Toxicology, 8(1977) 135--141 ©Elsevier/North-Holland Scientific Publishers, Ltd.
ORGAN DISTRIBUTION AND NERVOUS SYSTEM BINDING OF STYRENE A N D S T Y R E N E OXIDE
H. SAVOLAINEN and H. VAINIO Department of Industrial Hygiene and Toxicology, Institute of Occupational Health, Haartmaninkatu 1, SF-00290 Helsinki 29 (Finland)
(Received September 30th, 1976) (Revision received February 10th, 1977) (Accepted March 17th, 1977)
SUMMARY Ten adult male rats were injected intraperitoneally with 460 gmol of styrene oxide with radioactive label. Fifteen similar rats were injected similarly with 577 ~mol of styrene. The distribution of styrene in central nervous system, blood, liver, lungs, kidneys and d u o d e n u m was studied 3, 6 and 24 h after the injection while the same studies were done with styrene oxide 3 and 6 h after the injection. The liver, brain, kidney and duodenal contents of styrene and styrene oxide were higher than that in blood, lungs and spinal cord while the macromolecule-associated styrene oxide in the central nervous system was small. The removal of injected compounds was slow between 3 and 6 h after the injection in the organ systems although lipid-soluble compounds tended to diminish in brain more rapidly than the total radioactivity.
INTRODUCTION Styrene has been used widely since the 1940s in the chemical polymerization industry [1]. Despite the great number of exposed workers, no major outbreak of disease has occurred due to styrene toxicity while reports have been published on sporadic cases with optic atrophy or neuromuscular disease [2,3]. However, electrophysiological investigations on larger number of workers with low-grade styrene exposure describe subtle electroencephalo-
Abbreviations: ACHE, acetylcholine esterase; CNP, 2',3'~yclic nucleotide 3'-phosphohydrolase; CNS, central nervous system; TCA, trichloracetic acid.
135
graphic changes as well as decreased conduction velocity in the peripheral nerves [4,5]. Styrene is metabolized in the mammalian body by oxidation to styrene oxide with subsequent conjugation with glutathione or further oxidation [6]. Styrene oxide and phenylethylene glycol are more potent central nervous system (CNS) depressants than styrene itself [7]. Other studies emphasize the role of styrene oxide in the generation of hepatic lesion as well as its carcinogenic action (for review see ref. 8). These findings prompt us to carry out experiments on styrene and styrene oxide distribution and their uptake in vulnerable organs. MATERIALS AND METItODS Ten adult male rats of Sprague-Dawley strain with average body weight of 306 ± 14 g (+- S.D.) received an intraperitoneal injection of 460 /~mol of styrene oxide in 1 ml of olive oil with 3H-labelled compound as a tracer (radioactive concentration 187.5 pCi/ml, NEN Chemicals Ltd., Dreieichenhain, G.F.R.). Fifteen other rats of the same strain with body weight of 302 ~ 12 g (± S.D.) were injected similarly with 577 tlmol of styrene in I ml of olive oil with '4C-labelled tracer (radioactive concentration 16.5 t~Ci/ml, NEN Chemicals Ltd., Dreieichenhain, G.F.R.). Five rats in both experiments were killed 3 h, five rats 6 h and remaining five in the latter experiment 24 h after the injection. The animals were bled at the sacrifice. Samples were taken into heparinized tubes; the brain, spinal cord, liver, duodenum, lungs and kidneys were removed and placed in an ice bath. The lungs were washed extensively with physiological saline to remove blood. Tne tissue samples of the parenchymal organs were weighed and homogenized in 10 vol. of 8 M urea. The homogenates were analyzed for protein [9] ; and the radioactivity was measured using a liquid scintillation counter technique. The left cerebral hemispheres were homogenized in 10 vol. of chlorof o r m - m e t h a n o l (2 : 1, v/v} mixture and the organic phase was analyzed for radioactivity. The right cerebral hemispheres were homogenized in 10 vol. of 1 mM EDTA buffer (pit 7.4) and centrifuged at 9000 g for 30 min at 40°C. The supernatant and pellet were analyzed for protein and radioactivity as before. In addition, trichloracetic acid (TCA) was added to an aliquot of the EDTA homogenate before the centrifugation as well as to an aliquot of the supernatant after the centrifugation, to reach a concentration of 10% (w/v) in respect of the acid. The ensuing protein precipitate was separated by centrifugation at 3500 g for 15 min at room temperature. The precipitates and TCA extracts were analyzed for protein and radioactivity. The spinal cord samples were homogenized in 0.85 M sucrose -- 0.05 M potassium phosphate -- 0.01 M sodium chloride buffer (pit 6.0) for the isolation of an axonal fraction [10]. An aliquot of the spinal cord homogenate, as well as the axonal fractions were analyzed for protein and radioactivity. The purity of the axonal preparations was examined by analysis for myelin
136
o
t
Fig. 1. P h o t o m i c r o g r a p h o f r a t spinal c o r d a x o n a l f r a c t i o n as s t a i n e d w i t h 0.1% a m i d o black. N o t e t h e clear b a c k g r o u n d o u t s i d e t h e aggregated a x o n bundles. A x o n s t h a t deviate frorp t h e b u n d l e s are devoid of m y e l i n s h e a t h a n d t e r m i n a t e freely. A C h E activity is increased 38-fold in this f r a c t i o n w h i l e o n l y low specific CNP activity is f o u n d . × 60.
|
\
1
P
b-a
0o
975 n.s. 1012 ---
, S.D. (n = 5)
404 + 34 P< 0.005 321 ± 42 104 ~ 21 , 146
± 35
8
442 ~ 56 P< 0.0025 313 ± 31 34 ± 8
Brain Styrene
Styrene oxide
34 ±
STYRENE
714 n.s. 736 --
82 , 104
t
Styrene oxide
--
864 ~ 52 P< 0.05 723 ' 123
Styrene oxide
INJECTED
334 ± 25 n.s. 339 ± 41
Styrene
Styrene
--
186 + 13 n.s. b 179 , 22
Styrene oxide
Liver
OF INTRAPERITONEALLY
Duodenum
5 ± 1
23 ± 3 P< 0.0005 49_, 5
anmol/ml, others nmol/g bn.s., not significant.
6 24
3
24
6
3
Styrene
(h)
Injection
DISTRIBUTION
Blood a
I
Time after
ORGAN
TABLE
2 6 6 ~. 4 8 n.s. 231 ± 37 33± 7
Styrene
Spinal cord
7
, 13
, t7
4 2 "_
172 n.s. 140
OXIDE
, 44
, 40
437 n.s. 439
, 68
' 60
Styrene oxide
--
409 n.s. 423
Styrene oxide
STYRENE
Styrene
Lungs
AND
63
845 ± 102 P
ORGAN DISTRIBUTION AND NERVOUS SYSTEM BINDING OF STYRENE A N D S T Y R E N E OXIDE
H. SAVOLAINEN and H. VAINIO Department of Industrial Hygiene and Toxicology, Institute of Occupational Health, Haartmaninkatu 1, SF-00290 Helsinki 29 (Finland)
(Received September 30th, 1976) (Revision received February 10th, 1977) (Accepted March 17th, 1977)
SUMMARY Ten adult male rats were injected intraperitoneally with 460 gmol of styrene oxide with radioactive label. Fifteen similar rats were injected similarly with 577 ~mol of styrene. The distribution of styrene in central nervous system, blood, liver, lungs, kidneys and d u o d e n u m was studied 3, 6 and 24 h after the injection while the same studies were done with styrene oxide 3 and 6 h after the injection. The liver, brain, kidney and duodenal contents of styrene and styrene oxide were higher than that in blood, lungs and spinal cord while the macromolecule-associated styrene oxide in the central nervous system was small. The removal of injected compounds was slow between 3 and 6 h after the injection in the organ systems although lipid-soluble compounds tended to diminish in brain more rapidly than the total radioactivity.
INTRODUCTION Styrene has been used widely since the 1940s in the chemical polymerization industry [1]. Despite the great number of exposed workers, no major outbreak of disease has occurred due to styrene toxicity while reports have been published on sporadic cases with optic atrophy or neuromuscular disease [2,3]. However, electrophysiological investigations on larger number of workers with low-grade styrene exposure describe subtle electroencephalo-
Abbreviations: ACHE, acetylcholine esterase; CNP, 2',3'~yclic nucleotide 3'-phosphohydrolase; CNS, central nervous system; TCA, trichloracetic acid.
135
graphic changes as well as decreased conduction velocity in the peripheral nerves [4,5]. Styrene is metabolized in the mammalian body by oxidation to styrene oxide with subsequent conjugation with glutathione or further oxidation [6]. Styrene oxide and phenylethylene glycol are more potent central nervous system (CNS) depressants than styrene itself [7]. Other studies emphasize the role of styrene oxide in the generation of hepatic lesion as well as its carcinogenic action (for review see ref. 8). These findings prompt us to carry out experiments on styrene and styrene oxide distribution and their uptake in vulnerable organs. MATERIALS AND METItODS Ten adult male rats of Sprague-Dawley strain with average body weight of 306 ± 14 g (+- S.D.) received an intraperitoneal injection of 460 /~mol of styrene oxide in 1 ml of olive oil with 3H-labelled compound as a tracer (radioactive concentration 187.5 pCi/ml, NEN Chemicals Ltd., Dreieichenhain, G.F.R.). Fifteen other rats of the same strain with body weight of 302 ~ 12 g (± S.D.) were injected similarly with 577 tlmol of styrene in I ml of olive oil with '4C-labelled tracer (radioactive concentration 16.5 t~Ci/ml, NEN Chemicals Ltd., Dreieichenhain, G.F.R.). Five rats in both experiments were killed 3 h, five rats 6 h and remaining five in the latter experiment 24 h after the injection. The animals were bled at the sacrifice. Samples were taken into heparinized tubes; the brain, spinal cord, liver, duodenum, lungs and kidneys were removed and placed in an ice bath. The lungs were washed extensively with physiological saline to remove blood. Tne tissue samples of the parenchymal organs were weighed and homogenized in 10 vol. of 8 M urea. The homogenates were analyzed for protein [9] ; and the radioactivity was measured using a liquid scintillation counter technique. The left cerebral hemispheres were homogenized in 10 vol. of chlorof o r m - m e t h a n o l (2 : 1, v/v} mixture and the organic phase was analyzed for radioactivity. The right cerebral hemispheres were homogenized in 10 vol. of 1 mM EDTA buffer (pit 7.4) and centrifuged at 9000 g for 30 min at 40°C. The supernatant and pellet were analyzed for protein and radioactivity as before. In addition, trichloracetic acid (TCA) was added to an aliquot of the EDTA homogenate before the centrifugation as well as to an aliquot of the supernatant after the centrifugation, to reach a concentration of 10% (w/v) in respect of the acid. The ensuing protein precipitate was separated by centrifugation at 3500 g for 15 min at room temperature. The precipitates and TCA extracts were analyzed for protein and radioactivity. The spinal cord samples were homogenized in 0.85 M sucrose -- 0.05 M potassium phosphate -- 0.01 M sodium chloride buffer (pit 6.0) for the isolation of an axonal fraction [10]. An aliquot of the spinal cord homogenate, as well as the axonal fractions were analyzed for protein and radioactivity. The purity of the axonal preparations was examined by analysis for myelin
136
o
t
Fig. 1. P h o t o m i c r o g r a p h o f r a t spinal c o r d a x o n a l f r a c t i o n as s t a i n e d w i t h 0.1% a m i d o black. N o t e t h e clear b a c k g r o u n d o u t s i d e t h e aggregated a x o n bundles. A x o n s t h a t deviate frorp t h e b u n d l e s are devoid of m y e l i n s h e a t h a n d t e r m i n a t e freely. A C h E activity is increased 38-fold in this f r a c t i o n w h i l e o n l y low specific CNP activity is f o u n d . × 60.
|
\
1
P
b-a
0o
975 n.s. 1012 ---
, S.D. (n = 5)
404 + 34 P< 0.005 321 ± 42 104 ~ 21 , 146
± 35
8
442 ~ 56 P< 0.0025 313 ± 31 34 ± 8
Brain Styrene
Styrene oxide
34 ±
STYRENE
714 n.s. 736 --
82 , 104
t
Styrene oxide
--
864 ~ 52 P< 0.05 723 ' 123
Styrene oxide
INJECTED
334 ± 25 n.s. 339 ± 41
Styrene
Styrene
--
186 + 13 n.s. b 179 , 22
Styrene oxide
Liver
OF INTRAPERITONEALLY
Duodenum
5 ± 1
23 ± 3 P< 0.0005 49_, 5
anmol/ml, others nmol/g bn.s., not significant.
6 24
3
24
6
3
Styrene
(h)
Injection
DISTRIBUTION
Blood a
I
Time after
ORGAN
TABLE
2 6 6 ~. 4 8 n.s. 231 ± 37 33± 7
Styrene
Spinal cord
7
, 13
, t7
4 2 "_
172 n.s. 140
OXIDE
, 44
, 40
437 n.s. 439
, 68
' 60
Styrene oxide
--
409 n.s. 423
Styrene oxide
STYRENE
Styrene
Lungs
AND
63
845 ± 102 P
