Distribution and elimination of in rat.

CARLSSON A. Disl:ribution and elimination of 14C-styrene in ralt. Scand j work environ health 7 (1981) 45-50. Male rats were exposed to about 45 ppm of radioactively 1abeled styrene in the inspired air 1ior 1-8 h. The di'sttibution and elimination of styrene and its metabolites in different organs and tissues were studied. To get an idea of the ratio between unmetabolized and metabolized styrene in organs and tissues, a supplementary study was performed in whkh five male rats were exposed to about 250 ppm of radio actively labeled styrene. The largest amounts of styrene and its metabolites were found in the subcutaneous fat, and the concentoo'ti'on displayed a sharp rise during the first 4 h of exposure in comparison with the foUowing 4 h. The concentration of styrene and rts metabolites in the cerebrum, cerebellum and muscles paralleled the arterial blood concentration throughout the entire period of exposure and amounted to about 70 % -of the arterial blood value. In the 9Upplementary study an accumulation of styrene metabolites in subcutaneous fat was discl-osed; it ranged from 15 to 20 0/0 during exposure.

A<bout 3 % of the styrene taken up in man is exhaled in an unchanged form via the lungs (7'). A<bout 95 % is excreted in the urine as mandeli<: ac'id and phenylglyoxyhc acid (2).
In rat styrene is mainly excreted as hippuric acid (12). About 3 Ofo of the radioa-C'tively labeled styrene administered to rat in a subcutaneous !inj-ection is eX'haled in an unchan,ged form via the lungs, whereas the remaiinrder i's metabolized and excreted mainly via the urine (6).
In the present study rats were exposed to styrene in inspiratory air. The purpose was to study the distribuJ:ion and elimination of styrene and its metabolites.

Experimental design and methods
Radioa'Ctively labeled styrene (7_ 14 C), with a specific activity of 0.35 mei/mmol, was injected with a microliter syringe (Hamilton-Boyd, Switzerland) into a polyesterlaminated a'luminium f()il bag containing a known quantity of air. Male rats (Sprague-Dawley, 200 g) were exposed to the mixture of styrene vapor and air for 1-8 h in a metabolism cage (1'50 mm inner diameter, Kelbo-Grave, Sweden). 'Dhe air mixture from the aforementioned bag flowed via a teflon tube into the cage and then passed a flow meter (model 1350 V, AB Max Sievert, Va'llinglby, Sweden) and a capillary tube which induced a constant flow of 0.45 l/min. The concentration of styrene in the metabolism cage was analyzed with the charcoal sampling method (9). The air mixture contained 184 ± 13 (SD) mg/m 3 (43.5 ppm) of styrene.
After the exposure was concluded, some rats breat!hed ordinary air during a decay phase. Table 1 lists the exposure durations, decay phases and the number of rats exposed to styrene.
After the rats had been killed by luxa·· tion of the cervical spine, d'Ouble sampies were taken from organs and tissues for the assay of radioa'Ctively labeled styrene and radioactive metaboIi'tes. Samples were taken from the liver, ktdneys, subcu'tane-ous fat, cerebrum, cerebellum, muscles, 'lungs, spleen, isch!iadic nerve, and arterial blood. 'I'he arterial bllood (0.1-0.4 ml) was mixed with 1.5 ml of Soluene-100® (Packard Instrument AB): isopropanol (1: 1) in glass scintillation vialls (Packard). Mter 10-min digestion at room temperature, 0.5 ml of 35 Ofo hydrogen peroxide was added. After 60 min at room temperature, 15 ml of 0.5 N hydrochloric acid: Insta-Gel® (Packard) (1: 9) was added. The other samples, weighing 10-80 mg, were isolated and placed in glass scintillation vials conta'ining 1 ml of SoIl.uene-350® (Packard). They were then digested for 2-8 h at +50°C before the addition of 9 ml of scintillation liquid consisting of Permablend III® (Packard) dissolved in toluene. Table 1. Org'an and tis,sue concentrations (nmol/g tissue) of styrene equivalents (styrene and its metabolites) in rat followi'ng exposure to a styrene concentration of 184 mg/m 3 (43.5 ppm) in inspiratory air -Means ± standard errors (determined from the mean of two samples for eaoh rat) and the method 'error.
Duration of exposure + time after exposure (h) Method error a Organ 1 + 0 2+0 4+0 8+0 1 + 1 1 + 3 1 + 6 (% of mean  Table 2. Mean organ and tissue concen,trations of styrene (S) and its metabolites (M), specified as nanomoles of styrene equ,ivalel1its per gram of tissue, in rat after exposure to a styrene concentration of 1,014 mg/m 3 (240 ppm) in inspiratory air, the proportion of metabol·ites as percentage, and the error of the method for styrene.
Duration of exposure + time after exposure (h) Method error Organ 0.25 + 0  The assays were made with a liquid scin1Jillation counter (Tri Carlb 3002, Packard). Ea'ch s'ample was measured in two cycles either lasting 50 min or comprising a maximum of 50,000 impulses. The activity value for eaCh individual assay was calculated as the mean value of these two determinations. The results are reported as the mean va~ue of the two samples.
The error o'f the method for the scintillation counter was ± 0.7 % at 50,000 impulses. '11he error of the method was calculated for each organ and tissue according to the method for doub'1e determinations with systematic difference.
After the rats had been killed, it took about 15 min before an the organ and tissue samples ha'd been transferred to sealed scintIllation vials. So that it could be determined whether any styrene had evaporated 'from the samples into the ambient air during this period, a rat was exposed to styrene for 2 h with the previously described procedure. Samples were taken from different organs and tissues at the fdllowing three times: immediately after, 10 min after and 20 min after the rat was kiHed. The concentrations of styrene in the samples at the times cited did not differ significant!ly during the 20-min period of observation. Thus styrene did not evaporate in measurable quantities from the examined organs during the 1'5min prepar,ation period.
To get an idea of the ratio between unmetabolized and metabolized styrene in the organs and tissues, a supplementary study was performed. A total of five male rats (Sprague-Dawley, 200 g) were utilized in 'this study. The animals were exposed to radioactively labeled styrene (7_ 14 C) for 0.25-8 h in the same way as already descri'bed, with one rat at each exposure time (table 2). The specifiic activity was 0.18 mCi/mmol, and the air mixture contained 1,014 ± 90 (SD) mg/m 3 (240 ppm) of styrene. Organ and tissue samples were isolated from the Tiver, subcutaneous fat, muscles, cerebrum and arterial blood for the assay of the total content of styrene and its metabolites. 'I1he assays were made with liquid scintillation counting as already described.
The concentration of unmetaboTized styrene in the arterial blood was determined with a head-space method (I). The content of unmetabolized styrene in the organs and other tissues was determined with gas chromatography after evaporation into nitrogen at 150°C (7). Three samples were taken from eaCh organ and tissue, and the results are reported as the mean value of the three samples. The error of the method was calculated for ea,ch organ and tissue according to the method for triple determinations with systematic difference. Organ and tissue con-centraUons of styrene equivalents (styrene and its metabolites) in rat immediately after the conclusion of exposure as a function of the exposure duration in hours. The animals were exposed to a styrene concentration of 184 mg/m 3 (43.5 ppm).

In
(subc. = subcutaneous, n. = nerve) 8 Exp. hours Organ and tissue concentrations of styrene equivalents (styrene and -its metabolites) in rat as a function of the ttme elapsed after the conclusion of exposure. The animals were exposed ·to a styrene concentration of 184 mg/m 3 (43.5 ppm) for 1 h.
(subc. = subcutaneous, n. = nerve) 6 hours postexposure .Nfter 8 h of exposure tlhe conoentration had increased to 123 nmal/g. The concentration in tJhe ischiadic nerve following 8 h of exposure was 103 nmol of styr,ene equiivalents/g of tJissue. 'I'he corresponding value for tlhe cerebrum was 34 nmol. 'I'he concentration of styrene equivalents in the cerebrum, cerebellum and muscles paralleled tJhe arterial blood concenltration throughout the entire period of exposure and amounted to about 70 % of the arterial blood value. The lungs and spleen contained 68 anid 63 nmol of styrene equivalents/gill tissue, respectively, fol'lowlng 8 h of exposure, whereas the concentration in the arterial blood at the same time was 49. Throughout the entire period of exposure, the Il{rdneys displayed high concentrations of styrene equivalents with a value of 728 nmdl!g after 8 h of exposure.
The concentrations during a 6-h decay period following an init!iaiJ. exposure to styrene for 1 h are shown in fig 2. The concentrations in t!he cerebrum, cerebellum and muscles paral1e'1ed the blood concentration also during t!he decay phase and amounted to about 70 % ill the blood va'lue. After a 1-h decay phase, the concentration 'in the liver was relatively constant. The subcutaneous fat contained about 8 % of the initial value after t!he 6-ih decay Phase. The half-time for styrene equivalents in the subcutaneous fat fdllowing 1 h of exposure was estimated to be 2.2 h. The accumulation of styrene equiv,alents in the organs and tissues at the various exposure alterna'tives is descri'bed completely in taiblle l.
The relative concentration of metabolized styrene in relation to the total quantity of styrene and its metaboli:tes, ie, styrene equivalents, is shown in fig 3 '(supplementary study). After a period of exposure of 1 h or more, the liver contained %Styrene metabolites 100 Fig 3. The concentration of metabolized styrene in rat, expressed as ,a percentage of the total quantity of styrene and its metabolites in different organs and tissues during an exposure lasting up to 8 h, followed by a 3-h decay period. The animals were exposed to a styrene concentration of 1,014 mg/m 3 (240 ppm). 95. The muscles and cerebrum contained high concentrations of metabolites, ranging from 58 to 91 0/0. In the subcutaneous fat the metabolites amounted to 16-20 % during exposure lasting up to 8 h. It decreased to about 9 % 3 h after the conclusion of exposure. Throughout the entire period of exposure, the proportion of metabolites in the arterial 'blood was high, about 80 010. It increased after a 3-h decay period to about 95 010.
The quotients 'between the concentration of unmetabolized styrene in SUbcutaneous fat and the concentration in blood or air was ca1culated. After 4 h of exposure the quotient was found to be 36 for subcutaneous fat/blood and 197 for subcutaneous fat/air. The accumulation of styrene and metabol'ites is listed completely in table 2.

Discussion
Styrene is mainly metabolized in the liver. First, styrene is oxidized into styrene oxide by fue microsomal monooxygenase system (10). Styrene oxide is further metabolized by hydration or conjugation, reactions which mainly take Place in the liver, but also in the lungs, kidneys, intestinal mucosa and skin {l5). The following diagram shows the proposed ma'in pathways of styrene metabolism (12): In the present styrene study a concentration of about 85 nmol of styrene equivalents/g of tissue was found in subcutaneous fat after 1 III of exposure to ap-proximat~ly 44 ppm of styrene (table 1). A!iter adm'inistering a single oral dose of ra!dioactiv~y labeled styrene (192 nmo1/g of body weight) to 400-g male rats, Plotnick & Weigel (13) found a concentration of 97 nmol of styrene equivalents/g of subcutaneous fat. In exposure of man to 50 ppm of styrene for 2 h (7), the concentration of styrene in subcutaneous fat amounted to only 34 nmol/g after the conclusion of exposure.
The concentration of styrene equivalents in subcutaneous fat declined relatively quickly after exposure was concluded in the presen't study. The half-time for styrene equiva'lents in subcutaneous fat after 1 hof exposure was estimated to be 2.2 h. Engstromet al (7) estimated the half-time for styrene in the subcutaneous fa't of man to be about 72 h. Probably, both the distribution and the elim'ination of styrene to and from subcutaneous fa't is faster in rat than in man. The reason may be a faster rate of energy metabol'ism per gram of tissue in rat which results in a greater brood perfus'ion per gram of tissue. Man also has a larger vdlume of subcutaneous fat 1!han rat. 'I1he blood perfusion of subcutaneous fat in man deClines as the volume ,of the tissue increases (11).
From the supplementary study the co-eMicient between the concentration of unmebbolized styrene in subcutaneous fat and inspiratory air after 4 h of exposure was calculated to be 197. This value is much lower than the one calculated from in vitro experiments witJh oN/alt. van Rees (14) reported an oil/air ,partition coefficient of 4,100 for styrene. The reason for f'he different values is, above all, that the present study was undertaken in vivo and subcutaneous fat cannot be equated with oil.
The percentage of styrene metabolites in the liver was very high during exposure. It ranged Ifrom about 80 to 90 010 (ta!l:ile 2). In a Coorresponiding supplementary study on xylene r(5), the values ranged from albout 50 to 65 0/0. Be-s1des, the concentration of styrene equivalents in the liver was albout three times higher (table 1) than that of xylene. Bend et al (3) added He-styrene oxide to isolated, perfused rat liver and showed 1!hat the biliary excretion of metahdlites was dose-dependent, w'ith a decrease at high doses. At a dose of 10 ,umol of styrene oxide/Ever, the hiliiary excretion accounted for 40 % of the administered dose. Plotnick & Weigel (13) showed that, a/fter Ithe oral administra~ion of 14C-styrene to rats, fecal e~cretion accounted for less than 2 % of the dose. The greater proportion of styrene metabolites than xylene metabolites in the liver could be expla'ined by an enterohepatic circu1ation of styrene metabolites.
The percentage of styrene metabolites in the cerebrum ranged from about 60 to 75 during exposure and increased to 85 0/0 after the 3-h decay period. Savolainen & Vainio (16) also found relatively high levels of styrene metabolites in the brain after an intraperitoneal injection of radioactive styrene or radioactive styrene oxide.
In previous studies no accumulation of styrene metabol'ites 'in subcutaneous fat has been found (4). The results !rom our supplementary study indicate such an accumulation, rangling from about 15 to 20 Ofo during exposure. One possi'ble explanation for this finding may be the presence of polar styrene metabolites in the water content of adipose tissue (8). However, before a final c·oncl'llsion can be drawn, further investigation is needed, as the resuHs are based on only one rat at each exposure time.