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     

Genotoxicity of styrene-7,8-oxide and styrene in Fisher 344 rats: a 4-week inhalation study

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.     

Relationship between lead concentration in blood and biological response for porphyrin metabolism in workers occupationally exposed to lead

Summary The biological responses of the heme biosynthesis pathway in male workers moderately exposed to lead are discussed in relation to the concentration of lead in the blood. The level of erythrocyte delta-aminolevulinic acid dehydratase (ALA-D) activity in the group of lead-exposed workers was remarkably reduced while the level of erythrocyte protoporphyrin (Proto) in them was strikingly increased, compared to normal levels. On the other hand, the amounts of hemoglobin (Hb) and urinary delta-aminolevulinic acid (ALA) in the group of lead-exposed workers kept the normal levels.In the workers moderately exposed to lead, the log of erythrocyte Proto level was closely correlated to the blood lead level and the sensitivity of the Proto test was almost equal to that of erythrocyte ALA-D test. It was observed that the erythrocyte Proto was remarkably increased even in lead-exposed workers whose ALA excretion into the urine was in the range of normal level.     

Relationship between activation of delta-aminolevulinic acid dehydratase by heating and blood lead level

Both blood lead and erythrocyte delta-aminolevulinic acid dehydratase (ALA-D) activity were determined for workers with and without an occupational lead exposure.In workers occupationally exposed to lead, it was demonstrated that the erythrocyte ALA-D is markedly activated by heating the hemolysate at 60° C for 5 min and there is a good positive correlation between the ratio of heated to nonheated ALA-D activity and the blood lead level (r = 0.799). In addition, by heating the hemolysate, the ALA-D activity of the lead-exposed workers appears to be returned into the normal range regardless of the extent of lead absorption. However, in normal workers without the occupational lead exposure, no significant correlation was found between the ratio of heated to nonheated ALA-D activity and the blood lead level, although the normal ALA-D also can be slightly activated by heating the hemolysate at 60° C for 5 min.     

Induction of epoxide hydrolase in cultured rat hepatocytes and hepatoma cell lines

The expression of epoxide hydrolases was studied in cultured rat hepatocytes and hepatoma cell lines. Styrene 7,8-oxide and benzo[a]pyrene 4,5-oxide were used as substrates for microsomal epoxide hydrolase and trans-stilbene oxide for the cytosolic form of this enzyme. In freshly isolated hepatocytes from control rats, microsomal epoxide hydrolase activity was 7.7 and 10.8 nmoles/mg cellular protein/min with benzo[a]pyrene 4,5-oxide and styrene 7,8-oxide as substrates respectively. This enzyme activity increased by more than 2-fold in hepatocytes after 24 hr in culture and remained elevated throughout 96 hr using both substrates. In cultured hepatocytes from rats pretreated in vivo with phenobarbital, trans-stilbene oxide, 2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene, both benzo[a]pyrene 4,5-oxide and styrene 7,8-oxide hydrolase activities were increased greater than 1.8 relative to controls. Hepatocytes from 2-acetylaminofluorene-pretreated animals at 24 hr in culture had approximately 9-fold higher activities than control hepatocytes. In marked contrast to microsomal epoxide hydrolase activity, the cytosolic enzyme showed an initial activity of 191 pmoles/mg cellular protein/min in freshly isolated hepatocytes, decreased by 75% after 24 hr in culture, and was barely detectable at 96 hr. A similar trend was apparent in hepatocytes from the pretreated animals. In vitro treatment of hepatocytes with trans-stilbene oxide and phenobarbital increased microsomal epoxide hydrolase, while this activity was refractory to 2-acetylaminofluorene treatment. Styrene 7,8-oxide hydrolase activity was increased in the McA-RH-7777 rat hepatoma cell line by phenobarbital, trans-stilbene oxide and 2-acetylaminofluorene treatment. Similarly, benzo[a]pyrene 4,5-oxide hydrolase activity was also increased in this cell line by treatment with phenobarbital and trans-stilbene oxide but not by 2-acetylaminofluorene. Microsomal epoxide hydrolase activity in rat H4-II-E hepatoma cells was refractory to induction, except by trans-stilbene oxide treatment, which caused a 70% increase in benzo[a]pyrene 4,5-oxide hydrolase activity.     

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.     

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.     

Comparative study of effects of lead on the activity of erythrocyte pyrimidine 5′-nucleotidase and delta-aminolevulinate dehydratase in vivo and in vitro

The effect of lead on the activities of erythrocyte pyrimidine 5′-nucleotidase (Py5N) and delta-aminolevulinate dehydratase (ALA-D) was studied in the mice which were given ad libitum a drinking water containing lead of 10, 50 and 250 ppm, for 27 days. The erythrocyte Py5N activity was not decreased in all groups of lead-exposed mice. However, the erythrocyte ALA-D activity was markedly decreased in the groups exposed to 50 and 250 ppm lead. These data indicate that erythrocyte ALA-D is more sensitive than Py5N to lead in vivo. On the other hand, from the in vitro study, it was demonstrated that the human erythrocyte Py5N is moderately inhibited by zinc and tin, and markedly by mercury, cadmium, silver, copper, and lead, at 10⁻⁴ molar concentrations. In addition, it was observed that the erythrocyte Py5N is most remarkably inhibited by mercury while the ALA-D by lead, among metals tested.     

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.     

Interaction of zinc and other metal on the activity of erythrocyte delta-aminolevulinic acid dehydratase in vitro.

The interaction of zinc and other metal such as lead, mercury (II), cadmium, silver (II) on the activity of erythrocyte delta-aminolevulinic acid dehydratase (ALA-D) was investigated in vitro. At the concentrations ranging from 0.01 to 1.0 mmol/l, lead, mercury (II), cadmium and silver (II) strikingly inhibited the erythrocyte ALA-D activity. The in vitro addition of zinc having an activating effect for the erythrocyte ALA-D, was observed to eliminate the lead-induced inhibition of the erythrocyte ALA-D activity. And the degree of the elimination seemed to depend on the molar ratio (Zn/Pb) of both metal concentrations in the ALA-D assay. However, the inhibition of erythrocyte ALA-D activity caused by mercury (II), cadmium and silver (II) was not removed by the in vitro addition of zinc.     

Glutathione S-epoxidetransferase in the human placenta at different stages of pregnancy

Glutathione S-transferase activity with styrene 7,8-oxide as substrate was studied in the soluble fraction of human placentas. Measurable enzyme activities were noted in all placental specimens studied. The mean activity was 5.61 +/- 0.59 (SE) nmol of conjugate formed per min per mg of microsomal protein in midgestational placentas obtained between the 8th and 25th weeks of gestation. The corresponding activity was only 2.07 +/- 0.34 (SE) in four term placental specimens. In this material no relation between the enzyme activities and the smoking habits of the women was observed. Linear kinetics of the enzyme were observed both with respect to styrene 7,8-oxide and glutathione as substrates. Our results show that placental tissue catalyzes not only the formation of previously demonstrated reactive metabolites but also the further metabolism of such metabolites via conjugation with glutathione. The clinical significance of our findings is unclear, but one may speculate that the balance between the placental formation of toxic metabolites and their further degradation is important in regulation of the quantities of such metabolites that reach the fetal compartment.     

Relationship between lead concentration in blood and the activities of erythrocyte pyrimidine 5′-nucleotidase and delta-aminolevulinate dehydratase in the mice exposed to lead

The relationship between the activities of both pyrimidine 5-nucleotidase (Py5N) and delta-aminolevulinate dehydratase (ALA-D) in erythrocytes and the concentration of lead in blood was investigated in the mice which were given ad libitum a drinking water containing lead of 10 to 500 ppm, for 30 days.From these results, it was demonstrated that the erythrocyte PySN activity is inhibited by lead and its activity level is negatively correlated with the concentration of lead in blood (r=–0.78). In addition, it was suggested that the erythrocyte Py5N activity is a better indicator in the exposure to relatively high lead concentration while the ALA-D is a more sensitive indicator for evaluating the lead exposure of low and moderate levels.     

Influence of the level of cytosolic epoxide hydrolase on the induction of sister chromatid exchanges by trans-beta-ethylstyrene 7,8-oxide in human lymphocytes

trans-beta-Ethylstyrene 7,8-oxide, a substrate of cytosolic epoxide hydrolase, and 4-fluorochalcone oxide, an inhibitor of this enzyme, were investigated on induction of sister chromatid exchanges (SCE) in human lymphocytes. Both epoxides enhanced the frequency of SCE. 4-Fluorochalcone oxide at low concentration (2.5 microM) inhibited cytosolic epoxide hydrolase activity towards trans-beta-ethylstyrene 7,8-oxide in lymphocytes by 74% and had no effect on glutathione transferase activity using this substrate. At this concentration it did not induce SCE itself, but it potentiated the effect of trans-beta-ethylstyrene 7,8-oxide several fold. In lymphocytes from different subjects, the number of SCE induced by a low concentration of trans-beta-ethylstyrene 7,8-oxide correlated negatively with the individual cytosolic epoxide hydrolase activity (r = -0.72; -0.73 in two series of experiments). The number of SCE induced by a high concentration of trans-beta-ethylstyrene 7,8-oxide did not correlate with cytosolic epoxide hydrolase activity (r = 0.004; -0.24), but a negative correlation was found with glutathione transferase activity (r = -0.50). This finding is consistent with the results of biochemical studies in lymphocytes in which we determined the relative contribution of cytosolic epoxide hydrolase and glutathione transferase to the metabolism of trans-beta-ethylstyrene 7,8-oxide at varying substrate concentrations. The study demonstrates that the level of genotoxic effects induced in human lymphocytes is influenced by the individual level of detoxifying enzymes. At low concentrations, cytosolic epoxide hydrolase was more important than glutathione transferase activity.     

Effect of lead on the activity of erythrocyte porphobilinogen deaminase in vivo and in vitro

The effect of lead on the activity of erythrocyte porphobilinogen deaminase (PBGD) in vivo and in vitro was investigated using blood specimens obtained from controls and lead-exposed workers. When lead nitrate was added to the incubation mixture at a final concentration of 10⁻⁴ M, 83% inhibition of erythrocyte PBGD activity was found. However, in workers occupationally exposed to lead, no inhibition of erythrocyte PBGD activity was detected. This finding indicates that the erythrocyte PBGD test is not useful for evaluating exposure to lead in workers. In addition, the in vitro study confirmed that mercuric chloride strongly inhibits erythrocyte PBGD activity.     

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.     

In vivo regulation of delta-aminolevulinate dehydratase activity

The interrelationships among purified delta-aminolevulinate dehydratase (ALA-D; EC 4.2.1.24) activity, Pb, and ALA-D concentrations were studied in vitro. The ratio of Pb to the ALA-D subunit, but not the Pb concentration, determined the relative activity of ALA-D, indicating the significance of the amount of ALA-D in studying the mechanism of enzyme inhibition by Pb. To elucidate the in vivo mechanisms of ALA-D regulation, male Wistar rats, 6 months old, were treated with Pb, Zn, and glutathione (GSH) separately or in combination for 130 days. After Pb administration, the amount of ALA-D, as determined by radioimmunoassay, increased in the presence and in the absence of the Zn-GSH pretreatment, even if the enzyme activity were higher (the Zn-GSH-Pb-treated rats) than that of the control. Zn and GSH restored the enzyme activity in vivo synergistically. Since immunochemical study showed the identity of the liver ALA-D with the erythroid ALA-D, the liver and erythroid data were pooled to quantify the interrelationship among ALA-D activity, and Pb, Zn, SH, and the enzyme concentrations. The equation was the relative activity of ALA-D (%) = 0.256.[Pb/ALA-D subunit]2 - 9.56.[Pb/ALA-D subunit] - 0.000281.[square root Zn.SH/ALA-D subunit]2 + 0.0898..[square root Zn.SH/ALA-D subunit] + 33.6 (multiple correlation coefficient = 0.909, n = 108, p less than 0.01). The result indicated that 83% of the in vivo regulation of ALA-D activity is explained when the four factors, Pb, Zn, SH, and ALA-D concentrations, are considered in combination.     

Effect of aluminum on delta-aminolevulinic acid dehydratase from mouse blood

The objective of this study was to investigate aluminum deposition in whole blood and plasma of mice and the activity of blood delta-aminolevulinic acid dehydratase (ALA-D) after in vitro and in vivo exposure to this element. In vitro experiments showed activation and inhibition of the enzyme activity when 0.01-5.0 mM of aluminum sulphate were used (IC(50): 1.31 mM). Treatment with citrate and aluminum plus citrate increased ALA-D activity in vivo and the increase in enzyme activity was parallel to the increase in aluminum content in blood and plasma. These results show that aluminum has a distinct effect on ALA-D activity: first, at relatively lower concentrations it activated, and at high concentration it inhibited, blood ALA-D in vitro; second, it activated the enzyme when administered to drinking water. One important toxicological finding of the present report is that the apparent irrelevant addition of citrate to the drinking water significantly increased the level of aluminum in blood and plasma. Thus, in order to predict more accurately the extent of human exposure to aluminum it would be advantageous to consider the level of citrate ingestion and not exclusively the aluminum level in water or food.     

Effect of lead on electrophoretic mobility of rat erythrocytes

Lead often affects the erythrocyte membrane. The relationship between the changes in erythrocyte membrane and the anemia caused by lead is still unclear. Initially, the effect of lead injected intraperitoneally on the electrophoretic mobility of rat erythrocytes was investigated in order to study the relationship between them. As indices of lead exposure, hemoglobin (Hb) levels, hematocrits (Ht), δ-aminolevulinic acid dehydratase (ALA-D) activities and blood lead (blood Pb) levels in the injected rats were also examined. Exposure to lead significantly decreased the mobility of rat erythrocytes. The changes in mobility seemed to be less sensitive than those in ALA-D activity, however, the decreases in mobility were simultaneous with or prior to those in Hb level and Ht. The decreases in mobility were evident to some extent below a blood Pb level of 100 μg/100 ml and generally present at a level of 100 μg/100 ml and over. In the rats exposed to lead a significant negative correlation was found between the mobilities and the logarithms of blood Pb level.     

Cytogenetic activity of methyl isocyanate in vivo in the mouse micronucleus test

The ability of methyl isocyanate (MIC) to induce mutagenic and cytotoxic effects in vivo in the mouse micronucleus test was evaluated by assessing the induction of micronuclei and depression of polychromatic erythrocytes in bone marrow and peripheral blood smears. Animals were exposed to MIC through intraperitoneal injection for 2 and 5 days in separate experiments, and bone marrow and peripheral blood were sampled 6 and 48 h after the last injection, respectively. MIC did not significantly increase the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) and micronucleated normochromatic erythrocytes (MN-NCE) in bone marrow and peripheral blood samples respectively in either twice or multiply treated mice. However, a dose-dependent depression in percentage PCE observed was significant. This indicates that MIC exposure led to the cytotoxic effect by inhibition of bone marrow cell proliferation.     

Interactions of styrene and styrene oxide with partially purified cytochrome P-450 and P-448 from rat liver microsomes

Styrene and styrene 7,8-oxide were able to bind both to partially purified cytochrome P-450 isolated from phenobarbital (PB)-treated rat liver and to cytochrome P-448 from liver microsomes of 3-methylcholanthrene (3-MC)-treated rats.In the presence of either purified preparation or “fresh” microsomes from PB- or 3-MC-treated animals, styrene produced a characteristic Type I difference spectrum as did styrene 7,8-oxide with “fresh” microsomes from PB rats. In other experiments, the addition of styrene oxide produced spectra which resembled Type I spectra but were somewhat shifted to longer wavelengths.A comparison of the binding parameters for the interaction of styrene or styrene 7,8-oxide with partially purified preparations and “fresh” microsomes indicated that the binding is catalyzed by more than one type of P-450 hemoprotein and that the binding affinity is slightly reduced by the purification procedure. The addition of phosphatidylcholine was unable to restore the binding parameters.