronic inflammationwas present in livers of most female B6C3F1 mice. Degenerationof centrilobular hepatocytes was observed in one male B6C3F1mouse after 3 exposures to 200 ppm, and no significant lesionswere observed in livers of exposed Swiss mice. Significant dose-relateddecreases in hepatic GSH were observed in both sexes of bothstrains throughout the 2-week exposure. In general, hepaticGSH depletion was greatest in female B6C3F1 mice. Exposure to200 ppm caused 60–70% GSH depletion in female B6C3F1 miceat each time point. GSH depletion generally decreased in B6C3F1mice and increased in Swiss mice with continued exposure to150 ppm styrene. With continued exposure to 200 ppm, GSH depletiongenerally decreased in all mice. Blood styrene and SO levelsincreased in all groups with the number of exposures. Styrenelevels were significantly higher in B6C3F1 mice than in Swissmice; however, within each strain gender differences were notsignificant. These data suggest that the transient hepatotoxicityin female B6C3F1 mice was related to greater hepatic GSH depletionand/or slower GSH regeneration in these animals.     

Physiological modeling of the relative contributions of styrene-7,8-oxide derived from direct inhalation and from styrene metabolism to the systemic dose in humans.

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.     

4-Vinylphenol excretion suggestive of arene oxide formation in workers occupationally exposed to styrene

The toxicity of styrene has often been attributed to the formation of reactive epoxide intermediate, styrene-7,8-oxide. It has been suggested that in addition, an arene oxide, styrene-3,4-oxide, is a metabolite of styrene. Styrene-3,4-oxide is easily converted to corresponding phenols. In this study the presence of 4-vinylphenol in the urine is verified by gas chromatography/mass spectrometry and its quantity compared to mandelic acid excretion. Both 4-vinylphenol and mandelic acid were detected in the urine samples of workers occupationally exposed to styrene. No 4-vinylphenol was found in urine samples of unexposed individuals. The correlation between mandelic acid and 4-vinylphenol was fairly good (r = 0.93); increasing excretion of mandelic acid was also accompanied by increasing amounts of 4-vinylphenol in the urine. The interindividual variation of the 4-vinylphenol/mandelic acid excretion ratio was small, the mean ratio being about 0.3%. The presence of 4-vinylphenol in the urine of workers exposed to styrene suggests that, in man, styrene is also metabolized via arene oxidation. However, when the arene oxidation of styrene is compared to vinyl group oxidation the latter appears to be at least quantitatively by far the more important metabolic pathway.     

DNA adducts and strand breaks in workers exposed to vapours and aerosols of bitumen: associations between exposure and effect

To study the associations between exposure to vapours and aerosols of bitumen and genotoxic effects, a cross-sectional and cross-shift study was conducted in 320 exposed workers and 118 non-exposed construction workers. Ambient air measurements were carried out to assess external exposure to vapours and aerosols of bitumen. Hydroxylated metabolites of naphthalene, phenanthrene and pyrene were measured in urine, whereas (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide ((+)-anti-BPDE), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodGuo) and DNA strand breaks were determined in blood. Significantly higher levels of 8-oxodGuo adducts and DNA strand breaks were found in both pre- and post-shift blood samples of exposed workers compared to those of the referents. No differences between exposed workers and referents were observed for (+)-anti-BPDE. Moreover, no positive associations between DNA damage and magnitude of airborne exposure to vapours and aerosols of bitumen could be observed in our study. Additionally, no relevant association between the urinary metabolites of PAH and the DNA damage in blood was observed. Overall, our results indicate increased oxidative DNA damage in workers exposed to vapours and aerosols of bitumen compared to non-exposed referents at the group level. However, increased DNA strand breaks in bitumen workers were still within the range of those found in non-exposed and healthy persons as reported earlier. Due to the lack of an association between oxidative DNA damage and exposure levels at the workplaces under study, the observed increase in genotoxic effects in bitumen workers cannot be attributed to vapours and aerosols of bitumen.     

Perspectives on the genotoxic risk of styrene

Styrene is a highly reactive monomer widely used in the plastics industry. The potential for styrene to produce genotoxic effects has been studied extensively in experimental systems. Styrene can induce sister chromatid exchanges (SCE) and chromosome aberrations (CA) in vitro under test conditions that enhance metabolism of styrene to styrene 7,8-oxide (SO)or reduce detoxification of 50 by epoxide hydrolase. The in vivo animal data indicate that styrene is not clastogenic at concentrations (doses) likely encountered by humans under ambient or occupational exposure conditions. DNA binding studies with styrene in rats and mice demonstrated no increased adducts in mice compared to rats or in mouse lung compared to liver. As a result, DNA adducts in the lungs are unlikely to be the sole explanation of the development of lung tumors in mice exposed to styrene for 2 yr. Some epidemiological studies reported that DNA and/or protein adducts and DNA strand breaks result from occupational exposure to styrene and/or 50. Results of some of these studies, how-ever, are difficult to interpret, given that the statistical significance of reported effects (SCE, CA, and micronucleus formation) was often near or at p values of .05; dose and/or temporal response relationships often were missing; confounding variables could not be excluded; and, concomitant exposures to other industrial chemicals that are potentially genotoxic may also have occurred. These studies suggest that styrene, through metabolism to SO, could be clastogenic in humans at workplace levels in excess of 125 mg/m3. However, results from controlled animal studies involving in vivo exposure to styrene alone do not show clastogenic effects at exposures of up to 1500 mg/m3/d. In any event, these studies show that there is an apparent threshold for styrene-mediated effects.     

A recombinant model for assessing the role of GSTM1 in styrene-7,8-oxide toxicity and mutagenicity

Styrene-7,8-oxide (SO) is a highly reactive epoxide able to undergo reactions with endogenous nucleophiles, such as DNA. SO is inactivated by glutathione-S-transferase M1 (GSTM1). This detoxification enzyme is absent in approximately one-half of Caucasian (49%) populations. A GSTM1 recombinant human lymphoblastoid cell line (FB7) was generated from a GSTM1 negative parental cell line (WIL2NS). GSTM1 status was determined using RT-PCR and immunochemistry. Cells were challenged with a range of SO doses and subsequent toxicity (population growth in flasks) and genotoxicity (mutations at the HPRT locus) were monitored. FB7 (GSTM1 positive) exhibited greater cell survival after SO exposure relative to the GSTM1 negative parental line. The IC50 following a 1 h exposure to SO was 0.5 mM for WIL2NS, compared to greater than 2.5 mM for FB7. The extrapolated IC50 for FB7 was 5.5 mM. Significantly fewer mutant cells were induced by SO for FB7 than for WIL2NS at equivalent doses of SO. These findings suggest that the sensitivity of cells to styrene-7,8-oxide is influenced by GSTM1 status and that a recombinant GSTM1 positive cell line can efficiently detoxify styrene-7,8-oxide.     

Physiologically based modeling of the inhalation kinetics of styrene in humans using a bayesian population approach

Animal studies have implicated styrene as toxic to the central nervous system and its major metabolite styrene-7,8-oxide as a carcinogen. Therefore, a reliable estimate of the metabolic capacity for styrene in humans is of interest. However, the available models describing styrene kinetics in humans lack rigorous statistical validation and also ignore the population variability in metabolism. The population variability may be estimated by the use of population models. Furthermore, the statistical validation of pharmacokinetic models may be improved by use of Bayesian methods. These two approaches may be combined and recently have been gaining interest in the toxicology literature. A population-based physiologically based pharmacokinetic (PBPK) model for styrene was developed. The model was calibrated to extensive human toxicokinetic data from three previous studies in which 24 volunteers were exposed to 50-386 ppm of styrene at rest and various levels of exercise. Model fitting was performed in a Bayesian framework using Markov chain Monte Carlo simulation. The uncertainty around the partition coefficients and metabolic parameters for styrene was reduced. The metabolic capacity for styrene in humans was estimated to be 0.92 micromol/l kg(-1), with a lognormal standard deviation of 1.66. The estimated Vmax is 40% higher than previously estimated, whereas the population standard deviation is estimated for the first time.     

Microsomal and cytosolic epoxide hydrolase in Drosophila melanogaster

Subcellular fractions from Drosophila melanogaster and rat liver were investigated on their epoxide hydrolase activity. Both microsomes and the post-microsomal supernatant of Drosophila appeared to contain epoxide hydrolase activity using styrene-7,8-oxide as the substrate. Based on body weight, these activities were in the same order of magnitude. Rat liver cytosol was able to catalyze the hydrolysis of styrene oxide only if the glutathione S-transferase activity was blocked.     

Protein adducts as dosimeters of human exposure to styrene, styrene-7,8-oxide, and benzene.

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.     

Styrene oxide in blood, hemoglobin adducts, and urinary metabolites in human volunteers exposed to (13)C(8)-styrene vapors

Styrene is used in the manufacture of plastics and polymers and in the boat-building industry. The major metabolic route for styrene in rats, mice, and humans involves conversion to styrene-7,8-oxide (SO). The purpose of this study was to evaluate blood SO, SO-hemoglobin (SO-Hb) adducts, and urinary metabolites in styrene-exposed human volunteers and to compare these results with data previously obtained for rodents. Four healthy male volunteers were exposed for 2 h during light physical exercise to 50 ppm (13)C(8)-styrene vapor via a face mask. Levels and time profiles of styrene in exhaled air, blood, and urine (analyzed by GC) and urinary excretion patterns of mandelic acid and phenylglyoxylic acid in urine (analyzed by HPLC) were comparable to previously published volunteer studies. Maximum levels of SO in blood (measured by GC-MS) of 2.5-12.2 (average 6.7) nM were seen after 2 h, i.e., in the first sample collected after exposure had ended. The styrene blood level in humans was about 1.5 to 2 times higher than in rats and 4 times higher than in mice for equivalent styrene exposures. In contrast the SO levels in human blood was approximately fourfold lower than in mice. The level of hydroxyphenethylvaline (determined by GC-MS-MS) in pooled blood collected after exposure was estimated as 0.3 pmol/g globin corresponding to a SO-Hb adduct increment of about 0.003 pmol/g and ppmh. NMR analyses of urine showed that a major portion (> 95%) of the excreted (13)C-derived metabolites was derived from hydrolysis of SO, while only a small percentage of the excreted metabolites (< 5%) was derived from metabolism via phenylacetaldehyde. Signals consistent with metabolites derived from other pathways of styrene metabolism in rodents (such as glutathione conjugation with SO or ring epoxidation) were not detected.     

Styrene-7,8-oxide burden in ventilated, perfused lungs of mice and rats exposed to vaporous styrene.

Styrene (ST) is an important industrial chemical. In long-term inhalation studies, ST-induced lung tumors in mice but not in rats. To test the hypothesis that the lung burden by the reactive metabolite styrene-7,8-oxide (SO) would be most relevant for the species-specific tumorigenicity, we investigated the SO burden in isolated lungs of male Sprague-Dawley rats and in-situ prepared lungs of male B6C3F1 mice ventilated with air containing vaporous ST and perfused with a modified Krebs-Henseleit buffer (37 degrees C). Styrene vapor concentrations were determined in air samples collected in the immediate vicinity of the trachea. They were almost constant during each experiment. Styrene exposures ranged from 50 to 980 ppm (rats) and from 40 to 410 ppm (mice). SO was quantified from the effluent perfusate. Lungs of both species metabolized ST to SO. After a mathematical translation of the ex-vivo data to ventilation and perfusion conditions as they are occurring in vivo, a species comparison was carried out. At ST concentrations of up to 410 ppm, mean SO levels in mouse lungs ranged up to 0.45 nmol/g lung, about 2 times higher than in rat lungs at equal conditions of ST exposure. We conclude that the species difference in the SO lung burden is too small to consider the genotoxicity of SO as sufficient for explaining the fact that only mice developed lung tumors when exposed to ST. Another cause is considered as driving force for lung tumor development in the mouse.     

Styrene Inhalation Toxicity Studies in Mice: III. Strain Differences in Susceptibility

Inhalation toxicity studies were conducted to evaluate mousestrain differences in the susceptibility to styrene vapors.Male and female B6C3F1, C57BL/6, Swiss, and DBA/2 mice (8 weeksold) were exposed to 0, 125, 250, or 500 ppm styrene 6 hr/day,for 4 days (20/sex/dose). Histopathological changes and changesin liver weights were evaluated as a measure of hepatotoxicity.Styrene uptake and styrene-7,8-oxide (SO) formation were estimatedby measuring levels of styrene and SO in blood. An estimateof SO detoxification by conjugation with GSH was obtained bymeasuring hepatic GSH depletion. In general, mortality, increasedliver weights, and hepatocellular necrosis were observed inthe 250 and 500 ppm dose groups for all strains and both sexes.Considerable sex and strain differences were observed. Mortality,increased liver weights, and hepatocellular necrosis were greatestin B6C3F1 and C57BL/6 mice in the 250 ppm dose group and inmales; hepatotoxicity was similar in both strains. Swiss miceexhibited dose-dependent increases in mortality, liver weights,and in hepatocellular necrosis, with only slight sex differencesat early time points. Hepatotoxicity in DBA/2, B6C3F1, and C57BL/6strains was greater at 250 than 500 ppm; however, toxicity wasless severe in DBA/2 than in other strains based on absenceof mortality in either sex and less extensive liver necrosisat both 250 and 500 ppm. Blood styrene and SO levels did notcorrelate well with strain differences in toxicity. The relativetoxicity (mortality and hepatotoxicity) was B6C3FI>Swiss>DBA/2;however, relative blood styrene and SO levels were B6C3F1DBA/2>Swiss.Hepatic GSH depletion, (B6C3F1DBA/2>Swiss) correlated withblood SO levels as expected. These results demonstrate significantbiologic variability in the susceptibility of mouse strainsto styrene toxicity. These differences are presumably due tostrain and sex differences in metabolism; however, toxicitydid not correlate well with blood SO levels, suggesting thatother reactive metabolites may contribute to styrene toxicityin mice.