Mutagenesis vol. 19 no. 6 © UK Environmental Mutagen Society 2004; all rights reserved.
Styrene monomer does not induce unscheduled DNA synthesis in the mouse liver following inhalation exposure
Syngenta CTL, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK
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Abstract |
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Styrene monomer is a commercially important chemical used extensively in the production of plastics. It has been shown to induce lung tumours in the mouse via the inhalation route. Styrene monomer has shown a low reactivity with DNA and also a lack of genotoxic response in the mouse lung in vivo. Together with the fact that the mouse lung tumours were late occurring and mostly benign, which suggest a promotional effect rather than initiation, these factors have led to a suggestion that the tumours may not be of genotoxic origin. The studies examining the genotoxicity of styrene monomer in vivo have to date been predominantly cytogenetic assessments, although low levels of DNA adducts have been reported in the mouse liver and lung. In order to extend this evaluation, a mouse liver unscheduled DNA synthesis study has been performed to assess the ability of styrene monomer to induce DNA damage/repair. The negative response observed in this assay is consistent with the theory that tumours observed in mouse oncogenicity studies are non-genotoxic in origin.
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Introduction |
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Styrene monomer is produced globally on a large scale with a world wide production in excess of 20 million metric tonnes according to the Styrene Producers Association and is used extensively in the manufacture of styrene-based polymers. They are used in the manufacture of plastic and rubber products, including polystyrene, expandable polystyrene, acrylonitrile butadiene styrene, styrene-acrylonitrile, styrene butadiene rubber, unsaturated polyester resins and styrene butadiene latices. Polystyrene-based polymers are used as packaging materials, disposable cups, food containers and as insulation in cars, boats, computers, etc. Human exposure occurs at levels of up to 12 p.p.m. during production and industrial use and at higher levels in the glassfibre-reinforced plastics industry (up to 60 p.p.m.; Dalton et al., 2003
). The exposure levels in the general population are much lower and derive mainly from the inhalation of cigarette smoke and from the intake of food that has been in contact with styrene-containing polymers (Tang et al., 2000).
Styrene monomer has been tested for carcinogenicity in mice via the inhalation route (Cruzan et al., 2001). In male and female mice there was an increase in the incidence of pulmonary adenomas and, in female mice only, an increase in bronchiolo-alveolar carcinomas in the high dose group. Four previous studies in mice by oral gavage, four in rats by oral gavage, two in rats by inhalation and one in rats by drinking water were concluded by the IARC to be either negative or inadequate for the assessment of tumour incidence. Overall, the IARC concluded that there is limited evidence for the carcinogenicity of styrene monomer in humans and experimental animals. Styrene is considered by the IARC as possibly carcinogenic to humans (Group 2B) (IARC, 2002).
Styrene monomer has been tested in a range of genotoxicity assays. It has been shown to be predominantly negative in bacterial mutation assays, although some effects have been reported in the presence of metabolic activation. The majority of clastogenicity studies showed no increases in the frequency of micronuclei or chromosome aberrations (Scott and Preston, 1994), although positive responses have been reported for both sister chromatid exchange in rodents in vivo and chromosomal aberrations in human lymphocytes in vitro (IARC, 1994).
In order to provide an evaluation of the genotoxic potential of styrene monomer in vivo using a different end-point to a cytogenetic one, an in vivo unscheduled DNA synthesis (UDS) assay in the mouse was undertaken. This is a well-established assay for the detection of general DNA damage/repair activity and is an important assay used as a complementary assay to cytogenetic evaluations in testing strategies. The liver was selected as the tissue for examination since the methodology is well established for the UDS assay and this is a tissue that is a target organ for styrene monomer-induced toxicity.
Although some differences in toxicity between male and female CD-1 mice have been reported (Cruzan et al., 1997), the difference was not substantial and therefore the use of a single sex was considered justifiable (OECD, 2002). As the tumours seen in the carcinogenicity study were malignant in female mice but benign in males, the present study was performed using females only.
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Materials and methods |
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Chemicals
Styrene monomer was supplied by Shell Chemicals Europe B.V. as a colourless liquid with a stated purity of 99.9%. N-Nitrosodimethylamine (N-DMA) (Sigma) was used as a positive control. N-DMA was dissolved in sterile double-deionized water and atmospheres of styrene monomer were generated in clean dry air. Atmosphere samples were taken between 8 and 12 times from the exposed and control groups during each exposure period to quantify the test substance. All mean analysed atmosphere concentrations were within 10% of nominal concentrations.
Animals and husbandry
Female CD-1 mice in the age range 5–9 weeks were used for the study. The animals were supplied by Charles River (Margate, UK). On arrival the mice were individually housed and given food (Rat and Mouse No. 1 Maintenance diet; Special Diets Services) and water ad libitum. Animals were housed in plastic cages on woodflake bedding with nestlets for environmental enrichment. The animal rooms were maintained within the temperature range 19–25°C, with a relative humidity range of 30–70%. Lighting was controlled to provide 12 h artificial light followed by 12 h darkness. The animal room was under positive pressure with respect to the access corridor and had at least 15 air changes per hour. Animals received no food or water during the exposure period.
Dosing regime
Groups of randomly allocated, non-fasted female mice were exposed to atmospheres of the test substance or vehicle control. Clean dry air was supplied to the exposure chamber via the atmosphere generation system and directly, if appropriate, as diluting air. Flow rates through the chamber gave a minimum of 12 air changes per hour. The positive control substance (N-DMA) was prepared for each test as a solution at the appropriate concentration in sterilized double-deionised water in order to achieve a final dose level of 10 mg/kg. The positive control groups were dosed via the oral route at 10 ml/kg. Animals in the vehicle control and test substance groups received a single 6 h whole body exposure.
In order to select the relevant doses to be used in the UDS study, a preliminary study was conducted in which groups of five animals were exposed to a range of concentrations (160–1000 p.p.m.) of styrene monomer and observed for 4 days. After the observation period, the animals were killed by overexposure to halothane followed by cervical dislocation, given a gross necropsy and liver samples were taken for micropathological examination. No adverse clinical signs were observed for animals exposed to 160 p.p.m. Slightly decreased activity was observed in animals exposed to 250 p.p.m. Piloerection, reduced breathing rate and slightly decreased activity were observed in animals exposed to 500 p.p.m. All animals were fully recovered within 24 h. A single animal from the group exposed to 1000 p.p.m. was found dead on day 2. All other animals in this group showed clinical signs, including reduced breathing rate, decreased activity, shaking and hunched posture. This dose level was therefore considered to be in excess of the MTD (maximum tolerated dose). Micropathological examination of the livers from animals exposed to 500 p.p.m. showed minimal to slight glycogen deposition, focal/multifocal mixed inflammatory cell infiltration, increased number of mitoses, portal inflammation and multifocal single cell necrosis. Based on the significant findings at this dose level, including necrosis, 500 p.p.m. was also considered too high to be used for the main UDS assay due to evidence of toxicity in the target organ. Microscopic findings in livers from animals exposed to 160 and 250 p.p.m. included slight to moderate glycogen deposition and focal/multifocal mixed inflammatory cell infiltration. Such findings were considered acceptable for a maximum dose level in this assay. The main UDS assay was therefore performed using styrene dose levels of 250 and 125 p.p.m.
In the main UDS test the positive control group received a single oral administration and the vehicle control and test substance groups received a single 6 h whole body exposure. All animals were anaesthetized using halothane and the livers perfused either 2 or 16 h after dosing as described below. All animals were examined internally for organ/tissue abnormalities.
Slide preparation
The procedures used were in accordance with the OECD Guidelines for this assay (OECD, 2002). A V-shaped incision was made through both skin and muscle from the centre lower abdomen up through the rib cage. The hepatic portal vein was cannulated with an appropriate gauge catheter and the superior vena cava and inferior vena cava (superior to the kidney) were both cut. A buffer solution (150 mM NaCl, 3.73 mM NaHCO3, 4.84 mM Na2HPO4, 4.97 mM KCl, 1.24 mM KH2PO4, 0.62 mM MgSO4.7H2O and 0.62 mM MgCl2.6H2O) was used to flush the liver free of blood and to remove calcium from the desmosomes. A second buffer (142 mM NaCl, 24 mM NaHCO3, 4.37 mM KCl, 1.24 mM KH2PO4, 0.62 mM MgSO4.7H2O and 0.62 mM MgCl2.6H2O) to which calcium chloride and collagenase were added was used to cause desegregation of the liver tissue. The gall bladder was removed and the liver was then removed, finely chopped and filtered through 150 µm mesh nylon bolting cloth prior to hepatocyte preparation by low speed centrifugation and resuspension in Williams' E medium supplemented with 10% foetal calf serum. The viability of the hepatocytes was determined using trypan blue. The hepatocyte suspensions were diluted with Williams' E medium supplemented with 10% foetal calf serum to give a final cell count of 1.5 x 105 cells/ml and transferred onto coverslips placed etched side up in six-well plates. The cultures were placed in a humidified 37°C incubator with a 95% air:5% CO2 (v/v) atmosphere for at least 90 min to allow cell attachment. The culture medium was aspirated and the hepatocytes washed with Williams' E medium. Williams' E medium containing [3H]thymidine was added to each well and the dishes were incubated for 
4 h in a 37°C incubator. Incubation with [3H]thymidine commenced 3 h after the nominal sampling times. Cultures were then washed three times with Williams' E medium plus thymidine. The cultures were then incubated overnight (at least 12 h) with the same culture medium. The cultures were then washed once with Williams' E medium prior to being fixed three times with freshly prepared 1:3 glacial acetic acid:absolute alcohol (v/v), followed by four washes with double-deionized water. The coverslips were mounted, cell side up, on labelled microscope slides. Three slides from each animal were coated with photographic emulsion (Ilford K2) and stored at 4°C for 14 days. After the exposure period, slides were developed using Kodak D19 developer and Ilford Hypam fixer. The cells were stained using Meyers Haemalum and eosin.
Slide analysis
The slides were coded and analysed for UDS induction using a PC-based UDS data capture system (Perceptive Instruments, Haverhill, UK). For each cell, the number of silver grains over the nucleus (N) was determined. Then an equivalent area of cytoplasm tangential to the nucleus and with the highest apparent number of silver grains was analysed (C). The difference between these two values (N – C) was the net nuclear grain count. One hundred cells were scored from each animal.
Data evaluation
The computer system calculated the mean nuclear grain count (N), the mean cytoplasmic grain count (C), the mean net nuclear grain count (N – C) and the percentage of cells in repair (i.e. cells with N – C values of 
5) for each slide and animal. The data were interpreted using concurrent and, if appropriate, historical control data.
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Results |
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No adverse clinical reactions to treatment were observed in mice exposed to styrene monomer at 250 or 125 p.p.m. Examination of the internal organs/tissues showed no abnormalities. Hepatocytes prepared from all animals were examined microscopically and no signs of cytotoxicity were observed on slides from animals dosed with styrene monomer. Therefore, slides from animals treated with styrene monomer at both dose levels were assessed for UDS. Styrene monomer caused no significant increases, compared with the vehicle control, in mean net nuclear grain count or in percentage of cells in repair at either dose level or time point investigated (Tables I and II). Hepatocytes from all styrene monomer-treated animals had mean net nuclear grain values of <0. The positive control substance, N-DMA, induced appropriate increases in the mean net nuclear grain counts and percentage of cells in repair and proved the study to be valid. These data clearly provided no evidence for induction of UDS by styrene monomer. Therefore, it can be concluded that styrene showed no genotoxic activity in the UDS assay.
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Discussion |
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Styrene monomer shows at most a weak genotoxic activity, as demonstrated by its profile in a number of genotoxicity tests. It is either negative or weakly positive in mutagenicity assays in plants, V79 cells, lower eukaryotes and Drosophila, although in vitro assays for chromosome effects have generally shown positive responses in human lymphocytes and Chinese hamster lung cells (IARC, 1994
). Genotoxicity tests in vivo have generally not shown increases in chromosome aberrations or micronuclei but do show consistently small increases in sister chromatid exchanges (IARC, 1994). Increases in DNA alkali-labile sites and strand breaks have been reported, but it has been suggested that these may be due to oxidative damage rather than direct genotoxicity (Marczynski et al., 1997a,b). No increases in chromosome aberrations were reported in the lungs of mice exposed to up to 500 p.p.m. styrene monomer for 14 days (Kligerman et al., 1992), again suggesting a lack of genotoxic response in the target organ for tumour formation.
The tumours observed in the mouse study (Cruzan et al., 2001) were late occurring, mostly benign and were observed in an organ with a high spontaneous incidence. Such a profile suggests tumour promotional effects rather than tumour initiation (US EPA, 1996). This is supported by the lack of activity of styrene monomer in a lung tumour initiation assay (Brunnemann et al., 1992).
Although the present study assesses the genotoxicity of styrene in a different tissue from that in which tumours have been observed, the end-point chosen is a general one for detecting DNA damage and repair which has been shown to be a reproducible, reliable and sensitive technique for the evaluation of genotoxicity in vivo (Kennelly, 1995). The tissue investigated has been shown here to be a target tissue for toxicity following the administration of styrene monomer in the mouse by inhalation and is therefore considered relevant. The present study increases the in vivo genotoxicity database for styrene monomer by providing an evaluation using a different end-point to the cytogenetic end-points examined to date.
The positive control was administered by a different route (oral gavage) to the test substance (inhalation) for technical simplicity. In this instance the positive control is included in the study design as a system control to indicate the integrity of the post-cell isolation procedures. The use of N-DMA as a positive control substance for UDS in the liver has been demonstrated previously via both the oral route in this laboratory (Ashby et al., 1996) and the inhalation route in another laboratory (Doolittle et al., 1984).
The lack of induction of UDS in the study reported here using the same sex and strain of animals which showed the highest tumour incidence is consistent with the theory that tumours observed in the mouse following inhalation exposure to styrene monomer are not caused by a genotoxic mode of action.
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Acknowledgments |
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This work was sponsored by CEFIC Styrenics Steering Committee, who also paid for the preparation of the manuscript.
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Notes |
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1 Tel: +44 1625 515461; Fax: +44 1625 590249; Email: phil.clay{at}syngenta.com
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References |
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Received on March 18, 2004; accepted on October 11, 2004.
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