Immunohistochemical Localization of Paraquat in Lungs and Brains

Chapter 23

Immunohistochemical Localization of Paraquat in Lungs and Brains 1

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Masataka Nagao , Takehiko Takatori , Kazuaki Inoue , Mikio Shimizu , and Koichi Terazawa Downloaded by UNIV OF TEXAS AT ARLINGTON on January 6, 2018 | http://pubs.acs.org Publication Date: December 26, 1990 | doi: 10.1021/bk-1990-0451.ch023

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Department of Legal Medicine, School of Medicine, Hokkaido University, Sapporo 060, Japan Department of Pathology, Hokkaido University Hospital, Sapporo 060, Japan 2

Immunohistochemistry was used to investigate the localization and dynamics of paraquat in lung and brain in paraquat-poisoned rats. Rats were sacrificed at 3 h, 12 h, 24 h, 3 days, 7 days and 10 days after the intravenous injection of paraquat (5 mg/kg). In lung tissues, paraquat was localized in walls of blood vessels and bronchiolar epithelial c e l l s from 3 h to 10 days after the paraquat exposure. Furthermore, histiocytes containing paraquat were observed. Interstitial pulmonary fibrosis containing paraquat developed with time. These results indicate that histiocytes are the probable cause of the pulmonary fibrosis in paraquat-poisoned rats. On the other hand, in brain tissues, paraquat was localized only in capillary walls and g l i a l c e l l s but was not observed in nerve c e l l s 10 days after the injection of paraquat, providing evidence that paraquat cannot pass through the blood -brain barrier.

0097-6156/91/0451-0264$06.00/0 © 1991 American Chemical Society Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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Localization of Paraquat in Lungs and Brains

Downloaded by UNIV OF TEXAS AT ARLINGTON on January 6, 2018 | http://pubs.acs.org Publication Date: December 26, 1990 | doi: 10.1021/bk-1990-0451.ch023

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Paraquat ( 1 ,1 - d i m e t h y l - 4 , 4 - b i p y r i d i n i u m ) i s a widely used herbicide, and i s one of major causes of m o r t a l i t y i n human s e l f - p o i s o n i n g s (1 , 2 ) . Pulmonary f i b r o s i s i s one of the most harmful complications of paraquat p o i s o n i n g . Although s e v e r a l s t u d i e s concerning the mechanism of development of pulmonary f i b r o s i s have been performed ( 1 ,3-6 ) , there has not been a study on the l o c a l i z a t i o n and dynamics of paraquat i n lung. Also, parkinsonism can be induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an analog of paraquat (7,8) , and paraquat may a l s o be a cause of parkinsonism. In t h i s paper, we d e s c r i b e the l o c a l i z a t i o n and dynamics of paraquat i n lung and b r a i n tissues with immunohistochemical techniques using p r e v i o u s l y developed r a b b i t a n t i s e r a (£). M a t e r i a l s and Methods 1

Chemicals Paraquat d i c h l o r i d e (1,1 -dimethyl-4,4'-bipyridinium d i c h l o r i d e ) was purchased from A l d r i c h Co., U.S.A. Stravigen (biotin-streptavidin amplified system (peroxidase)) was purchased from BioGenex L a b o r a t o r i e s , U.S.A. 3-Amino-9-ethylcarbozole (AEC) and 3,3 -diaminobenzidine (DAB) were obtained from Sigma Chemical Company, U.S.A. A l l other reagents were purchased from Nakarai Chemical Co., Japan. 1

Animal Treatment Male Sprangue-Dawley r a t s (200-250g) were i n t r a v e n o u s l y administered paraquat d i c h l o r i d e (5 mg/kg) d i s s o l v e d i n s a l i n e . They were s a c r i f i c e d 3 h, 12 h, 24 h, 3 days, 7 days and 10 days a f t e r i n j e c t i o n and the t i s s u e s (lung and brain) were removed. Tissue P r e p a r a t i o n The t i s s u e s were f i x e d i n 0.1 M phosphate b u f f e r (pH 7.4) c o n t a i n i n g 4 % paraformaldehyde f o r 6 h. A f t e r f i x a t i o n , they were cut to a thickness of 2 mm and these s l i c e s were f i x e d again i n the paraformaldehyde f o r 3 days; the s l i c e s were then dehydrated i n upgrading s e r i e s of ethanol, c l e a r e d i n xylene, and embedded i n p a r a f f i n . Sections of 3 um in thickness were cut and used for the immunohistochemical a n a l y s i s of paraquat. Antibodies Against Paraquat The p o l y c l o n a l a n t i b o d i e s against paraquat reported previously (9J were used. After these a n t i s e r a were prepared a 1:10 d i l u t i o n with 0.01 M phosphate-buffered s a l i n e (pH 7.4, PBS), they were absorbed with bovine serum albumin (BSA, 1 mg/ml) overnight a t 4°C before use. Immunohistochemical Procedures f o r L i g h t Microscopy The immunohistochemical s t a i n i n g using a b i o t i n - s t r e p t a v i d i n peroxidase complex was performed according to the

Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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method as described p r e v i o u s l y ( 1_0). The s e c t i o n s from lungs and b r a i n s were s t a i n e d with AEC and with DAB, respectively. Histological Staining In order to evaluate the morphological changes of lung and brain, a hematoxylin-eosin stain was performed. A Masson trichrome s t a i n was a l s o used i n order to evaluate the c o l l a g e n generation i n lungs.


Results Following paraquat exposure, i n f i l t r a t i o n of inflammatory c e l l s i n t o lung t i s s u e and f i b r o s i s of i n t e r s t i t i a l lung t i s s u e developed with time (Figure 1). Pathological changes were not observed i n b r a i n t i s s u e s . In all immunohistochemical sections of the lungs during the experiments, antibody binding was observed i n w a l l s of blood v e s s e l s , h i s t i o c y t e s and bronchiolar epithelial cells (Figure 2). It was also observed that the paraquat localized i n the b r o n c h i o l a r e p i t h e l i a l c e l l s seemed to be secreted i n t o bronchiole (Figure 2-A). On the other hand, the paraquat-localization in brain was found only in capillary walls and glial cells (Figure 3-A). However, these positive reactions were completely i n h i b i t e d when the antiserum was absorbed with 3 mg/ml paraquat overnight at 4 °C before use (Figures 2-D and 3-B), and i n c o n t r o l s e c t i o n s of lung and b r a i n antibody b i n d i n g was not observed. Discussion The l o c a l i z a t i o n of paraquat i n lung and b r a i n of the paraquat-exposed rats was demonstrated immunohistochemically. Since paraquat i s water-soluble, i t would be removed by the f i x a t i o n and s t a i n i n g processes except f o r the i n t r a c e l l u l a r l y l o c a l i z e d paraquat. It is not clear whether paraquat is localized i n t r a c e l l u l a r l y as a f r e e form, or bound to c e l l u l a r components. However, the antiserum used i n t h i s study selectively recognizes both the methyl group and bipyridyl ring of paraquat, and dose not bind even s l i g h t l y to analogs of paraquat (9) , i n d i c a t i n g that the antiserum s p e c i f i c i c a l l y binds to the i n t r a c e l l u l a r l y localized paraquat which keeps the original structure. The further investigations for the intracellular localization of paraquat should be performed. In the lung 3 h a f t e r paraquat exposure, h i s t i o c y t e s containing paraquat already had infiltrated into i n t e r s t i t i a l lung t i s s u e and could be found there 10 days a f t e r the exposure. I n t e r s t i t i a l f i b r o s i s was a l s o observed to increase with time. Schoenberger et


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F i g u r e 1. Masson t r i c h r o m e s t a i n i n g s o f l u n g ; 3 h ( A ) , 24 h ( Β ) , 3 d a y s (C) and 10 d a y s ( D ) .

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Figure 2. Immunohistochemical localization of paraquat i n the lung t i s s u e . (x400) 3 h (A), 3 days (B), 10 days (C) and c o n t r o l (D).


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Figure 3. Immunohistochemical localization of paraquat i n the b r a i n t i s s u e . (x400) 3 days (A) and control (B).



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a l . (_3) reported that a f t e r paraquat exposure i n v i v o , a l v e o l a r macrophages produce f i b r o n e c t i n and r e l e a s e a fibroblast growth f a c t o r and Wong and Stevens ( 1 1 ) reported that a f t e r exposure i n v i t r o , paraquat induces not only c y t o t o x i c i t y i n a l v e o l a r macrophages but a l s o e x t r a c e l l u l a r superoxide anion generation from a l v e o l a r macrophages. From these f i n d i n g s i t i s l i k e l y that the development of the pulmonary f i b r o s i s induced by paraquat i s due t o the r e l e a s e of the b i o l o g i c a l l y a c t i v e substances produced by macrophages, which are a c t i v a t e d by phagocytizing h e r b i c i d e damaged c e l l s , i n a d d i t i o n t o the d i r e c t t o x i c i t y of paraquat. The mechanism of migration o f macrophages t o the i n t e r s t i t i a l lung t i s s u e i s not c l e a r . However, i t i s possible that the increase i n vascular permeability a f t e r the paraquat exposure i n v i v o (12) r e s u l t s i n the production of a macrophage chemotactic f a c t o r from plasma p r o t e i n s (13,14) a t the i n t e r s t i t i a l lung t i s s u e . Webb (J_5) reported that i n an autoradiographic study paraquat i s l o c a l i z e d i n b r o n c h i o l a r epithelium 24 hafter the paraquat exposure and i m p l i c a t e s the bronchiolar epithelium as the s i t e of paraquat uptake. In our immunohistochemical study, we not only observed that paraquat was s e l e c t i v e l y localized i n b r o n c h i o l a r e p i t h e l i a l c e l l s , but a l s o observed that paraquat was secreted i n t o the b r o n c h i o l e . In the b r a i n , even 10 days a f t e r the paraquat exposure, paraquat was not found i n nerve c e l l s but observed only in glial cells and capillary walls. These findings i n d i c a t e that paraquat i s unable t o pass through the b l o o d - b r a i n - b a r r i e r , suggesting that paraquat itself i s not a cause of Parkinson's disease ( 1 6 ) . Acknowledgments This work was supported i n part by the Shimabara Promotion Foundation.

Science

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7. Langston J . W.; Ballard P. Α.; Tetrud J . W.; Irwin I. Science 1983, 219, 979-80. 8. Burns R. S.; Chiueh C. C.; Markey S. P.; Ebert M. H.; Jacobowitz D. M.; Kopsin I. J . Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 4546-50. 9. Nagao M.; Takatori T.; Terazawa K.; Wu B.; Wakasugi C.; Masui M.; Ikeda H. J . Forens. Sci. 1 989, 34, 547-52. 10. Nagao M.; Takatori T.; Inoue K.; Shimizu M.; Terazawa K.; Akabane H. Toxicol. (in press) 11. Wong R. C.; Stevens J . B. J . Toxicol. Environ Health 1985, 15, 417-29. 12. Tanaka R.; Fujisawa S.; Kawamura K.; Harada M. J. Toxicol. Sci. 1983, 8, 147-59. 13. Honda M.; Hirashima M.; Hayashi H. Virchows Arch. Β Cell Pathol. 1978, 27, 317-33. 14. Ishida M,; Honda M.; Hayashi H. Immunol. 1978, 35, 167-76. 15. Webb D. B. Br. J. exp. Pathol. 1980, 61, 217-21. 16. Roller W. C. Neurol. 1986, 36, 1147. RECEIVED 1990

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Immunohistochemical Localization of Paraquat in Lungs and Brains

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