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In the UK RAR (June 2008) the data on biokinetics and metabolism were summarized as follows (the references are given in brackets):

Summary of toxicokinetics:

A substantial amount of information is available on the toxicokinetics of styrene in humans, following exposure by the inhalation route; information on percutaneous absorption in humans is also available. 

In humans, inhaled styrene vapour (at concentrations of 10-200 ppm) is well absorbed across the respiratory tract (Engstöm et al., 1978a; 1978b; Fiserova-Bergerova and Teisinger, 1965; Fernandez and Caperos, 1977; Teramoto and Horiguchi, 1979; Wigaeus et al, 1983; 1984; Norström et al, 1992). Thus, a value of 100% for absorption via the inhalation route of exposure is taken forward to the risk characterisation.

Dermal absorption of the liquid has been estimated to be approximately 2% of the applied dose in anin vitrostudy using human skin samples (Gedik and Roper, 2003). This value is taken forward to the risk characterisation. Dermal uptake of the vapour appears to make only a small contribution (5% or less) to the total body burden arising from combined inhalation and dermal exposure to the vapour (Wieczorek, 1985; Riihimäki and Pfäffli, 1978; Limasset et al., 1999).

No information is available on oral absorption in humans, but from the physicochemical properties of styrene and experimental animal information (Plotnick and Weigel, 1979), one would expect extensive absorption from the gastrointestinal tract. Thus, a value of 100% for oral absorption is taken forward to the risk characterisation. Following absorption, it can be predicted from experimental animal data (Withey, 1978; Savolainen and Pfäffli, 1978; Plotnick and Weigel, 1979; Boogaard et al., 2000) that styrene is widely distributed in humans, and needle biopsy investigations have shown that styrene certainly locates in adipose tissue (Engström et al., 1978a; 1978b); there was a correlation between the amount of body fat and the total body burden of styrene. Data on styrene blood levels in human volunteers following single inhalation exposures and in rats exposed via inhalation show that at identical exposure concentrations, styrene blood levels are very similar (Ramsey and Young, 1978)(e.g. aprox. 0.8mg/ml in rats and humans at 80 ppm styrene).

The rate of absorption following inhalation is much higher (2-3 fold) in mice than in rats. The absorption rate in humans is approximately the same as in rats. The rate of styrene uptake in the upper respiratory tract is partly dependent on its metabolism, and was decreased when animals were pre-treated with a P450 inhibitor (Morris, 2000).

In humans, styrene is eliminated from the body relatively rapidly, primarily in the urine (Guillemin and Bauer, 1978; Caperos et al., 1979). However, there is some evidence for modest biopersistence in human adipose tissue on repeated daily exposure (Engström et al., 1978a). Styrene clearance from blood is biphasic. Half-lives for inhaled styrene were reported at 0.6 hours for the first elimination phase and 13 hours for the second elimination phase (Ramsey et al., 1980). From studies in mice, there is evidence that styrene is also rapidly eliminated from blood following either single or repeated inhalation exposure (Vodicka et al, 2001). A study in pregnant mice has shown that styrene and/or its metabolites can cross the placenta into the foetus (Kishi et al., 1989). 

The metabolism of styrene has been studied thoroughly in mice, rats and humans. A number of metabolic pathways have been identified (the quantitatively most important being side chain oxidation to styrene oxide, cleavage by epoxide hydrolase to styrene glycol and further oxidation to mandelic acid and phenylglyoxylic acid, GSH conjugation by glutathione transferase to mercapturic acids). The evidence suggests that these pathways are active in mice, rats and humans, although there are species differences in their relative importance.

Styrene is metabolised extensively in all species. According to a PBPK model developed by Ramsey and Andersen (1984), saturation of styrene metabolism in humans occurs at blood levels exceeding 0.85 mg/ml styrene or 200 ppm styrene in air. Below these concentrations, the rate of styrene metabolism is limited by the rate of blood perfusion in liver or other organs involved in styrene elimination. The first step in the metabolism of styrene involves oxidation of the aromatic ring or side-chain. The main route in each species is the oxidation of the side chain to give the epoxide, styrene-7,8-oxide (SO) (Kessler et al., 1992; Morgan et al., 1993c; Korn et al., 1994; Mendrala et al., 1993; Carlson et al., 2000; Filser et al., 2000; Hoffmann et al., 2006) . A number of studies have demonstrated the involvement of P450 in this step and have provided information on the specific P450 isoforms involved in the production of SO (CYP2E1, CYP2B6 and CYP2C8 in the liver and CYP2F2/1 and CYP2E1 in the lung) (Nakajima et al., 1994a; 1994b; Kim et al., 1997; Carlson 1997b; Carlson et al., 1998). Pre-treatment of rodents with diethyldithiocarbamate, a specific cytochrome P450 monooxygenase inhibitor, effectively inhibited the metabolism of styrene (Filser et al., 1993), and reduced toxicity in the mouse lung (Green, 1999d; Green et al., 2001a) and nasal tissue (Green 1999c; Green et al., 2001b) was observed when the animals were pre-treated with 5-phenyl-1-pentyne, a cytochrome P4502F2 inhibitor. In isolated Clara cells and microsomes, an inhibitor of 2F2 reduced the production of SO by around 30-50%; a 2E1 inhibitor showed less inhibition indicating a lower importance of this isoform in the lung. The SO produced is enantiomeric and is produced in the R- and S-forms, probably as a result of metabolism by different P450 isoforms. Different ratios of R-SO to S-SO are found in different tissues and different species. Mouse Clara cells produce about 3 times more of the R-enantiomer than the S-enantiomer, while rat produces more of the S-enantiomer, and humans, like rats, produce more of the S-form (Hynes et al., 1999).

SO is either metabolised further by conjugation with glutathione to give mercapturic acids, or is hydrolysed by epoxide hydrolase (EH) to phenylglycol (Ryan et al., 1976; Truchon et al., 1998). This is subsequently metabolised to mandelic, phenylglyoxylic and hippuric acids (Truchon et al., 1990; Leibman, 1975; Guillemin and Bauer, 1979; Caperos et al., 1979; Philippe et al., 1971; Sumner and Fennell, 1994). P450 and EH are both microsomal enzymes in the endoplasmic reticulum. Therefore, SO producedin situby P450 may potentially be rapidly detoxified if there is sufficient EH present.

Other metabolic pathways can lead to phenylacetaldehyde (PA) and phenylacetic acid (PAA) (via side-chainb-oxidation and hydroxylation), to phenylethanol and acetophenone (via side-chaina-oxidation and hydroxylation) (Sumner et al., 1995; Johanson et al., 2000; Cruzan et al., 2002), oxidation of the aromatic ring to give 4-vinylphenol (4-VP) (Bakke and Scheline, 1970; Pantarotto et al., 1978; Watabe et al., 1984; Manini et al., 2003), and products of ring opening (Cruzan et al., 2002). These metabolites are excreted in the urine. There are studies which have demonstrated that P450 enzymes are also involved in both the side-chain and ring oxidation of styrene and that 4-VP is further metabolised in lung microsomes by specific P450 isoforms to extremely reactive downstream products (e.g. an epoxide and a hydroquinone derivative) (Carlson et al., 2001; Bartels et al., 2004). Subsequently these derivatives are conjugated with glutathione (Bartels et al., 2004), but at present there is no information on the relative rates of 4-VP metabolites detoxification between different species.

There are some data from rodents (rats and mice) and humans to indicate the relative extent of flux through these various pathways. The approximate relative contribution of each metabolic pathway in each species, as determined from urinary metabolites, is shown in Table (a). The urinary metabolites are an indication of the overall metabolism of styrene, which is considered to occur largely in the liver.

It is clear that metabolism involving SO as an intermediate is a major route in rodents and humans. However, there are some notable species differences. In humans, almost all of styrene (95%) is metabolised to SO and further metabolised by EH; approximately 5% of styrene is metabolised via the phenylacetaldehyde pathway. No more than trace amounts of SO-GSH conjugates or ring-oxidized metabolites of styrene (4-VP) occur in humans exposed to styrene. Further metabolism of SO by EH is important but less extensive in rodents than in humans (68-72% in rats and 49-59% in mice). In rodents, conjugation of SO with GSH is an important route accounting for up to a third of the SO removal. The most significant difference between mice and rats is in relation to the production of phenylacetaldehyde (12-22% in mice against 3-5% in rats) and products of ring-oxidation (4-VP; 4-8% in mice against <1% in rats).

Table (a). Approximate relative contribution of metabolic pathways for styrene indicated by urinary metabolites (Cruzanet al., 2002; Johansonet al., 2000)

Metabolic route

Urinary metabolites (%)

 

Rat

Mouse

Human

Products of action of EH on SO

68-72

49-59

95

Conjugation of SO with GSH

23-26

20-35

Very low

Phenylacetaldehyde

3-5

12-22

5

Ring opening

ND

ND

<1

Products of 4-vinylphenol conjugation

<1

4-8

<1

ND, not determined.

In the literature update up to Oct. 01, 2015, the following additional information was obtained on the profile of metabolites excreted in rat urine:

Cosnier et al. (2012) developed a gas chromatographic method for simultaneous determination ofbenzoic (BA), phenylacetic (PAA), mandelic (MA), phenylglyoxylic (PGA), hippuric (HA) and phenylaceturic (PAUA) acids in rat urine. They exposed rats at 25 and 75 ppm styrene oxide and at 75 ppm styrene (over 4 days, Fisher 344 rats) and to 250 ppm styrene (over 1 day, Sprague Dawley rats), each 6 h/d and collected the urine over the following 18 h directly after exposure. The results obtained for the different urinary metabolites are given in table b (in mg/g creatinine):

Exposure

BA

PAA

MA

PGA

HA

PAUA

SO 25

15

37

103

125

815

249

SO 75

34

42

279

294

1619

263

Styrene 75

31

40

256

419

1714

200

Styrene 250

46

67

256

672

1366

485

Controls

4

58

 

 

996

379

In summary,this is the first systematic investigation on the concentration of urinary metabolites in rat urine after exposure to styrene and styrene oxide.

 

In the literature update up to Oct. 01, 2015, the following additional information was obtained on PBPK modelling:

Valcke and Krishnan (2011) evaluated the impact of the exposure duration and intensity on the human adjustment factor for biokinetic variability (HKAF). A PBPK model was used to compute the blood concentration and the amount metabolized by the liver in adults, neonates (030 days), toddlers (13 years), and pregnant women following inhalation exposure to several oganic chemicals, including styrene. Exposure scenarios simulated involved the RfC and six of U.S. EPA’s Acute Exposure Guideline Levels (AEGLs) (high), for durations of 10 min, 60 min, 8 h, and 24 h, as well as at steady-state. The distributions for several physiological parameters were used to model the HKAFs by Monte Carlo simulations [95th percentile in each subpopulation/median in adults]. Here only the results will be given for continuous exposure (steady state) conditions that may be comparable to the exposure situation of the general population calculated for the RfC of the US EPA (1 mg/m³). The highest and the lowest HKAF for the blood concentration were 1.4 (adults) and 2.5 (neonates) and for the rate of liver metabolism 1.3 (neonates) and 1.9 (pregnant women).

Verner et al. (2012) used PBPK modelling to determine optimal sampling times for biomonitoring of styrene exposure by styrene concentrations in venous blood and expired air. To this aim, the inter-individual variability of physiological parameters and of exposure and workload scenarios at an exposure level of 20 ppm were modelled by Monte Carlo simulations. It was concluded that the best time for sampling venous blood is at the end of shift for poorly ventilated and 15 min after shift for highly ventilated workplaces. Exhaled air concentrations are most informative 15 min after shift. For high workloads, median styrene concentrations in venous blood are calculated to be 0.4 mg/l (5th-95thpercentiles: 0.2-0.6) at the end of shift and in exhaled air 0.5 ppm (5th-95thpercentiles: 0.3-0.8) 15 min after shift. This study supprts the BEI of ACGIH of 0.2 mg/l in venous blood.

Mörk et al. (2014) derived assessment factors for human toxicokinetic variability (AFHK) for styrene using PBPK modelling in a population framework. Monte Carlo simulations were applied to cover the influence of age and gender for toxicokinetic variability as well as ventilation rates and fluctuations in exposure leves and workload at the workplace. The AFHK were based on the 95thpercentile for all subpopulations (with only minor differences for the 90thand 95thpercentiles). The exposure of the general population was assumed to be continuous at the RfC of the US EPA (0.24 ppm) and different age groups (adults, 15, 10, 5, 1 year and 3 months) were modelled. For workers, an exposure at the TLV of ACGIH (20 ppm) with 5 d/week, 8 h/d (with 1 h at rest in the middle) and light physical workload were assumed modelling in addition slow and rapid air exchange at the workplace. The following AFHK were obtained:

-         For the whole population 1.5 with some age related influence, the highest AFHK for 5 year old children (2.1)

-         For workers with continuous exposure 1.7.

These AFHK are lower than the default AF for intrahuman variability in toxicokinetics, namely 3.3.

In summary:some new publications were identified taking into account intrahuman verability to derive the toxicokinetic subfactor for intrahuman variability. These subfactors may be used to substitite the default factor of ECHA (3.3).

Metabolism in specific tissues:

The metabolism of styrene in specific tissues, lung, liver and nasal tissue, has been investigated in detail. These studies have demonstrated significant differences in the metabolism of styrene between these tissues and between species.

Liver

Styrene is metabolised to a significant extent in the liver in all species. Results from PBPK modelling suggest that metabolism of styrene by the liver is probably mainly responsible for the production of the blood level/body burden of SO. These models predict that following inhalation exposure to styrene concentrations of up to 250 ppm, the toxicokinetic processes follow first order kinetics, hence, SO blood levels are similar in both the mouse and rat (Kessler et al., 1992). At higher styrene concentrations, SO production reaches a plateau in the rat, whereas in the mouse, SO blood levels increase with increasing styrene concentration such that at an exposure of 800 ppm (Kessler et al., 1992), SO blood levels in the mouse are 20 times higher than the predicted values in the rat. The kinetics of styrene in humans are predicted to be similar to those of the rat (Csanady et al., 2003). The enzyme kinetics for hepatic P450 and EH suggest that human liver has a 3-5 times lower ability to produce SO and a higher ability to hydrolyse it with EH than rodents, resulting in half-lives for SO hydrolysis of 38, 9-17 and 1.8 min for mice, rats and humans, respectively (Mendrala et al., 1993). Human liver microsomes produced approximately equal proportions of R- and S-SO (Carlson et al., 2000).

Lung

The available data suggest that the Clara cell is mainly responsible for metabolising styrene in the lung. In cells isolated from the lungs of mice and rats it was evident that the Clara cells were responsible for the metabolism of styrene, and that type II cells did not metabolise it to any significant extent (Hynes et al., 1999). This is consistent with the fact that Clara cells contain the main metabolising enzymes, 2E1 and 2F2/1, and type II cells do not (Forkert, 1995; Buckpitt et al., 1995 ).

There are species differences in the number and structure of Clara cells. In mice, Clara cells are relatively numerous (approx. 89% of bronchiolar epithelium) and are spread throughout the airways which will impact significantly on the metabolism of styrene. In rats, they are significantly fewer in number (approx. 25% of bronchiolar epithelium) and they are found mainly in the terminal bronchiolar region. In contrast to rodents, in the human lung, Clara cells are rare and are found in small numbers in the distal bronchioles. They also are morphologically different, not possessing the extensive endoplasmic reticulum (on which P450s are localised) that is apparent in mouse Clara cells (Plopper et al., 1980). However, it should be noted that in humans the pulmonary cell types with the most significant CYP expression are cells of the bronchial and bronchiolar epithelium and not the Clara cells (Foth, 1995), and that cell-type specific expression of CYP enzymes in human lung has not been thoroughly investigated up to now. The distribution of Clara cells was mirrored by the pattern of immunohistochemical staining for two isoforms of P450, 2E1 and 2F2. In the mouse, these were found at high levels in the terminal bronchioles and to a lesser extent in the larger bronchioles. In rats, the activity of these enzymes was found at only low levels in the terminal bronchioles, and in humans, it was below the level of detection in one study (Green, 2000a) and it was found at levels (~12 pmol/mg protein/min) 400 times lower than those in mice in another study (Nakajima et al., 1994b). A more recent investigation (Bernauer et al., 2005) has reported that CYP2E1 activity was found among 86 samples of human lung tissue at levels (range between 1.1 and 23.9 pmol/mg protein/min) which are 3 orders of magnitude lower than those reported for the human liver (in the range of nmol/mg protein/min). There are also other studies, in which the expression of CYP2F1 in human lung tissue has been demonstrated (Runge et al., 2001; Hukkanen et al., 2002; Ding and Kaminski, 2003).

Studies in microsomes from lung cells have indicated significant species differences in the rate and extent of both the production of SO, its subsequent detoxification and in the production of the ring-oxidized metabolite, 4-VP and its downstream products. The enzyme kinetics for P450 in microsomes indicated that mouse and rat microsomes metabolise styrene to SO at approximately the same rate at high concentrations (i.e. at saturation) but, due to the much lower Kmfor mouse P450, mice will produce SO around 3-10 times more rapidly than rats at lower concentrations (Filser, 2000; Csanady et al., 2003). The maximum rate of hydrolysis by EH is similar in rats and mice, but it occurs to a greater extent in rats at lower concentrations due to the lower Km. The extent of SO conjugation with GSH was similar in mice and rats (Filser, 2000). Recent investigations have also shown that styrene is metabolised to 4-VP which is further metabolised to reactive downstream products, an epoxide and a hydroquinone derivative, and that in rats these reactive 4-VP derivatives are produced to a lesser extent than in mice (19-87% of the mouse concentrations) (Bartels et al., 2004). In marked contrast, in most human microsome samples tested there was no styrene-metabolising activity (Filser, 2000; Carlson et al., 2000), although in some individual samples, very low levels (around two orders of magnitude lower than in mouse and rat microsomes) of styrene metabolism to SO and styrene glycol were detected (Nakajima et al., 1994b). No detectable levels of styrene metabolism to 4-VP were also found, most likely as the consequence of the high reactivity of 4-VP. Indeed, in experiments conducted in the presence of excess GSH to trap the reactive downstream metabolites of 4-VP, the epoxide and the hydroquinone derivatives of 4-VP were isolated. However, these represented only 1.5-5% of the concentrations determined in mouse lung (Bartels et al., 2004). Human microsomes also had a substantially higher EH activity than rodents (Filser, 2000; Csanady et al., 2003), indicating that any SO producedin situor migrated from the blood would be rapidly hydrolysed.

In addition to differences in the extent and rate of production of SO, there are also clear differences in the ratio of R- and S-SO formed. Mouse Clara cells produce approximately 2-4 times more R-SO than S-SO, whilst rats produce a more equal ratio or preferentially S-SO (Hynes et al., 1999). Using PBPK modelling, it has been shown that R-SO predominates in the terminal bronchioles of mice, while S-SO predominates in rats (Sarangapani et al., 2002). The Sarangapani model predicts a maximum level of 2mM R-SO in the terminal bronchioles of rats due to saturation of styrene metabolism at 500-600 ppm. This is only half the level of R-SO achieved in the terminal bronchioles of mice exposed to 20 ppm styrene. The model also predicts that at a given airborne concentration, the level of total SO in the terminal bronchioles of mice is approximately 100-fold higher than in the terminal bronchioles of humans. In humans, the maximum concentration of SO in the terminal bronchioles (approx. 0.09mM) is reached at an airborne concentration of 200 ppm; this same concentration is found in the lungs of mice exposed only to 0.1 ppm styrene. Similarly, the Csanady model (Csanady et al., 2003) predicts that the pulmonary SO burden in humans exposed to 20 ppm of styrene for 8-hours is 17- and 50- fold lesser than the corresponding values in rats and mice. This model also predicts that glutathione depletion occurs in the lungs of mice exposed to styrene concentrations as low as 30 ppm; in contrast, rats are predicted to have less glutathione depletion while no glutathione depletion is estimated to occur in human lungs at styrene concentrations of up to 200 ppm.

Nasal tissues

In rats and mice, the uptake of styrene in the upper respiratory tract is partly dependent on its metabolism. The percentage of styrene absorbed decreased with increasing airborne concentration, demonstrating saturation of metabolism. Saturation of uptake occurred at a lower airborne concentration of styrene in rats than in mice, indicating a greater metabolic capacity in mice than in rats (Morris, 2000). From investigations conducted on microsomes from olfactory and respiratory nasal tissue (Green, 1999c; Green et al., 2001b), it has been found that nasal olfactory epithelium produces SO from styrene at about the same rate in both rats and mice with an R:S ratio of about 3. There is approximately twice as much metabolism occurring in the olfactory cells compared to the respiratory region. However, there is a signficant difference in the activity of EH, with rats having up to 10-fold higher activity than mice. The Kmfor EH was much lower in both species in respiratory epithelium than in olfactory epithelium, especially for R-SO (Green et al., 2001b) In contrast, human microsomes did not appear to have any ability to produce SO, even though 2E1 and 2F2 were found at low levels in some specific regions of the human nasal region (Green, 1999c; Green et al., 2001b). Human microsomes had around 3-times higher EH activity than mice (Green et al., 2001b).

There were some differences in the distribution of 2E1 and 2F2 within the nasal region in mice and rats. Mice generally had higher levels in the olfactory apical cytoplasm and basal cells (Green, 1999c)

Overall

These data indicate significant differences in the metabolism of styrene between species and between tissues. It should be noted that, although these data arise fromin vitrostudies and PBPK modelling, they clearly mirror the toxicodynamic picture of styrene obtainedin vivo. The tissue-specific metabolism of styrene suggests thatin situmetabolism within each tissue may be a more important determinant of toxicity than the overall systemic metabolism and blood levels of styrene metabolites. The implication of this is that the specifics of the local metabolism in a target tissue must be considered when extrapolating findings in animals to assess the likely hazard and risks in the equivalent human tissues.

A general observation is that the human tissues investigated – apart from the liver - produce very little SO, if any, and have a greater capacity to hydrolyse SO with EH than rodents. This difference is most pronounced in human nasal and lung tissues where production of SO is minimal or undetectable, and is also associated with a greater capacity to hydrolyse SO by EH. The mouse lung and nasal tissues produce the greatest amount of SO among the species tested, and, in general, have less EH activity, suggesting that significantly high local concentrations of SO will be present in these tissues. It is also evident that other toxic metabolites, particularly 4-VP and its reactive downstream products, are produced to a far higher extent in mouse lung than in rat (14-79% of the mouse concentrations) or human lung (1.5-5% of the mouse concentrations). Although it cannot be ascertained whether or not these species differences in the formation of 4-VP metabolites in the lung may be a reflection of the different numbers of Clara cells (the metabolically active lung cells) present in the different species, since 4-VP metabolites are produced by the same cytochrome P450 enzymes involved in the production of SO, it is most likely that the species differences in the formation of 4-VP metabolites observed reflect species differences in metabolic capability.

 

A literature search from 1998 to January 2010 overlapping the date of the UK RAR (June 2008) was conducted. Overall, this did not lead to a modification of the conclusions reached by the UK RAR and reinforced some of the aspects elaborated above.

 

Fustinoni et al. (2008) correlated various urinary metabolites to the airborne exposure in two small groups of styrene exposed workers (mean exposures: 0.8 and 4.2 ppm). Small amounts of 4-vinylphenol conjugates (glucuronides and sulfates) were detected in pre- and post-shift urine. The sum of the 4-vinylphenol conjugates was in the range of about 2% of the sum of mandelic acid and phenylglyoxylic acid demonstrating the aromatic ring hydroxylation is also a minor metabolic pathway in humans. Similar results were reported by De Palma et al. (2003).

 

Some investigations focused on the urinary excretion of mercapturic acids that are formed as diastomeric glutathione adducts in the course of one detoxification pathway of styrene oxide. Fustinoni et al. (2008) showed that urinary mercapturic acids represented only about 0.2% of the total of all urinary metabolites. In addition polymorphism of glutathione transferase (GSTM1 but not GSTT1) significantly correlated with the amount of mercapturic acids excreted in urine (higher levels of mercapturic acids were found in persons with active GSTM1). These results were confirmed by Haufroid et al., (2001; 2002) investigating workers exposed at 18.2 ppm or volunteers exposed for 8 h at 12 ppm styrene. These results confirm that detoxification of styrene oxide via glutathione transferase is only of minor quantitative importance in humans.

Ma et al. (2005) studied the influence of genetic polymorphism (Cyp 2E1, CYP2B6, epoxide hydrolase, glutathione transferase) on the urinary excretion of mandelic acid and phenylglyoxylic acid in workers of the reinforced-plastics industry. The median exposure to styrene was 55.9 ppm with a range of 0.3-133.5. The concentrations of the urinary metabolites were not significantly affected by the genotype. However, there was a significant interaction between the CYP2E1 genotype and smoking indicating that lifestyle may have an influence.

 

Prieto- Castello et al.-, (2010) studied the influence of the expression and polymorphism of CYP2E1 on the urinary excretion of mandelic acid and phenylglyoxylic acid in workers exposed to styrene (average level 362.7 mg/m³). The CYP2E1 mRNA expression was statistically significantly higher in exposed workers as compared to controls. A significant positive correlation between the urinary metabolite concentrations and the expression of CYP2E1 in blood was observed. Furthermore, there was an indication for an influence of polymorphism of CYP2E1 on the urinary metabolite concentrations that was, depending on the alleles, either at the limit of significance or just only showed a tendency.

 

Measured and calculated glutathione concentrations in the lung at different styrene exposure concentrations are given by Filser et al., (2002) substantiating the modeling results of Csanady et al. (2003). Glutathione depletion was much higher in mice than in rats, while in humans virtually no depletion is to be expected. Furthermore, Filser et al., (2002) presented measured and calculated styrene oxide concentrations in blood at different airborne styrene exposures. Up to air concentrations of 250 ppm blood concentrations were very similar in rats and mice, but in humans the blood concentrations were by a factor of 20 lower (at concentrations up to 80 ppm). When exposure was increased to 250-800 ppm the blood concentrations of styrene oxide rose steeply in mice while in rats a plateau was maintained. Styrene oxide concentrations were also predicted for the whole lung. At exposure concentrations up to 160 ppm styrene oxide concentrations were highest in mice, somewhat lower in rats (by about a factor of 2) and by far the lowest for humans, by about two orders of magnitude as compared to mice.

 

The metabolism of styrene in relation to the mode of action for lung carcinogenicity in mice and nasal toxicity in mice and rats has been reviewed by Cruzan et al. (2009). This review included a comparison with other chemicals with a similar toxicological profile (coumarin, naphthalene, ethylbenzene, cumene, divinylbenzene, benzofuran). The authors concluded that the effects noted for these chemicals, especially also for styrene, is driven by CYP2F isoforms of the cytochrome P450 family, namely CYP2F2 in nasal and lung tissues of mice and CYP2F4 in nasal tissues of rats. Importantly, the CYP2F1 isozyme expressed in humans has a low/no capacity to metabolize styrene. Metabolites resulting from the action of CYP2F2 (in mice) and CYP2F4 (in rats) are cytotoxic leading to nasal toxicity (in rats and mice) and regenerative hyperplasia and finally to lung tumors in mice. Inhibition of these CYP2F isozymes by 5-phenyl-1-pentyne prevented these effects.

 

Vodicka et al. (2006a) reviewed the metabolism and genotoxicity of styrene in relation to its potential carcinogenicity for humans. They conclude that styrene oxide that is formed by more than 95% by metabolic activation contributes quantitatively by far the most to the genotoxicity, while the minor ring oxidation products are potent pulmonary cytotoxins and contribute to local toxicities. The authors point to the importance of metabolic detoxification of styrene oxide that is efficiently carried out by the microsomal epoxide hydrolase (EH). The broad substrate specificity requires - as is the case for practically all xenobiotic metabolizing enzymes - a rather loose fit of the substrate to the catalytically active site of the enzyme. This leads to a relatively low overall turnover of the xenobiotic epoxides to the terminal product diols by EH. However, the enzyme very quickly removes, in a first step, genotoxic epoxides from the system by the formation of a covalent intermediate, an enzyme-substrate ester. This intermediate is subsequently hydrolyzed in a second, slow, rate-limiting step to give the free diol plus free enzyme. Mechanistic considerations and epidemiological studies have shown that only a very small fraction of the styrene oxide formed from styrene in the metabolically most active organ, the liver, reaches the systemic circulation, whereas the vast majority is immediately enzymatically hydrolyzed. As long as the local concentration of styrene oxide is lower than the local “concentration” of the epoxide hydrolase, detoxification of styrene oxide is practically instantaneous. In liver epoxide hydrolase is highly abundant. Only at higher local styrene oxide concentrations the slow regeneration of free epoxide hydrolase becomes important. In all species, including humans, the lung has lower epoxide hydrolase expression than the liver. However, the styrene activating activity of lung microsomes is very low, especially in humans. In contrast to rodents, detoxification of styrene oxide by glutathione conjugation is of minor importance. The efficiency of this cytosolic detoxification pathway is less than that of epoxide hydrolase.

Overall the author conclude that for equal exposures to styrene steady state concentrations of styrene oxide in blood are highest in mice, lower in rats, and still lower in humans.

Carlson (2008) reviewed the expression of cytochrome P450 enzymes in human lung and compared metabolic activation rates between liver and lung, and for the lung, between mice rats and humans. In general, pulmonary metabolism is very slow compared to hepatic metabolism. Furthermore, metabolic rates in humans are slow compared to those in rats and mice. Pharmacokinetic and PBPK models have indicated that very little styrene oxide produced in liver would be expected to be found in the lung by distribution via blood circulation. Therefore focus is on the in situ generation of active metabolites in human lung. The ability of human lung to metabolize styrene to styrene oxide is extremely limited and has only been demonstrated in rare cases. While the complete picture of the different cytochromes P450 involved in lung metabolism of styrene is not yet completely understood, CYP2F2 has been identified in mice as being primarily responsible for styrene metabolism in the lung. CYP 2F1, the corresponding human homolog of CYP 2F2, has a very low activity in the human lung. According to the authors, this argues against the hypothesis that human lung would produce enough styrene oxide (or other reactive metabolites) to damage pulmonary epithelial cells.

A new literature search up to March 2013 was carried out. The new studies identified are also mentioned in the section on carcinogenicity (“IPCS Conceptual Framework for Evaluating a Mode of Action” for Styrene Induced Mouse Lung Tumors) and were related to

               GSH depletion in mice after styrene exposure

               Aromatic hydroxylation

               Relevance of CYP 2F2 in mouse lung

               Other aspects of styrene metabolism

GSH depletion in mice after styrene exposure:

Carlson (2010a) investigated the effect of styrene and of the two enantiomers of styrene oxide (R-SO and S-SO) on glutathione levels in plasma and bronchioalveolar lavage fluid (BAL) of mice after a single intraperitoneal application. Styrene (600 mg/kg bw) caused a significant fall in GSH levels in both BAL and plasma within 3 h. These returned to control levels by 12 h. R-SO (300 mg/kg bw), the more toxic and genotoxic enantiomer, also produced significant decreases of GSH in both body fluids, but S-SO was without marked effect. The author concludes that the reduced GSH concentrations may significantly impair the ability of the lung to buffer oxidative damage. This study supports that styrene and the more toxic isomer R-SO lead to a reversible depression of GSH.

However, these findings are not supported by a 5 -day study in which wild type mice showed a similar degree of lung toxicity after ip injection of R-SO or S-SO (Cruzan et al., 2012).

Richter et al. (2011) developed a histochemical / fluorimetric method to measure GSH in the lung of mice after inhalation exposure (40 and 60 ppm, up to 6 h/d over up to 3 d). GSH was determined in the whole lung and in the bronchial system in parallel to cell proliferation. Exposures to 40 and 160 ppm styrene resulted in a decrease of GSH in lung homogenates during each exposure period (6 h) of 0 - 29% and 31 - 47%, respectively. At both exposures, a rebound of GSH to 110% and 135% of the control concentrations was observed at the beginning of the exposures on days 2 and 3, respectively. There was a gradient of the GSH concentration within the airway epithelium decreasing from the proximal bronchi to the terminal bronchioles. In the latter, cell proliferation was significantly increased at both styrene concentrations. Generally, GSH was reduced in proliferating cells. The authors concluded that the imbalance of the GSH status seems to be a major determinant in styrene-induced cell proliferation in the mouse lung. In summary, this study shows a temporary GSH depletion in the bronchial/bronchiolar epithelium of mice after styrene exposure in parallel with increased cell proliferation.

Aromatic hydroxylation:

Linhart et al. (2010) analyzed urine from mice exposed to styrene vapors (600 and 1200 mg/m3, 6 h) for ring-oxidized metabolites, namely 2-, 3-, and 4-vinylphenol (2-, 3-, and 4-VP), 4-vinylpyrocatechol, and 2-, 3-, and 4-vinylphenylmercapturic acid (2-, 3-, and 4-VPMA). Three isomers, 2-, 3-, and 4-VP, were found and three novel minor urinary metabolites, the arylmercapturic acids 2-, 3-, and 4-VPMA. Excretion of the most abundant isomer, 4-VPMA represented approximately 0.047 and 0.043% of the absorbed dose. In model reactions of styrene 3,4-oxide (3,4-STO) with N-acetylcysteine in aqueous solutions, 4-VP was always the main product, while no 3-VP or mercapturic acids were found. The in vivo formation of 2- and 3-isomers of both VP and VPMA, neither of which was formed from 3,4-STO in vitro, strongly suggests that another arene oxide, styrene 2,3-oxide, might be a minor metabolic intermediate of styrene. In summary some new urinary metabolites in mice were identified stemming from aromatic hydroxylation. There is an indication that styrene 2,3-oxide is formed as an intermediary metabolite to a minor extent.

Linhart et al. (2012) identified 4-VPMA together with traces of 2- and 3-VPMA in the urine of workers exposed to styrene vapors at concentrations ranging from 23 to 244 mg/m3. The excretion of 4-VPMA accounted for only about 3.5×10−4% of the absorbed dose of styrene. Despite this very low metabolic yield, formation of VPMAs clearly indicates ring oxidation of styrene. In summary, several metabolites derived from aromatic hydroxylation were isolated from urine of exposed workers.

Shen et al. (2010) compared aromatic and side chain metabolism of styrene in mice lung and liver microsomes and compared lung toxicity in mice evoked by some of the metabolites. After incubation of styrene with liver microsomes styrene glycol (SG), 2-, 3-, and 4-vinylphenol (VP) were identified as well as in incubation with lung microsomes. The amounts of these metabolites formed by lung microsomes was less that those obtained by liver microsomes. After large scale incubations, in addition vinyl-1,4-hydroquinone and 4- and 2-hydroxystyrene glycol were found with liver microsomes, and only 2-hydroxystyrene glycol with lung microsomes. The ratios of VPs/SG production were calculated to determine the relative contribution of vinyl/aromatic oxidation: larger VPs/SG ratios were obtained for lung as compared to liver microsomes. Among the phenolic metabolites, 2-VP was formed at a rate 10-fold faster than that of 3-VP and 4-VP in both mouse liver and lung microsomes and the production rate of SG was by a factor of 100 greater than that of 2-VP. Metabolism of styrene was slower in mouse lung than in liver microsomes. Disulfiram and 5P1P inhibited VPs and SG production both in liver and lung microsomes. Importantly, the formation of VPs and SG was almost completely inhibited by 5P1P in lung microsomal incubations. Incubation of VPs with liver microsomes led to a substantial disappearance of all of them and 4-VP was lost the fastest. The following downstream metabolites (vinylcatechols and vinyl-1,4-hydroquinone) were identified: 3-vinylcatechol and vinyl-1,4-hydroquinone from 2-VP; 3-vinylcatechol and 4-vinylcatechol from 3-VP; 4-vinylcatechol from 4-VP. In addition the 3 VPs were metabolized to the corresponding glycols by side chain oxidation. Thus, VPs undergo further aromatic hydroxylation as well as vinyl oxidation. In addition, the toxicity of styrene, SO and of the 3 VPs to the lung of mice (as determined by LDH activity and cell number in bronchioalveolar lavage) was determined after a single ip dose of 100 mg/kg bw. Significant increases of cell number and LDH activity in the lavage fluid were only found after application of 4-VP.

In summanry, this study extends the knowledge of aromatic hydroxylation of styrene by microsomes from mouse lung and liver. New styrene metabolites, namely 2-VP, 3-VP, vinyl-1,4-hydroquinone,and 2-hydroxystyrene glycol were identified in mouse liver microsomal incubations. CYP2F2 and CYP2E1 were found to catalyze the formation of VPs and SO from styrene in mouse liver and lung microsomes. The detected 2-VP, 3-VP, and 4-VP were further oxidized to the corresponding catechols and/or hydroquinone by aromatic hydroxylation and to the corresponding oxides by vinyl epoxidation. 2- and 3-VP were not as toxic as 4-VP to the pulmonary system in mice. However, according to the authors, the potential toxicity of 10-fold higher production of 2-VP than that of 4-VP in mouse liver and lung microsomal incubations of styrene should not be neglected.

Shen et al. (2011) studied oxidative metabolism of styrene in CYP2F2-null mouse liver and lung microsomes. An approximately 50% reduction was observed in the rates of the formation of SO (styrene glycol was monitored) in liver and lung microsomes prepared from CYP2F2-null mice relative to the wild-type mice. Substantial decreases in the formation of vinylphenols (VP), vinyl-1,4-hydroquinone, and 2-hydroxystyrene glycol were also found in the CYP2F2-null microsomal incubations with styrene. In addition, incubation of 2-, 3-, and 4-VPs with liver and lung microsomes showed a tremendously decreased metabolism of the VPs to the corresponding catechols/hydroquinone and oxides (vinyl epoxides) in CYP2F2-null mouse lung microsomes, compared with that of the wild-type mice. In summary, these results show that CYP2F2 is not only involved in the primary aromatic and side chain metabolism of styrene but also in further downstream toxification of the aromatic hydroxylated metabolites.

Relevance of CYP 2F2 in mouse lung:

Carlson (2012) studied lung and liver metabolism and toxicity in knockout mice. Mice deficient in cytochrome P450 reductase in the liver (that attenuates the total cytochrome P450 activity) but not in other tissues were used as well as CYP2F2 deficient mice. Mice deficient in hepatic cytochrome P450 reductase had much less metabolism of styrene to SO by liver microsomes but not in lung microsomes. Styrene induced hepatotoxicity after 600 mg/kg bw (once ip) was markedly attenuated (as measured by sorbitol dehydrogenase activity) in the deficient mice as was the reduction of glutathione in the liver in comparison to the wild-type mice. Unexpectedly this enzyme deletion also led to some protection against pneumotoxicity (measured by different parameters in bronchioalveolar lavage fluid). According to the author, the protection against pneumotoxicity in hepatic cytochrome P450 reductase deficient mice may need to be clarified by further pharmacokinetic studies. When comparing CYP2F2 knockout mice with the wild-type the deficiency led to a large decrease in metabolism of styrene to SO in lung microsomes but only to a small decrease in liver microsomes. No difference in hepatotoxicity and a decrease in pneumotoxicity were observed in the knockout mice as compared to the wild-type mice. Finally, pneumotoxicity of SO was markedly reduced in CYP2F2 knockout mice supporting a further toxification pathway via aromatic ring hydroxylation by CYP2F2. This study underlines the importance of CYP2F2 for styrene induced lung toxicity. Liver toxicity depends on CYPs located in the liver. There is indication that CYP2F2 lung toxicity is also mediated by downstream metabolites of SO, possibly by aromatic hydroxylation.

Cruzan et al. (2012) studied the toxicity of styrene and SO in CYP2F2(-/-) knock out in comparison to wild-type mice. Mice were treated with styrene (400 mg/kg bw/d by gavage or 200 or 400 mg/kg bw/d ip) or with S- or R-SO (200 mg/kg bw/d ip) for 5 days. Wild-type mice displayed a significant necrosis and exfoliation of Clara cells after treatment with styrene. The cumulative BrdU-labelling index of S-phase cells was markedly increased in the terminal bronchioles after treatment with styrene or S- or R-SO. In contrast, no toxicity to Clara cells or terminal bronchioles was observed in CYP2F2(-/-) knockout mice exposed to styrene or S- or R-SO. This study clearly demonstrates that lung toxicity of styrene and also of SO depends on the metabolism by CYP2F2, most probably by production of ring hydroxylated downstream metabolites. Importantly, the human isoform of CYP2F2, namely CYP2F1, is expressed at much lower levels in humans. Therefore, styrene-induced mouse lung tumors do not predict lung tumor potential in humans. In conclusion, this study shows that lung toxicity of styrene and SO depends on CYP2F2 that is specific for the mouse lung. Most probably lung toxicity is mediated by ring hydroxylated metabolites of styrene and SO.

Zhang et al. (2011) investigated the metabolism of 4-vinylphenol (4VP), the glutathione (GSH) conjugation of the metabolites of 4VP and the cytochrome P450 (CYP) specificity in epoxidation in different microsomes (mouse lung and liver, rat lung, human lung). Aromatic ring cleavage of 4VP, as measured by formation of 14C02 was negligible. Incubations of 4VP with mouse lung microsomes afforded two major and several minor metabolites. The major metabolites resulted from ring hydroxylation and epoxidation of 4VP to 4VP catechol and the side chain epoxide (4VPO). By trapping with GSH, GSH conjugates of 4VP quinone and 4VPO were obtained. 4VPO was the most abundant metabolite of 4VP and formed two major GSH conjugates at the C-1 position and two minor ones at the C-2 position. The formation of the C-1- and C-2-GSH conjugates was inhibited with specific CYP inhibitors for 2F2 and 2E1 (5P1P and DDTC, respectively). The inhibitor 5P1P had a greater inhibitory effect in the mouse lung than DDTC (85% vs. 57%), suggesting that 2F2 was primarily responsible for 4VPO formation in the mouse lung. It is concluded that 4VP can be metabolized to the electrophilic metabolites, 4VPO and 4VP quinone. These metabolites could can form adducts with nucleophilic macromolecules and this would contribute to the toxicity of 4VP. The formation of GSH conjugates is much higher in rodent microsomal incubation than in those from humans, indicating that the possible toxicity caused by 4VP and styrene in humans will be much lower than in rodents. In summary, this study showed that downstream metabolism of 4-vinyphenol by aromatic and side chain oxidation is mainly effected by CYP2F2 and most prominent in mouse lung microsomes. These toxification pathways are much more effective in rodents than in humans.

Other aspects of styrene metabolism:

Carlson (2010b) studied the metabolism and toxicity of styrene in microsomal epoxide hydrolase-deficient mice (mEH -/-) in comparison to wild-type mice. mEH-/- mice metabolised styrene to SO at the same rate as wild-type mice, but there was minimal metabolism of SO to the glycol. mEH-/- mice were more susceptible to the lethal effects of styrene. After ip application of 200 mg/kg styrene hepatotoxicity (as measured by sorbitol dehydrogenase activity) and pneumotoxicity (by different parameters in bronchioalveolar lavage fluid) was higher in mEH-/- mice as compared to the wild-type. mEH -/-mice were also more susceptible to oxidative stress as indicated by greater decreases in hepatic glutathione levels. On the other hand, SO produced pneumotoxicity was similar in both strains. This was explained by the high exogenous SO dose overwhelming the detoxification capacity of mEH. Since the turnover number for mEH is low it can efficiently detoxify SO as long as it is locally produced in the endoplasmatic reticulum, but exogenously administered SO must be absorbed into the systemic circulation and distributed to the target tissue (cf Vodicka et al., 2006a). Overall, the author concludes that mEH plays an important role in the detoxification of styrene but not exogenously administered SO. This study demonstrates the importance of epoxide hydrolase for detoxification of styrene metabolites (SO) especially as long as SO is produced locally and not administered exogenously.

Hartman et al. (2012) investigated allostery of styrene oxidation to SO by recombinant CYP2E1 and human liver microsomes. At low styrene concentrations, oxidation is inefficient because of weak binding to CYP2E1. A second styrene molecule then binds with higher affinity and significantly improves oxidation. The transition coincides with reported styrene concentrations in blood from exposed workers; thus, this CYP2E1 mechanism may be relevant in vivo. Modeling showed that low styrene levels should be much less toxic than generally assumed. An allosteric interaction was also shown between styrene and 4-methypyrazole. Therefore the authors conclude that mixtures of styrene and other molecules can induce allosteric effects on metabolism by CYP2E1 and thus affect the efficiency of their metabolism and corresponding effects on human health.

In the literature update until Oct. 01, 2015, the following new information was obtained:

Gao et al. (2012) studied in vitro the metabolic mechanism of chiral inversion of S-mandelic acid. It is known that S-MA undergoesone-directional chiral inversion (S-MA to R-MA) in Wistar and Sprague-Dawley rats in vivo.S-MA was converted toR-MA in rat hepatocytes, whereas MA enantiomers remained unchanged in acidic and neutral phosphate buffers, HepG2 cells, and intestinal flora. In addition, the synthesizedS-MA-CoA thioester was rapidly racemized and hydrolyzed toR-MA by rat liver homogenate and S9, cytosolic and mitochondrial fractions. The data suggest that chiral inversion ofS-MA may involve the hydrolysis ofS-MA-CoA.

Some studies focused on polymorphism in humans, but they are considered to be of minor relevance for a hazard identification of styrene.

Rihs et al. (2008) studied 10 single-nucleotide polymorphisms (SNP) in genes of styrene metabolizing enzymes in 89 workers of a fiber-reinforced plastic boat building factory. The influence of 2 SNPs of CYP2E1, epoxide hydrolase and GSTP1, of 1 SNP of CYP2A1 and CYP3A4 and of the deletion of GSTM1 and GSTT1 was analyzed in relation to the concentrations of styrene in blood and of mandelic acid and phenylglyoxylic acid in urine. Effects were only observed for two SNPs (CYP2E1-71TT and GSTP1 2627AG), namely a decrease of urinary phenylglyoxylic acid concentrations without a significant effect on the styrene concentration in blood.

Wang et al. (2008) studied the relationship between polymorphisms of NAT2, CYP2B6 and GSTP1 and urinary metabolites of styrene in 58 styrene-exposed workers. The CYP2B6 mutation genotype was significantly associated with the profile of urinary metabolites, but not the genetic polymorphisms of GSTP1 and NAT2 M3. The authors propose that the genetic susceptibility of exposed workers should also be taken into consideration for interpretation of biomonitoring results. (Publication in Chinese language, only English abstract)

Wang et al. (2009) divided 58 workers occupationally exposed to styrene into a high exposure group (≥ 100 mg/m3) and a low exposure group (≤100 mg/m3). The level of urinary styrene metabolites was influenced by the genotypes of CYP2B6, CYP2D6 and GSTP1. (Publication in Chinese language, only English abstract)

Zhang et al. (2010) studied the influence of genetic polymorphisms of CYP2E1 on the excretion of urinary metabolites (mandelic acid and phenylglyoxylic acid) of styrene in 56 exposed workers. Single nucleotide polymorphisms (SNPs) on CYP2E1 (5-flanking region, RsaI, and intron 6, DraI) were determined. Time-weighted average (TWA) exposure was measured by personal sampling. There was a significant association between urinary metabolites and genotypes of CYP2E1 (5-flanking region, HsaI) in low-exposed group (P<0.05), but no information is given in the abstract regarding the high exposure group. The authors conclude that styrene metabolism could be influenced by genotypes of CYP2E1 and that this polymorphism may be a factor to be taken into consideration for assessment of individual susceptibility. (Publication in Chinese language, only English abstract)

Zhang et al. (2011a) studied the influence of genetic polymorphism of GSTM1 and GSTT1 on metabolism of styrene in 65 styrene-exposed workers. The 8-h-time-weighted average (8h-TWA) was determined by personal passive sampling and the concentration of the phenyl-hydroxyethyl-mercapturic-acid (PHEMA) in urine was measured. The results are not clear as it is stated in the abstract that “whether in high-exposed group (8h-TWA>50 mg/m3) or low-exposed group (8h-TWA£50 mg/m3), there was a significant association between the concentration of the PHEMA in urine and genotypes of GSTM1(P<0.05).” But obviously there was an association with GSTM1 as the authors conclude that “genetic polymorphisms of GSTM1 can influence metabolic process of styrene”. (Publication in Chinese language, only English abstract)

In the literature update up to Oct. 2015 a further study on polymorphism was identified:

Carbonari et al. (2015) studies the influence of polymorphic CYP2E1, EPHX1, GSTT1, and GSTM1 on the urinary excretion of MA and PGA in 30 workers employed in GFR industries. Exposure concentrations measured by personal air sampling were 28.75 mg/m³(median; 8 h TWA) and by biological monitoring urinary concentrations of MA+PGA were 47.15 mg/g creatinine (median: sampling time not given). Urinary MA+PGA were significantly reduced in individuals carrying the CYP2E1*5B or CYP2E1*6 heterozygote alleles in comparison the homozygote wild type and those exhibiting both heterozygous genes had the lowest level of urinary MA+PGA. Also the slow allele EPHX1 codon 113 caused a reduced excretion of MA+PGA.