* Department of Molecular Toxicology, Shell Research and Technology Center, Amsterdam, Shell International Chemicals B.V., P.O. Box 1030 BN Amsterdam, The Netherlands; and
Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709
Received April 19, 2000; accepted July 18, 2000
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ABSTRACT |
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Key Words: styrene; inhalation exposure; disposition; autoradiography; Sprague Dawley rats; CD1 mice; metabolism; CO2; urine; feces.
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INTRODUCTION |
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One of the most important pathways in the mammalian metabolism of styrene is oxidation to styrene-7,8-oxide (SO), an established bacterial and mammalian mutagen. Despite the fact that SO is rapidly detoxified by epoxide hydrolase catalyzed hydrolysis and glutathione-S-transferase catalyzed conjugation, the genotoxicity of SO has raised concern about the potential carcinogenicity of styrene (IARC, 1994a). A critical review of 11 long-term carcinogenicity studies in animals revealed a number of adverse effects in the mouse but not in the rat, and the reviewers concluded that the evidence for styrene carcinogenicity was, at best, equivocal (McConnell and Swenberg, 1994
). Some cases of leukemia and lymphoma in styrene workers were reported, but invariably these workers had also been exposed to chemicals that are suspect or proven leukemogens, such as butadiene and benzene. Several cohort studies were conducted in workers from a variety of styrene industries, but increased incidences of lymphatic and hematopoietic cancers were not significant or not correlated to styrene exposure levels. IARC evaluated these studies as inadequate in determining the carcinogenic effect of styrene in humans. Although the same review concluded that only limited evidence in experimental animals existed for the carcinogenicity of styrene, styrene was classified as a possible human carcinogen (2B), because it is metabolized to SO (IARC, 1994a
).
In recent bioassays in Sprague Dawley rats and CD1 mice, nasal toxicity and an increased incidence of masses in the bronchioalveolar region in lungs of mice were reported, but there were no increases in tumor incidence in rats exposed to up to 1000 ppm styrene (Cruzan et al., 1998; 2000). However, the tumorigenicity of styrene was not related simply to SO concentrations in blood. SO concentrations were lower in mice exposed to 160 ppm styrene than in rats exposed to 2001000 ppm styrene. However, at a concentration of 160 ppm, styrene is toxic in mice but not in rats.
Clearly, a detailed understanding of the fate and effects of styrene is an essential component in the risk assessment of styrene. Therefore, a series of studies was set up to obtain a more complete and detailed picture of the disposition, metabolism, and genotoxic potency of styrene in rats and mice during and following inhalation exposure. The present report describes the inhalation exposure and the overall disposition of styrene in rats and mice. In a companion paper (Boogaard et al., 2000), the DNA binding of styrene in liver, lung, and isolated lung cells is reported. A third report, on the metabolism of styrene, is being prepared as a separate publication.
[Ring-14C]styrene was used at a high specific radioactivity of approximately 1.85 TBq x mol1 (50 Ci x mol1) to enable a single inhalation exposure to a relatively low concentration of 160 ppm. Chronic exposure to this concentration led to the development of bronchioalveolar tumors and signs of toxicity in male mice, although no adverse effects were observed in male rats.
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MATERIALS AND METHODS |
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Animals.
Male Sprague Dawley rats and CD1 mice were purchased at the age of 910 weeks from Charles River Co. (Raleigh, NC, USA). Following acclimatization, the rats were used at an age of approximately 12 weeks (360 g) and the mice at an age of approximately 1012 weeks (
36 g). Animals were housed according to standard animal care procedures with free access to food (NIH-07; Zeigler Brothers, Gardners, PA, USA) and purified water in a climate-controlled (relative humidity 55%, temperature 22 ± 2°C) room on a 12-h light-dark cycle. This study was approved by the Institutional Animal Care and Use Committee and was performed in accordance with the Declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health. The inhalation exposures were short term and were performed in the absence of food and water.
Generation and control of exposure atmosphere.
The required atmosphere of 160 ppm 14C-styrene was generated using a stainless steel pressure vessel system (38.3 l) as shown schematically in Figure 2. After flushing the pressure vessel with nitrogen (zero grade), the 14C-styrene was transferred onto the bottom of the vessel, which was subsequently closed. The styrene was then allowed to evaporate and diffuse through the pressure vessel overnight. Prior to the exposure, the vessel was pressurized to a predetermined value by addition of nitrogen (300 l for the rat exposure and 209 l for the mouse exposures). To begin exposure, the nitrogen-styrene mixture was metered into the nose-only chamber inlet line and mixed with oxygen (USP grade) and dilution air prior to mixing with the recirculating flow and entering the nose-only chamber. The oxygen flow was monitored with a rotameter and the dilution air with a mass flow controller. An IR spectrophotometer (MIRAN 1A, Foxboro, MA, USA) served as both a mixing chamber and as a styrene concentration monitor (vide infra). A portion of the outlet atmosphere was recirculated back into the chamber inlet using a stainless steel diaphragm pump (Model MB-41, Metal Bellows Co., Sharon, MA, USA) to push the air flow through the recirculation line. A valve upstream of this pump was used to control the flow rate. A condenser was placed in the recirculation line. Chilled water was passed through the condenser jacket to condense moisture from the recirculating atmosphere. The condensate was collected in a trap in the bottom of the condenser. The recirculated atmosphere was also passed through a trap containing SodaSorb to absorb CO2 and through a 60-µm filter to remove particles. Styrene is not adsorbed to SodaSorb (Leavens et al., 1996
). The portion of the flow that was not recirculated was passed through KOH traps to remove CO2 and through sets of charcoal filters to absorb styrene. During exposure, this exhaust flow was adjusted to maintain the static pressure inside the nose-only chamber at a level approximately equal to or slightly less than ambient atmosphere pressure.
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Disposition studies.
Immediately after cessation of exposure, half the animals (6 rats and 15 mice in each exposure experiment) were killed for collection of tissues for macromolecular binding studies (Boogaard et al., 2000). Of the remaining animals, 4 rats and 8 mice (4 in each exposure experiment) were transferred to individual gas-tight metabolism cages. Feces and urine were collected over ice for 06, 624, and 2442 h following exposure. Radioactivity in samples was determined by scintillation counting as described below. The expired air from each cage was drawn by a vacuum pump at a rate of 500 ml x min1 for rats and 200 ml x min1 for mice through a series of three charcoal filters (Supelco ORBO, 0.9 g) for the collection of exhaled organic volatiles (14C-styrene and 14C-metabolites) and subsequently serially through two 1 M KOH solutions (500 ml each for rats and 250 ml each for mice) for determination of CO2. Traps for volatiles and CO2 were changed at intervals, 1, 3, 6, 24, and 42 h after cessation of exposure, and analyzed as described below. For the collection of larger urine volumes necessary for metabolite isolation and identification, 2 additional rats were housed individually and 22 mice (11 for each exposure experiment) in groups of 3 or 4 mice in polycarbonate metabolism cages. At the end of the collection period, all animals received a lethal dose of pentobarbital and were processed for autoradiography or DNA analysis (Boogaard et al., 2000
). The cages were washed with water, and the measured aliquots of the washings were collected and counted by liquid scintillation counting (LSC).
Urine, feces, and charcoal traps for exhaled volatiles were transferred to glass tubes, sealed, and stored individually at 80°C until transport and further analysis.
Quantitative whole-body autoradiography.
One rat and 2 mice (one from each inhalation experiment) received a lethal dose of pentobarbital 42 h after termination of exposure. Following confirmed death, the carcasses were stretched and mounted on a plastic support. The tails were clipped off, the eyes were moistened with 2% (w/v) aqueous carboxymethyl cellulose solution, and the carcasses were then frozen in a mixture of n-hexane and excess solid CO2. The frozen carcasses were stored at 80°C until dispatch on solid CO2 to Covance Laboratories, Harrogate, UK, for analysis. The frozen carcasses were set in blocks of 2% (w/v) aqueous carboxymethylcellulose and mounted onto the stage of a cryomicrotome (MPV 450 MP, Thermometric Ltd, Northwich, UK) maintained at 20°C. Sagittal sections (nominally 30 µm thickness) were obtained from five different levels to expose a range of smaller tissues that could not easily be dissected, such as nasal passages, central nervous system, and glandular organs. The sections were mounted on Syrom 90 tape (Milnes Packaging Group, Brighouse, UK), lyophilized (Lyolab B, Life Sciences Laboratories Ltd, Luton, UK), placed into contact with imaging plates (Fuji type BAS IIIs, Raytek Scientific Ltd, Sheffield, UK), and stored in a refrigerated lead-lined exposure box for 3 days. The distribution of radioactivity in the sections was measured using a radioluminography system (Fuji 1500, Raytek Scientific Ltd). Quantitation was obtained by comparison with a range of 14C-blood standards, which were included with each autoradiogram, using a PC-based image analysis package (Seescan Densitometric Software, Lablogic Ltd, Sheffield, UK). For all calculations, the density and quench characteristics of the tissues analyzed were assumed to be similar to those of blood.
Quantitation of radioactivity.
Liquid samples were taken up in Ultima Gold scintillation cocktail (Canberra-Packard, Groningen, The Netherlands) in antistatic scintillation vials and measured by LSC using TriCarb 2200 CA counters (Canberra Packard). The machines were calibrated using a commercial 14C internal standard kit for organic solvents (Wallac, Turku, Finland). The calibration was checked daily by counting a set of quenched standards commercially prepared in sealed glass vials. Counting efficiency was determined using the spectral index of the internal standard (SIE), and cpm values were automatically transformed to dpm. Samples were corrected for background.
The radioactivity in the KOH trap solutions containing exhaled 14CO2 was measured by LSC. Duplicate aliquots of 1 ml were added to preweighed 20-ml antistatic scintillation vials containing 3 ml purified water to which 16 ml scintillation cocktail was added before counting. In some cases, 1-ml aliquots of the KOH solution were evaporated to dryness to remove possibly dissolved styrene. The dry residue was subsequently taken up in 1 ml purified water and 19 ml scintillation cocktail was added before counting. The urine samples were diluted 100 times with purified water and duplicate 10-µl aliquots were counted by LSC. Cage washings were pooled for each cage, and a 1-ml aliquot was counted by LSC.
Charcoal filters containing exhaled volatiles were analyzed for radioactivity by emptying the charcoal from the filter into a conical centrifuge tube and adding 2 ml formamide. After extraction, the charcoal was pelleted by centrifugation (1500 x g, 5 min) and the supernatant removed. Aliquots of the supernatant were analyzed by LSC.
Feces were pulverized in liquid N2 using a hammer mill (6700 Freezer/Mill, Glen Creston Inc., Stanmore, UK), and aliquots (approximately 100 mg) were transferred to glass tubes with 1.0 ml tetraethyl ammonium hydroxide and closed. The samples were kept for 48 h at 60°C until dissolved. The samples were centrifuged for 5 min at 2500 x g, and the supernatants counted by LSC. Colored samples were treated with hydrogen peroxide prior to addition of the scintillation cocktail.
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RESULTS |
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Excretion of Radioactivity
A summary of the excretion of radioactivity is given in Table 2. From the radioactivity that was estimated to be taken up by the rats, 79 ± 7% was accounted for by excreta (urine, feces, CO2, and exhaled volatiles). For the two mouse exposures, 84 ± 10% and 77 ± 10% of the amount estimated to be taken up by the animals was accounted for by excreta.
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Small amounts of radioactivity were excreted in the feces. For the rats, the total amount of radioactivity excreted through the feces was 1.0 ± 0.6% of the retained radioactivity. In the second mouse experiment, a similar value was found, 1.3 ± 0.2%. In the first mouse experiment, a higher average value of 8.5 ± 5.6% was found, which was almost completely due to the higher value measured in a single feces sample collected between 6 and 24 h after cessation of exposure. Although contamination of this sample with urine cannot be completely ruled out, it is unlikely that such a contamination would increase the value to the extent measured. Although the production of 14CO2 was only a minor part of the metabolism, a major difference was seen between mice and rats in the amount of 14CO2 that was being exhaled. Mice exhaled 3 to 4 times more 14CO2 than rats. Virtually all of the 14CO2 was produced during the exposure. In rats, the average amount of 14CO2 measured for the period of 2442 h after exposure was relatively high, and higher than during the two previous periods. However, this value was not statistically different from the previous time points due to the large standard deviation associated with this value. At the end of the holding period in the metabolism cages, only a small percentage of the styrene metabolized by the animals was associated with the tissues (Table 3).
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DISCUSSION |
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The uptake of styrene in mice was on average 186 µmol x kg1 x h1, but a steady decrease in uptake to about a quarter of the initial uptake was measured during the 6 h of exposure. This reduction was caused partly by a reduction in the ventilation rate and partly by reduction of the overall metabolism, as oxygen consumption by the mice was reduced to about half of the initial value in the first h of exposure and remained at this low level during the rest of the exposure period. Some of the mice showed signs of toxicity at the end of the exposure period. The uptake in rats was lower than in mice (113 µmol x kg1 x h1) and virtually constant over the entire period of 6 h. There was no change in oxygen consumption by the rats, nor were any signs of toxicity observed.
The data from quantitative whole-body autoradiography show a slightly larger retention of radioactivity in mice, with an average concentration in blood in the two mice about 30% higher than in the single rat that was analyzed. Most organs had radioactivity levels that were equal to or less than the blood in both rats and mice. A notable exception was the nasal mucosa, in which higher levels of radioactivity than in any other tissue were measured in both rat and mouse. The rather large difference between the two mice is probably due to inconsistencies in the sectioning rather than a true reflection of animal variation. The disposition of radiolabeled styrene has been studied only once in rats (Carlsson, 1981) and a few times in mice (Ghantous et al., 1990
; Kishi et al., 1989
; Löf et al., 1984
). Accumulation of styrene in the nasal mucosa was also observed, but not quantified, in male C57BL mice 8 h after a 10-min inhalation exposure to an undefined concentration of [8-14C]-styrene (Ghantous et al., 1990
) and in pregnant CD-1 mice exposed to 2.3 µmol [8-14C]-styrene by tail vein injection (Kishi et al., 1989
). Kishi and coworkers proposed that the high concentrations of radioactivity in the nasal passages and lung might originate partly from 14CO2 through metabolic decarboxylation of the sidechain. However, our present results indicate that metabolism is virtually complete 45 h after cessation of exposure, which excludes the possibility that the radioactivity comes from 14CO2. In both the present study and the study by Kishi et al., the radioactivity more likely originates from tissue binding of styrene metabolites generated in situ. This would imply that rats and mice both form reactive metabolites from styrene in the nasal mucosa, but only mice form substantial amounts of reactive metabolites from circulating styrene in the lung; the tissue concentration of radioactivity in rat lung is well below the concentration in blood (Table 3
). This formation of reactive intermediates would be consistent with the recent observations in CD-1 mice and CD rats upon chronic exposure to styrene (Cruzan et al., 1998
; 2000). Styrene-related non-neoplastic histopathological changes, mainly respiratory metaplasia of the olfactory epithelium with changes in the underlying Bowman's gland, were observed in the nasal passages of both mice and rats. The severity of these changes increased with styrene concentration and duration of exposure, but occurred in mice at much lower styrene concentrations than in rats. In mice, the incidence of bronchioalveolar adenomas was significantly increased after 2 years of exposure, but in rat lungs no histopathological changes were observed (Cruzan et al., 1998
; 2000).
In liver and kidney cortex, again, higher concentrations of radioactivity were found in mice compared to rats. The concentration of radioactivity in mouse liver was 4.5 times the concentration in blood, whereas the concentration in rat liver was only slightly higher than in blood. In the kidney cortex of mice, 6.7 times the concentration in blood was measured, whereas the concentration in the kidney cortex of rats was only 1.4 times higher than in blood. In a previous study in Sprague-Dawley rats exposed to 43.5 and 240 ppm of [7-14C]styrene for 1 to 8 h, high concentrations of 14C were found in kidneys, liver, and subcutaneous fat. At all time points (i.e., up to 6 h postexposure) concentrations in kidneys were 3 to 14 times higher than in subcutaneous fat or liver (Carlsson, 1981). However, this high level of radioactivity in the rat kidney is a transient phenomenon and most likely related to the fact that almost all the retained radiolabeled styrene is cleared through the kidney. At the time points chosen in this study, renal excretion is at its maximum. Similarly, the high concentrations of styrene in adipose tissue reported by Carlsson (1981) were also transient. We measured levels of radioactivity in fat, 45 h after cessation of exposure, that were equal or slightly less than in blood, indicating that styrene is stored in fat during exposure but swiftly released after termination of exposure. Differences observed in distribution appear to be related to the timing of sampling rather than to the different ways of administration of the 14C-styrene as suggested previously (Sumner et al., 1995
). Observed differences between the present study and earlier studies may also be explained, at least partly, by the position of the radiolabel: [8-14C]styrene will easily lose its label through metabolic decarboxylation within a short period after the end of exposure and become undetectable in (auto-)radiography, whereas [ring-U-14C]styrene will not release 14CO2 through simple decarboxylation and may be detected in the tissues for a longer period.
The position of 14C-label in the ring in the present study implies that exhaled 14CO2 is a result of styrene metabolism involving opening and degradation of the aromatic ring. Ring opening is probably preceded by ringoxidation (Blaesdale et al., 1996). Evidence of ringoxidation of styrene was found in the formation of 4-vinylphenol, which was reported as a urinary metabolite of styrene (Bakke and Scheline, 1970
; Pfäffli et al., 1981
). Other evidence for ring-opened metabolites of styrene was recently reported (Sumner et al., 1995
). Formation of reactive metabolites through ringoxidation of styrene (or SO) and subsequent ring opening might be catalyzed by specific isoforms of cytochrome P450 present in specific cell types. Formation of 14CO2 was limited during exposure and almost absent after cessation of exposure in both rats and mice. Nevertheless, there was a substantial difference between rats and mice, with mice producing 34 times more 14CO2 than rats, accounting for 6.4 ± 1.0 and 8.0 ± 0.5% of the total styrene retained during the first and second mouse exposure, respectively. This larger production of 14CO2 in mice than in rats, in combination with the substantially higher tissue binding observed in the lungs, might be indicative of the formation of reactive ring-opened metabolites in the mouse lung, which in turn might be related to the observed development of bronchioloalveolar tumors and nasal effects in mice exposed to styrene.
One of the major metabolic pathways of styrene is the cytochrome P450-dependent oxidation to SO, which is rapidly detoxified by enzyme-catalyzed epoxide hydrolysis or glutathione (GSH) conjugation. High concentrations of cytochrome P450 are found in liver, lung, and renal cortex. The extensive cytochrome P450-dependent oxidation of styrene to SO and ring-opened metabolites, which also are likely to be detoxified for a substantial part through GSH conjugation, might lead to local depletion of GSH. Such a GSH depletion would not only explain the observed extensive tissue binding in liver, kidney, and lungs, but also the toxicity observed in mice, as more oxidative metabolism occurs in mice than in rats. Despite the high tissue binding in mouse lung, the DNA binding was very low ( 1 adduct per 108 nucleotides) and not significantly different from the DNA binding in rat lung (Boogaard et al., 2000
). This suggests that a nongenotoxic mechanism, possibly caused by a cytotoxic metabolite, underlies the observed bronchioalveolar tumor formation in mice.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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