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Arch. Toxicol. 38, 169--176 (1977)

TOXICOLOGY 9 by Springer-Verlag 1977

Toluene Concentrations in Various Tissues of Rats after Inhalation and Oral Administration Kaija Pyykk61., Hanna T/ihti 2, and Heikki Vapaatalo 2 i Department of Clinical Sciences, University of Tampere, 2 Department of Biomedical Sciences, University of Tampere, SF-33101 Tampere 10, Finland

Abstract. The uptake, distribution, and elimination of 3H-toluene in various tissues of rats were studied after inhalation or after gastric intubation. The maximum radioactivity was measured 2 - 3 h after gastric intubation in tissues, except in white adipose tissue, where the peak radioactivity was reached at5h. After inhalation exposure, the uptake to various tissues was very rapid. The maximum radioactivity in most tissues was reached in 1 5 - 3 0 min. The accumulation was slowest in white adipose tissue, where it took 1 - 2 h. The radioactivity in tissues decreased after inhalation exposure more rapidly than after gastric intubation. Brown adipose tissue and white adipose tissue were different from other tissues in their ability to retain toluene. Twenty-four hours after exposures, only 1% or less of the initial radioactivity was found in tissues other than white adipose tissue, in which the corresponding value was 3.5-5%. The results show a very rapid absorption and distribution of toluene after inhalation and a retention of radioactivity in white adipose tissue. After oral ingestion the distribution showed a similar but much slower pattern.

Key words: Toluene -- Absorption -- Distribution - Elimination. Zusammenfassung. Nach Inhalation und nach oraler Gabe wurden Aufnahme, Verteilung und Ausscheidung von 3H-Toluol in verschiedenen Geweben der Ratte untersucht. Bei oraler Gabe (mittels Schlundsonde) wurde das Maximum an Radioaktivit/it im weiBen Fettgewebe nach 5 Std erreicht, w/ihrend es in allen anderen untersuchten Organen bereits nach 2--3 Std auftrat. Nach Inhalation wurde 3HToluol wesentlich schneller aufgenommen. Die htchste Radioaktivit~it wurde hierbei im weil3en Fett nach 1 - 2 Std und in den meisten anderen Geweben nach *

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K. Pyykk6 et al. 1 5 - 3 0 min gemessen. Ebenso war die Abnahme der Radioaktivit/it nach Inhalation schneller als nach oraler Gabe, wobei sich auch in diesem Fall das braune und das weiBe Fettgewebe von anderen Organen unterschieden. Vierundzwanzig Stunden nach Applikation enthielt das Fett noch 3,5-5% der initialen Radioaktivit/it, w~ihrend in allen anderen Geweben nur noch 1% oder weniger gemessen wurden. Die Ergebnisse dieser Studie zeigen, dab Toluol nach Inhalation sehr viel rascher absorbiert und wieder ausgeschieden wird, als nach oraler Aufnahme, w/ihrend das Verteilungsmuster nach beiden Applikationsarten sehr /ihnlich ist.

Introduction

Toluene is widely used as an industrial solvent. The maximum allowable concentration of toluene has been set at 200 parts per million (ppm) (ACGIH, 1971). It has been shown that acute inhalation exposure of 200 ppm concentrations for 8 h causes slight but definite impairment of coordination and reaction time. Higher concentrations induce more severe effects, and with concentrations of 600-800 ppm these effects are observed after exposures for only a few hours (von Oettingen et al., 1942). Chronically toxic industrial exposures to toluene may involve concentrations of 4 0 0 - 5 0 0 ppm for weeks or months (Press and Done, 1967). Inhalation or "sniffing" of toluene contained in solvents, glues, and lacquer or paint thinners, has become increasingly common in recent years. Concentrations of toluene achieved by "sniffing" can be about 50 times the allowable concentration (Press and Done, 1967). Toluene is absorbed through the skin, mucous membranes, the gastrointestinal tract, and lungs. In the body it is oxidized to benzoic acid. Benzoic acid is mainly conjugated with glycine to form hippuric acid and less with glucuronic acid to yield benzoylglucuronic acid (Laham, 1970). Conjugates are excreted in urine. Some 10-20% of a dose of toluene is eliminated unchanged in the expired air (Nomiyama and Nomiyama, 1974a). Toxic effects of toluene have been suggested to be associated primarily with neurologic complications (von Oettingen et al., 1942). Industrial exposure of human beings to toluene has also caused enlargement of liver, macrocytosis, moderately decreased levels of erythrocyte counts, and absolute lymphocytosis (Greenburg et al., 1942). "Sniffing" of toluene contained in solvents has caused metabolic acidosis, which may be due to the capability of toluene to impair renal tubular acidification (Taher et al., 1974). Evaluation of the literature concerning the potential toxicity of toluene is complicated by the fact that industrial solvents also contain other agents. Though toluene is common as an industrial solvent, there are very few experimental studies about its effects (von Oettingen et al., 1942) and distribution in the body (Nomiyama and Nomiyama, 1974b). In the present work we have studied the uptake and elimination of toluene in rat tissues to establish a basis for our further investigations about its toxic mechanisms. We have compared two different routes of administration of 3H-toluene.

Toluene Concentrations in Rat Tissues

171

Materials and Methods Animals. Male Sprague-Dawley rats weighing 150--280 g were used. The animals fasted for 12 h before the experiment.

Chemicals. Labeled toluene (4-3H-toluene, with a specific activity of 230 mCi/mmol and 98% radiochemical purity by gas-liquid chromatography, the Radiochemical Centre Ltd., Amersham, England) was used. It was diluted with non-radioactive toluene (E. Merck, Darmstadt, Federal Republic of Germany) to obtain a final activity of 100 ~Ci/100 ~1 for the gastric intubation experiments and 200 ~Ci/130 ~1 for the inhalation experiments. The scintillator fluid contained 24 g of PPO (2,5-diphenyloxazole, E. Merck, Darmstadt, Federal Republic of Germany) and 600 mg of POPOP (l,4-bis-(5-phenyloxazol-2-yl)benzene, Beckman Instruments Inc., Geneva, Switzerland) in 1 1 of toluene. Exposure Procedures. Two different exposure procedures were used. In oral administration experiments, the animals were slightly anaesthetized with ether. Labeled toluene (100 ~1 corresponding 100 ~Ci in 400 ~1 peanut oil) was administered by stomach tube. Then the tube was rinsed with 1.5 ml peanut oil into the stomach. The total radioactivity of a dose was 222 x 106 DPM (disintegrations per minute) or 793,000--1,480,000 DPM/g of body weight. In the inhalation experiments, 130 ~1 (200 ~Ci) of toluene was introduced into a respiration chamber (volume 6.5 1). The calculated concentration of toluene vapour was 20 ppm in the chamber air. The exposure time was 10 min. The total radioactivity of inhalated toluene was calculated to be 30.7 x 106 DPM (Dost, 1968), and the initial concentration 153,500 DPM/g of body weight.

Sampling Procedures. The rats were killed from 15 min to 24 h after the oral administration or after the exposure in the respiration chamber. The sampling procedures were conducted at the temperature of +4 ~ C. The animals were under slight ether anaesthesia when the blood sample was taken by heart puncture. They were then decapitated and the tissue samples were removed rapidly in the following order: heart, thyroid gland, lungs, brain, liver, spleen, kidneys, adrenal glands, white adipose tissue around kidneys, stomach, right femoral muscle, and brown adipose tissue between scapulae. Bone marrow was taken from the thigh bone. The tissue samples were washed with cold 0.9% NaCI solution.

Extraction and Counting of Radioactivity. Weighed samples (2 g or less) of tissues were homogenized with 30 ml of cold toluene and 5 g of anhydrous sodium sulphate in a Sorvall Omni-Mixer homogenizer for 5 min by relative speed 7/10. The homogenates were shaken overnight to complete the equilibration of the labeled and unlabeled toluenes. The homogenates were then filtered through 1 g of anhydrous sodium sulphate on Schleicher & Sch/ill No. 595 filter paper. Then 10 ml of the clear filtrate was pipetted into a counting vial and 2 ml of scintillator fluid was added. The recovery of the initial radioactivity in the extraction was 99.0%. The radioactivities were measured in a LKB-WalIac Ultrobeta 1210 liquid scintillation counter by using the external standardization with 226Ra, automatic background subtraction and DPM calculation.

Results Absorption and Distribution T h e r e l a t i v e r a d i o a c t i v i t y is p r e s e n t e d in F i g u r e 1. I t is e x p r e s s e d as a p e r c e n t r a t i o : 100 • r a d i o a c t i v i t y in t h e t i s s u e ( D P M / g ) p e r t o t a l r a d i o a c t i v i t y o f t h e d o s e ( D P M / g o f b o d y weight). I n t h e gastric intubation e x p e r i m e n t s t h e r e l a t i v e r a d i o a c t i v i t y in b l o o d r e a c h e d its m a x i m u m level 2 h a f t e r t h e a d m i n i s t r a t i o n o f l a b e l e d t o l u e n e . S i m u l t a n e o u s l y , t h e r a d i o a c t i v i t y in t h e s t o m a c h t i s s u e b e g a n t o d e c r e a s e slowly. A t

172

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Fig. 1. Uptake and elimination of relative radioactivity in rat tissues after an oral administration (A--A) and inhalation ( 0 - - 0 ) of 3H-toluene. [Relative radioactivity % = 100 x radioactivity in the tissue (DPM)/sample weight (g or ml) per total radioactivity of dose (DPM)/body weight (g)]

tion was reached 15--30 min after the end of 10 min exposure. In white adipose tissue the highest relative radioactivity values were measured 1 h after the end of the exposure. At 1 and 2 h, the order of the tissues according to the increasing amounts of relative radioactivity (Table 2) was as follows: bone marrow < blood, muscle, spleen, lung < heart, thyroid gland < brain < liver, kidney < stomach, adrenal glands < brown adipose tissue < white adipose tissue. At 4 h, the relative amounts of radioactivity of stomach, kidney and brown and white adipose tissues increased further. This was due to a more rapid elimination rate from the blood than from other tissues. The ratios between tissue and blood radioactivity did not differ significantly after inhalation and after oral administration.

Elimination The rate of elimination of radioactivity during the first 9 h was regularly more rapid in all tissues after inhalation than after gastric intubation (Fig. 1). Twelve hours after

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Table 1. Distribution of radioactivity in rats after different intervals following an oral administration of all-toluene dissolved in oil. Mean • S.E.M. of 2 to 3 measurements of radioactivity in 1 g of wet tissue per radioactivity in 1 ml of blood Tissue

Tissue/blood ratio

Heart Lung Stomach Liver Kidney Brain Muscle Spleen Bone marrow White adipose t. Brown adipose t. an = 2;

1 ha

2 hb

1.10 • 0.03 0.56 • 0.08 4.06 • 0.89 2.19 • 0.30 1.94 • 0.05 1.71 • 0.22 0.81 • 0.28 0.70 • 0.04 1.58 _+ 0.36 23.85 c 13.33 • 1.10

1.23 0.62 4.35 2.86 1.84 1.29 0.68 0.54 1.59 27.21 14.81

bn = 3;

4 hb + 0.10 • 0.15 • 1.17 • 0.40 • 0.84 • 0.18 • 0.14 • 0.06 _+ 0.56 • 2.71 • 0.64

1.28 0.63 3.55 2.58 1.77 1.58 0.94 0.76 2.16 52.58 24.80

12 h a • 0.06 • 0.18 • 1.13 • 0.29 • 0.11 • 0.05 • 0.03 • 0.04 _+ 1.18 • 9.60 • 5.27

1.23 0.79 4.65 2.82 6.42 1.40 2.38 0.70 3.61 252.30 52.52

• • • • • • • • • • •

0.03 0.04 2.52 0.54 0.39 0.04 1.36 0.03 2.40 1.19 0.60

Cn = 1

Table 2. Distribution of radioactivity in rats after different intervals following inhalation of 3H-toluene. Mean • S.E.M. of 2 to 3 measurements of radioactivity in 1 g of wet tissue per radioactivity in 1 ml of blood Tissue

Tissue/blood ratio 1 ha

Heart Lung Stomach Liver Kidney Brain Muscle Thyroid gland Adrenal gland Spleen Bone marrow White adipose t. Brown adipose t. an = 3;

1.54 1.26 4.69 2.17 2.01 1.74 1.23 1.51 7.35 0.96 0.58 65.66 39.68

2 hb

+ + + + + • + • • • • • •

0.29 0.17 0.93 0.27 0.13 0.23 0.28 0.50 3.05 0.36 0.12 7.08 3.72

1.31 1.17 5.02 1.91 1.97 1.65 1.06 1.32 6.07 1.08 0.53 75.17 60.40

4

+_ 0.05 _+ 0.22 • 1.68 + 0.19 _+ 0.34 • 0.19 • 0.06 • 0.08 • 0.32 • 0.30 • 0.07 • 16.10 • 15.18

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1.49 1.08 6.68 2.29 3.89 1.42 2.61 1.39 4.84 0.98 0.27 215.90 87.92

12 h a + 0.14 + 0.05 + 0.53 + 0.59 + 1.39 + 0.03 + 1.56 _+ 0.08 • 0.91 _+ 0.09 • 0.20 • 29.19 • 2.02

2.39 2.00 9.55 2.72 8.04 1.72 2.05 12.84 15.21 2.05 11.45 257.90 39.16

_+ 1.50 _+ 0.41 • 6.13 • 0.55 +_ 4.97 • 0.13 + 0.13 • 10.38 • 9.99 + 0.91 • 7.65 • 131.7 _+ 23.27

bn = 2

the e x p o s u r e , t h e relative r a d i o a c t i v i t y o f e a c h tissue w a s a b o u t e q u a l in b o t h c a s e s , a n d t h e r a t e o f e l i m i n a t i o n b e t w e e n 12 a n d 24 h after the e x p o s u r e s w a s n e a r l y t h e s a m e . T h e s l o p e s o f t h e e l i m i n a t i o n c u r v e s w e r e v e r y similar, w i t h t w o e x c e p t i o n s ; t h e e l i m i n a t i o n o f r a d i o a c t i v i t y f r o m w h i t e a d i p o s e tissue w a s m u c h s l o w e r t h a n f r o m o t h e r tissues, a n d the b o n e m a r r o w s h o w e d o n l y a v e r y slight d e c r e a s e o f t h e relative r a d i o a c t i v i t y d u r i n g 24 h.

Toluene Concentrations in Rat Tissues

175

After inhalation exposure, the radioactivity in white adipose tissue decreased to Yl0 of the initial value in 12 h. In other tissues this took, on an average, 4 h. The radioactivity decreased to Yl00 of the initial value in white adipose tissue in 24 h, while in other tissues this took, on an average, 12 h. The radioactivity of white adipose tissue 24 h after gastric intubation was 5% of the initial value. After inhalation exposure, the corresponding value was 3.5%. In brown adipose tissue the radioactivity decreased to 0.2% of the initial value in both experiments. In other tissues, the concentrations decreased below 1% of the initial values, and in most tissues the percentage was even below 0.1.

Discussion In this study, the uptake, distribution and elimination of radioactivity in various tissues of the rat were examined after giving 3H-labeled toluene as a single exposure by two different routes: gastric intubation and inhalation. According to our results, the distribution rate of radioactivity to various tissues was faster after inhalation exposure than after oral administration. The relative radioactivities in various tissues, however, were about equal after these two exposures. Apparently the tissues have a capacity to retain toluene. Elimination occurred rapidly from the tissues in which the uptake was fast, such as the lungs. On the contrary, by the adipose tissue the uptake was slow, and, consequently, even the elimination was slow. The adipose tissue is poorly perfused, in contrast to more vascular tissue groups, and it has a large partition coefficient (tissue/blood) because of its high lipid contents (Fiserova-Bergerova et al., 1974). Compared to blood, the concentration of toluene in stomach and in kidneys was still high after 12 h in both administrations. The relatively high amount of toluene found in kidneys is important because of its toxic effects on kidneys. Toluene "sniffing", for example, causes renal tubular acidosis (Taher et al., 1974). The high concentrations of toluene in stomach after inhalation could be due to the secretion of toluene into gastric fluid. There are no reports on toxic effects of toluene on stomach. Our observations may have no direct practical significance, but they are pharmacokinetically interesting. In brain tissue, the concentration of toluene was higher than in blood 12 h after exposure. This may be in accordance with the neural effects of this solvent. Toluene also seemed to accumulate in thyroid and adrenal glands, the relative amount still being high 12 h after exposure. There is a very high correlation and linear relationship between toluene concentrations in alveolar air and arterial blood (Gamberale and Hultengren, 1972). According to Astrand et al. (1972), in man, the concentration of toluene in alveolar air and blood increases very rapidly during the first 10--15 min of exposure at rest as well as during exercise. We found that the maximum radioactivity was reached 1 5 - 3 0 min after the end of exposure in all rat tissues except in white adipose tissue. This finding agrees with the observations on man. The absorption from gastro-intestinal tract retarded the distribution rate. Thus, in most tissues maximal radioactivity was reached in 3 h and in white adipose tissue even later, in about 5 h. The uptake, distribution, and elimination of toluene seems to

176

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follow a complex pharmacokinetic model, in which, after a single exposure, absorption through the inhalation route to blood and well perfused tissues is rapid. The accumulation to depots of body fat with subsequent redistribution into blood takes several hours. The metabolism of toluene m a y modify this pharmacokinetic model. The metabolism increases the uptake but decreases the concentrations of solvents in tissues (Fiserova-Bergerova et al., 1974). Toxic exposure to organic solvents is usually evaluated by measuring their concentrations in expiratory air, in urine, and in blood. These measurements may, however, oversimplify the real situation unless simultaneous accumulation of the solvents in other tissues is taken into account. Our results imply the danger of accumulation of toluene in various organs. This accumulation is due mostly to the ability of the adipose tissue to retain toluene. The bone marrow also showed a very slow elimination of radioactivity. The concentration was relatively low, but this slowness of elimination m a y explain the accumulation of solvents in chronic exposures and, consequently, blood dyscrasias induced by industrial solvents.

References The American Conference of Governmental Industrial Hygienists. Threshold limit values 1966. In: Handbook of Laboratory Safety (N.V. Steere, ed.), p. 822. Cleveland: The Chemical Rubber Co. 1971 Dost, F. H.: Korrespondierende F1/ichen. In: Grundlagen der Pharmakokinetik, pp. 155-161. Stuttgart: Thieme 1968 Fiserova-Bergerova, V., Vlach, I., Singhal, K.: Simulation and prediction of uptake, distribution, and exhalation of organic solvents. Brit. J. industr. Med. 31, 45-52 (1974) Gamberale, F., Hultengren, M.: Toluene exposure II. Psychophysiological functions. Wk-Environ.Hlth 9, 131--139 (1972) Greenburg, L., Mayers, M. R., Heimann, H., Moskowitz, S.: The effects of exposure to toluene in industry. J. Amer. reed. Ass. 118, 573--578 (1942) Laham, S.: Metabolism of industrial solvents. 1. The biotransformations of benzene and benzene substitutes. Occup. Hlth Rev. 21, 3-4 (1970) Nomiyama, K., Nomiyama, H.: Respiratory eliminationof organic solvents in man. Benzene,toluene, n-hexane, acetone, ethyl acetate and ethyl alcohol. Int. Arch. Arbeitsmed. 32, 85-91 (1974a) Nomiyama, K., Nomiyama, H.: Respiratory retention, uptake and excretion of organic solvents in man. Benzene, toluene, n-hexane, trichloroethylene, acetone, ethyl acetate and ethyl alcohol. Int. Arch. Arbeitsmed. 32, 75--83 (1974b) von Oettingen, W. F., Neal, P. A., Donahue, D. D.: The toxicity and potential dangers of toluene. J. Amer. med. Ass. 118, 579--584 (1942) Press, E., Done, A. K.: Solvent sniffing. Physiologic effects and community control measures for intoxication from the intentional inhalation of organic solvents. I. Pediatrics 39, 451--461 (1967) Taher, S. M., Anderson, R. J., McCartney, R., Popovtzer, M. M., Schrier, R. W.: Renal tubular acidosis associated with toluene sniffing. New Engl. J. Med. 290, 765--768 (1974) Withey, R. J., Hall, J. W.: The joint toxic action of perchloroethylenewith benzene or toluene in rats. Toxicology 4, 5--15 (1975) Astrand, I., Ehrner-Samuel,H., Kilbom, A., t~vrum, P.: Toluene exposure. I. Concentration in alveolar air and blood at rest and during exercise. Wk-Environ.-Hlth 9, 119-130 (1972) Received July 23, 1976