Disposition of [Ring-U-14C]styrene in Rats and Mice Exposed by Recirculating Nose-Only Inhalation

P. J. Boogaard*,1, K. P. de Kloe*, S. C. J. Sumner{dagger}, P. A. van Elburg* and B. A. Wong{dagger}

* Department of Molecular Toxicology, Shell Research and Technology Center, Amsterdam, Shell International Chemicals B.V., P.O. Box 1030 BN Amsterdam, The Netherlands; and {dagger} Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709

Received April 19, 2000; accepted July 18, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The disposition of styrene was studied in a group of 12 Sprague Dawley rats and two groups of 30 CD1 mice exposed separately to 160 ppm [ring-U-14C]styrene of high specific radioactivity of 1.92 TBq x mol–1 (52 Ci x mol–1) for 6 h. A nose-only exposure system was successfully adapted to (1) recirculate a portion of the flow to limit the amount of 14C-styrene required, and (2) avoid any polymerization of the compound. The mean uptake of styrene in rats was 113 ± 7 µmol x kg–1 x h–1 and stable over time. The mean uptake in mice was higher, 189 ± 53 and 183 ± 76 µmol x kg–1 x h–1, for the first and second mouse inhalation experiment, but decreased steadily over time. Some of the mice, but none of the rats, showed signs of overt toxicity. The overall excretion of styrene and its metabolites was quantitatively similar in rats and mice. Urinary excretion was the primary route of excretion while fecal excretion accounted for only a very small part of the radioactivity. There was, however, a significant difference between mice and rats in the exhalation of 14CO2, which must have resulted from opening and subsequent breakdown of the aromatic ring. In mice the exhalation of 14CO2 accounted for 6.4 ± 1.0 and 8.0 ± 0.5% of the styrene retained during the first and second mouse inhalation experiment. In rats, exhalation of 14CO2 accounted for only 2.0 ± 0.7% of the retained styrene. Together with the results from the quantitative whole-body autoradiography (showing significantly higher binding in mouse lung and nasal passages compared to rat) the larger production of 14CO2 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 bronchioalveolar tumors and nasal effects in mice exposed to styrene.

Key Words: styrene; inhalation exposure; disposition; autoradiography; Sprague Dawley rats; CD1 mice; metabolism; CO2; urine; feces.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Styrene is one of the most widely used monomers in the polymer production industry. Its major use is in the production of polystyrene, resins, paints, and synthetic rubbers and in the reinforced plastic industry (Miller et al., 1994Go). Human exposure occurs mainly in the workforce handling styrene monomer. The highest occupational exposures have been measured in reinforced plastic industries. Styrene is an irritant that may depress the peripheral and central nervous systems and cause hepatotoxicity and pneumotoxicity in experimental animals (Bond, 1989Go; Gadberry et al., 1996Go; Sumner et al., 1997Go).

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, 1994aGo). 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, 1994Go). 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, 1994aGo).

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., 1998Go; 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 200–1000 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., 2000Go), 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 mol–1 (50 Ci x mol–1) 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Radiochemical synthesis.
[Ring-U-14C]styrene was prepared in three steps from [ring-U-14C]benzoic acid (Fig. 1Go). Initially, a 1.85-GBq (50 mCi) aliquot of [ring-U-14C]styrene with a specific radioactivity of 2.04 TBq x mol–1 (55.2 Ci x mol–1), a radiopurity of 96% (major contaminant 4% 14C-benzaldehyde), and a chemical purity of 90% (major contaminant 5% benzene) was synthesized. This material was tested for its stability under various conditions. When stored in capillary tubes at 4°C, rapid polymerization occurred, which was probably catalyzed by active hydroxyl groups on the large glass surface. DMSO solutions of the 14C-styrene were also unstable. Stored in glass vials in the dark as a neat liquid in the presence of 100–200 ppm 4-tert-butylcatechol (pTBC), the 14C-styrene was stable for a period of at least 3 weeks with high recovery of styrene monomer (94%) and no indication of polymerization. Subsequent storage at room temperature for 1 day did not cause any loss (Van Elburg, 1998Go). Based on the results of this stability test, 28.1 GBq (759 mCi) of [ring-U-14C]-styrene was synthesized (Chemsyn Laboratories, Lenexa, KA, USA), in two batches of 19.94 GBq (539 mCi) and 8.14 GBq (220 mCi). The specific radioactivity was 1.92 TBq x mol–1 (52 Ci x mol–1) as determined by GC and densitometric analyses (Van Elburg, 1998Go). The first batch had a radiochemical purity, as determined by high-performance liquid chromatography (HPLC), of 96.8% with 3.0% 14C-benzaldehyde as the major contaminant. The chemical purity, as determined by HPLC and GC, was 96.5%. This batch was used in two portions of 11.80 GBq and 8.14 GBq, for the rat and first mouse exposure, respectively. The second batch of 14C-styrene had a radiochemical purity of 96.7%, with 3.2% U-14C-benzaldehyde as the major contaminant. The chemical purity was 96.4%. This batch of 8.14 GBq was used for the second mouse exposure.



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FIG. 1. [Ring-U-14C]-styrene was synthesized from [ring-U-14C]-benzoic acid. The [ring-U-14C]-benzoic acid was reduced with lithium aluminum hydride to [ring-U-14C]-benzyl alcohol with 79% yield. The [ring-U-14C]-benzyl alcohol was subsequently oxidized to [ring-U-14C]-benzaldehyde using manganese(IV) oxide with 80% yield. Finally, the [ring-U-14C]-benzaldehyde was converted to [ring-U-14C]-styrene with triphenylphosphonium iodide in benzene in the presence of aqueous sodium hydroxide, and a trace of 4-tert-butylcatechol to prevent polymerization, with 40% yield.

 
Chemicals.
Unless stated otherwise, chemicals were of the highest purity available. Tetraethyl ammonium hydroxide was purchased as a 20% aqueous solution from Merck (Darmstadt, Germany). Nitrogen (zero gas, > 99.998%) was obtained from Sunox (Cary, NC, USA) and oxygen (USP grade, > 99.9%) from Holox (Norcross, GA, USA). Styrene for calibration (159 ppm styrene in N2, custom prepared) was purchased from Praxair (Danbury, CT, USA).

Animals.
Male Sprague Dawley rats and CD1 mice were purchased at the age of 9–10 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 10–12 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 2Go. 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., 1996Go). 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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FIG. 2. Schematic representation of the recirculating nose-only exposure system.

 
The styrene concentration in the system was monitored by two independent techniques. The primary monitoring was by IR spectrophotometry (Sumner et al., 1997Go). Prior to exposure, the IR spectrophotometer was calibrated, over the range of 0 to 302 ppm using a closed loop technique. In this technique, a stainless steel diaphragm pump connected to the inlet and outlet of the IR spectrophotometer circulates the contents of the IR spectrophotometer. Known volumes of styrene were injected into the known volume of the recirculating atmosphere, and the response of the IR spectrophotometer was recorded. The secondary monitoring was by gas chromatography. A gas chromatograph (GC) (HP5890 Series II, Hewlett Packard, equipped with a 7 ft x 1/8-inch stainless steel 35/60 Tenax column and a flame ionization detector kept at 250°C, eluted with helium at 20.3 ml x min–1) equipped with a multiport sampling valve was calibrated with standards prepared using Tedlar® bags filled with a known volume of nitrogen into which a known volume of styrene was injected. In addition, commercially prepared gas reference standards were used. The GC was used to monitor the concentration of styrene at the inlet (Fig. 2Go, 1) and the outlet (Fig. 2Go, 2) of the chamber and also after the first charcoal filter (Fig. 2Go, 3) to check for eventual breakthrough. The sensor of an oxygen monitor was placed in the outlet stream of the chamber to measure the oxygen. The monitor was calibrated against room oxygen content, which was assumed to be 21%, prior to each exposure. During the course of the exposure, styrene and oxygen concentrations were continuously recorded on a strip chart recorder. The styrene and oxygen flows were adjusted periodically to maintain 160 ppm of styrene and approximately 21% oxygen (Table 1Go). All flow settings and styrene and oxygen concentration readings were recorded approximately every 30 min over the course of the exposure.


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TABLE 1 Exposure Conditions and Rate of Uptake (values are means ± SE)
 
Inhalation exposure of rats and mice.
Exposures were conducted in Cannon-type nose-only exposure towers (Cannon et al., 1983Go) constructed from inert materials (stainless steel with Teflon delivery lines) with 52 positions (Lab Products, Maywood, NJ, USA). The tubes were blocked at unused exposure positions. Prior to starting the exposure, the recirculating pump was turned on and allowed to run for about 30 min to warm up. The recirculating flow was measured with a flow monitor and adjusted to give the desired recirculation. Oxygen flow was started prior to loading the animals, and all flows except for the pressure vessel flow were set to their predetermined values. Once the animals were installed in the exposure tower, the pressure vessel flow was started. As that flow increased, the dilution air was decreased proportionally. The exposure began when the styrene flow started. The exposure was ended by shutting off the pressure vessel flow and increasing the dilution air flow to maintain the pressure balance in the chamber. The flows were maintained until the styrene concentration dropped to zero. After the concentration reached nearly zero, the animals were removed from the exposure tower. The exposure groups consisted of 12 rats or 30 mice. Two separate inhalation experiments were conducted on mice (i.e., 60 mice total). These large numbers of animals were required to obtain sufficient material for DNA adduct analysis (Boogaard et al., 2000Go). In addition, 2 rats and 5 mice served as controls and did not receive treatment.

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., 2000Go). 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 0–6, 6–24, and 24–42 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 min–1 for rats and 200 ml x min–1 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., 2000Go). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nose-Only Inhalation Exposures
The exposure concentrations generated with the recirculating nose-only exposure system were within 93% of the target concentration of 160 ppm throughout the course of the exposures (Fig. 3Go). The concentrations of 14C-styrene averaged 159 ± 3 (SE) ppm for rats and 160 ± 3 and 158 ± 5 ppm for the first and second mouse exposure, respectively. In rats, the uptake of 14C-styrene was stable during the 6-h period and averaged 113 ± 7 µmol x kg–1 x h–1 (Table 1Go). In contrast to the rats, the uptake rate of styrene in mice decreased steadily over time (Fig. 4Go). The average 14C-styrene uptake was 189 ± 53 and 183 ± 76 µmol x kg–1 x h–1, for the first and second mouse exposure, respectively. The oxygen uptake was proportionally related to the styrene uptake in both rats and mice (Fig. 5Go).



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FIG. 3. 14C-styrene concentrations in the nose-only exposure chambers during the inhalation studies. The target concentration (160 ppm) is indicated by the dotted line.

 


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FIG. 4. Styrene uptake in rats and mice nose-only exposed to 160 ppm 14C-styrene.

 


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FIG. 5. Oxygen uptake in rats and mice nose-only exposed to 160 ppm 14C-styrene.

 
Animal Observations
No signs of toxicity were observed in rats during or after the exposure to 14C-styrene. Animals were damp after the inhalation exposure, either from urine or humidity in the air, but appeared otherwise well. Rats that were held in metabolism cages for 42 h after cessation of exposure appeared healthy and were eating and drinking normally. In contrast to the rats, some of the exposed mice showed signs of toxicity, such as a hunched and unkempt appearance, at the end of exposure. These symptoms persisted in some of the mice during the 42-h holding period in metabolism cages.

Excretion of Radioactivity
A summary of the excretion of radioactivity is given in Table 2Go. 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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TABLE 2 Disposition of Radioactivity in kBq
 
Radioactivity in the urine accounted for 75 ± 7% of the styrene retained by rats. Of the radioactivity that was taken up during exposure, 20 ± 5% was excreted in the urine during exposure and 55 ± 6% in the period from 0 to 42 h after cessation of exposure. The small volumes of urine produced by the mice during exposure (tube urine) could not be collected, but 6 ± 2% of the retained radioactivity in the first mouse experiment could be washed from the exposure tubes. This radioactivity is considered to be mostly due to urine contamination of the tubes. The radioactivity in the urine collected from mice in the period from 0 to 42 h after cessation of exposure was 63 ± 9%. The urinary elimination was virtually complete 42 h after termination of exposure, with the greatest proportion excreted in the first 6 h after cessation of exposure. In rats, the excretion of styrene metabolites in urine was faster than in mice. In rats, 37 ± 4% of the radioactivity taken up during exposure was excreted in the urine in the first 6 h after termination of exposure, and 15 ± 3% and 3 ± 1% in the subsequent two periods of 18 h, respectively. In mice, 34 ± 11% of the retained radioactivity was excreted in the urine in the first 6 h after cessation of exposure but still 25 ± 4% and 4 ± 1% in the subsequent two periods of 18 h.

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 24–42 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 3Go).


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TABLE 3 Tissue Concentrations of Radioactivity after Administration by Inhalation of 14C-Styrene to the Male Rat and Mouse
 
Quantitative Whole-Body Autoradiography
The concentrations of radioactivity measured in most tissues were below those present in blood for both rat and mouse (Tables 3 and 4GoGo). In addition, the qualitative distribution of radioactivity in the various tissues was similar when comparing species, although mice tended to contain significantly higher levels. High levels of radioactivity were noted in the livers of rat and mice (Fig. 6–8GoGoGo). However, the levels were more elevated in mice (approximately 4.5 times those found in blood when compared with 1.5 times in the rat). High levels were also present in the kidney cortex of each species. Concentrations were about 1.5 times that measured in rat blood but about 6- to 7-fold higher than the concentration in mouse blood. Concentrations in the renal medulla were more comparable between the species and much lower than in the renal cortex (in fact, lower than in the blood in both species). Special attention was paid to the distribution of radioactivity in the nasal passages, major airways, and lungs (Fig. 9Go). The level of radioactivity in the lungs of rats was lower than in the blood (about 0.6 times), whereas the levels were clearly higher in lungs of mice compared to blood (more than 2-fold higher). The radioactivity was mainly located in discrete regions of the lungs, presumably in the bronchi. The levels in the nasal mucosa were much higher than in the blood (more than 3-fold in the rat and 2- to 13-fold in the mice), and the radioactivity appeared to reside mainly in the olfactory mucosa as opposed to the respiratory mucosa (Fig. 10Go).


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TABLE 4 Relative Tissue Concentrations of Radioactivity after Administration by Inhalation of 14C-styrene to the Male Rat and Mouse
 


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FIG. 6. Whole-body autoradiograms of a male Sprague-Dawley rat 45 h after nose-only inhalation exposure to 14C-styrene (animal number MR005). Abbreviations: ad, adrenal; ao, aorta; bd, bile ducts; bdr, bladder; bl, blood; bm, bone marrow; br, brain; bf, brown fat; bug, bulbo-urethral gland; ca, caecum; ed, epididymis; elg, exorbital lachrymal gland; gb, gall bladder; Hd, harderian gland; ilg, intra-orbital lachrymal gland; kdc, kidney cortex; kdm, kidney medulla; kdp, kidney pyramid; li, large intestine; le, lens; lv, liver; lu, lung; lma, mandibular lymph nodes; mu, muscle; my, myocardium; nm, nasal mucosa; oe, oesophagus; pa, pancreas; pb, pineal body; pt, pituitary; pg, preputial gland; pr, prostate; re, rectum; sg, salivary glands; sv, seminal vesicles; sk, skin; si, small intestine; sc, spinal cord; sp, spleen; st, stomach; ts, testis; th, thymus; ty, thyroid; to, tongue; tp, tooth pulp; uvt, uveal tract; fa, white fat.

 


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FIG. 7. Whole-body autoradiograms of a male CD1 mouse 44 h after nose-only inhalation exposure to 14C-styrene (animal number MM105). For abbreviations, see Figure 6Go caption.

 


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FIG. 8. Whole-body autoradiograms of a male CD1 mouse 42 h after nose-only inhalation exposure to 14C-styrene (animal number MM205). For abbreviations, see Figure 6Go caption.

 


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FIG. 9. Enlargement of whole-body autoradiograms of a male Sprague-Dawley rat (animal RM005, top), and two CD1 mice (MR105, middle; MR205, bottom) mouse showing the lung 42–45 h after nose-only inhalation exposure to 14C-styrene.

 


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FIG. 10. Enlargement of whole-body autoradiograms of a male Sprague-Dawley rat (animal RM005, top), and two CD1 mice (MR105, middle; MR205, bottom) mouse showing the nasal cavity 42–45 h after nose-only inhalation exposure to 14C-styrene.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The high specific radioactivity of the styrene at ~1.85 TBq x mol–1 (50 Ci x mol–1) that was needed to attain the desired limit of detection for subsequent macromolecular binding studies (Boogaard et al., 2000Go) gave rise to two challenges in setting up the nose-only exposure. The first was the generation of the styrene atmosphere. A common approach is to introduce styrene into a stream of clean air using a syringe pump (Sumner et al, 1997Go). Because this requires heating of the styrene at the injection point to aid vaporization, this method could not be used because the risk of polymerization of the styrene was substantial. The alternative, to aid vaporization by spreading the styrene over glass wool, was not possible either, as the stability tests suggested that large glass surfaces also catalyzed polymerization. We therefore decided to use a vapor system in which the neat styrene was transferred into a pressure vessel at ambient temperature. The vessel was sealed and the styrene allowed to completely vaporize overnight. Prior to the exposure, the vessel was pressurized, resulting in a styrene and nitrogen mixture that was metered into the exposure chamber air stream to obtain the desired exposure concentration. The second challenge to overcome was due to the total amount of radioactivity required. For nose-only exposure of 12 rats to 160 ppm styrene during 6 h, more than 20 GBq (0.54 Ci) of 14C-styrene was needed. Because such a large amount of radioactivity could not be accommodated within the institutional license, the system was adapted to allow recirculation of a portion of the 14C-styrene. The designed system was successful in that no signs of polymerization were observed. In addition, a constant flow close to the target concentration of 160 ppm styrene could be maintained in the Cannon-tower for the full 6 h of the exposures, while the partial recirculation reduced the amount of styrene normally needed for a nose-only exposure of rats to 60%.

The uptake of styrene in mice was on average 186 µmol x kg–1 x h–1, 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 kg–1 x h–1) 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, 1981Go) and a few times in mice (Ghantous et al., 1990Go; Kishi et al., 1989Go; Löf et al., 1984Go). 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., 1990Go) and in pregnant CD-1 mice exposed to 2.3 µmol [8-14C]-styrene by tail vein injection (Kishi et al., 1989Go). 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 3Go). 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., 1998Go; 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., 1998Go; 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, 1981Go). 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., 1995Go). 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., 1996Go). 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, 1970Go; Pfäffli et al., 1981Go). Other evidence for ring-opened metabolites of styrene was recently reported (Sumner et al., 1995Go). 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 3–4 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., 2000Go). This suggests that a nongenotoxic mechanism, possibly caused by a cytotoxic metabolite, underlies the observed bronchioalveolar tumor formation in mice.


    ACKNOWLEDGMENTS
 
The authors thank John E. Murphy, Tim Moore, and Rod Snyder for technical assistance and the CIIT animal care staff for their assistance in the study. This study was sponsored in part by the Styrene Information and Research Center.


    NOTES
 
1 To whom correspondence should be addressed at P.O. Box 162, 2501 AN The Hague, The Netherlands. Fax: +31 70 377 6380. E-mail: peter.j.boogaard{at}si.shell.com. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bakke, O. M., and Scheline, R. R. (1970). Hydroxylation of aromatic hydrocarbons in the rat. Toxicol. Appl. Pharmacol. 16, 691–700.[ISI][Medline]

Blaesdale, C., Kennedy, G., MacGregor, J. O., Nieschalk, J., Pearce, K., Watson, W. P., and Golding, B. T. (1996). Chemistry of muconaldehydes of possible relevance to the toxicology of benzene. Environ. Health Perspect. 104(S6), 1201–1209.[ISI][Medline]

Bond, J. A. (1989). Review of the toxicology of styrene. CRC Crit. Rev. Toxicol. 19, 227–249.[ISI]

Boogaard, P. J., De Kloe, K. P., Wong, B. A., Sumner, S. C. J., Watson, W. P., and Van Sittert, N. J. (2000). Quantification of DNA adducts formed in liver, lungs, and isolated lung cells of rats and mice exposed to 14C-styrene by nose-only exposure. Toxicol. Sci. 57, 203–216.[Abstract/Free Full Text]

Cannon, W. C., Blanton, E. F., and McDonald, K. E. (1983). The flow past chamber: an improved nose-only exposure system for rodents. Am. Ind. Hyg. Assoc. J. 44, 923–928.[ISI][Medline]

Carlsson, A. Distribution and elimination of 14C-styrene in rat. (1981). Scand. J. Work Environ. Health 7, 45–50.[ISI][Medline]

Cruzan, G., Cushman, J. R., Andrews, L. S., Granville, G. C., Johnson, K. A., Hardy, C. J., Coombs, D. W., Mullins, P. A., and Brown, W. R. (1998). Chronic toxicity/oncogenicity study of styrene in CD rats by inhalation exposure for 104 weeks. Toxicol. Sci. 46, 266–281.[Abstract]

Cruzan, G., Cushman, J. R., Andrews, L. S., Granville, G. C., Johnson, K. A., Bevan, C., Hardy, C. J., Coombs, D. W., Mullins, P. A. and Brown, W. R. (2000). Chronic toxicity/oncogenicity study of styrene in CD-1 mice by inhalation exposure for 104 weeks. J. Appl. Toxicol., (in press).

Gadberry, M. G., DeNicola, D. B., and Carlson, G. P. (1996). Pneumotoxicity and hepatotoxicity of styrene and styrene oxide. J. Toxicol. Environ. Health 48, 273–294.[ISI][Medline]

Ghantous, H., Dencker, L., Gabrielsson, J., Danielsson, B. R. G., and Bergman, K. (1990). Accumulation and turnover of metabolites of toluene and xylene in nasal mucosa and olfactory bulb in the mouse. Pharmacol. Toxicol. 66, 87–92.[ISI][Medline]

IARC (1994a). Styrene. IARC Monographs on the evaluation of carcinogenic risks to humans 60, 233–320.[Medline]

IARC (1994b). Styrene-7,8-oxide. IARC Monographs on the evaluation of carcinogenic risks to humans 60, 321–346.[Medline]

Kishi, R., Katakura, Y., Okui, T., Ogaea, H., Ikeda, T. and Miyake, H. (1989). Placental transfer and tissue distribution of 14C-styrene: an autoradiographic study in mice. Br. J. Ind. Med. 46, 376–383.[ISI][Medline]

Leavens, T. L., Moss, O. R., and Bond, J. A. (1996). A dynamic inhalation system for individual whole-body exposure of mice to volatile organic chemicals. Inhal. Toxicol. 8, 655–677.[ISI]

Löf, A., Gullstrand, E., and Byfält-Nordqvist, M. (1984). Tissue distribution of styrene, styrene glycol and more polar styrene metabolites in the mouse. Scand. J. Work Environ. Health 9, 419–430[ISI]

McConnell, E. E., and Swenberg, J. A. (1994). Review of styrene and styrene oxide long term animal studies. Crit. Rev. Toxicol. 24, S49–S55.[ISI][Medline]

Miller, R. R., Newhook, R., and Poole, A. (1994). Styrene production, use, and human exposure. Crit. Rev. Toxicol. 24, S1–S10.[ISI][Medline]

Pfäffli, P., Hesso, A., Vainio, H., and Hyvönen, M. (1981). 4-Vinylphenol excretion suggestive of arene oxide formation in workers occupationally exposed to styrene. Toxicol. Appl. Pharmacol. 60, 85–90.[ISI][Medline]

Sumner, S. C., Asgharian, B., Moss, O., Cattley, R. C., and Fennel, T. R. (1995). Correlating styrene metabolism and distribution with hepatotoxicity. The Toxicologist. 15, 4.

Sumner, S. C. J., Cattley, R. C., Asgharian, B., Janszen, D. B., and Fennell, T. R. (1997). Evaluation of the metabolism and hepatotoxicity of styrene in F344 rats, B6C3F1 mice, and CD-1 mice following single and repeated inhalation exposures. Chem. Biol. Interact. 106, 47–65.[ISI][Medline]

Van Elburg, P.A. (1998). Synthesis and stability of [ring-U-14C]-labelled styrene to be used in animal exposure experiments. Report CA.98.20479. Shell International Chemicals BV, SRTCA, Amsterdam.





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