Life Sciences, Vol. Printed in the USA
50, pp.
PL-41 - PL-45
PHARMACOLOGY
Accelerated
Pergamon
LETTERS
Communication
IMMUNOCYTOCHEMICAL DETECTION OF THE 72-kDa HEAT SHOCK PROTEIN IN HALOTHANE INDUCED HEPATOTOXICITY IN RATS R.A. Van Dyke I, S. Mostafapour, Henry Ford Health
Press
H.M. Marsh,
-
Y. Li, M. Chopp
Sciences, 2799 West Grand Boulevard, Detroit, MI 48202
(Submitted November 12, 1991; accepted November 14, 1991; received in final form December 13, 1991) Abstract. Liver sections removed from phenobarbital induced rats 24 to 48 hours after a 2 hour exposure to 1.0% halothane with 10% oxygen and subjected to immunocytochemical treatment showed evidence of centrilobular damage as well as evidence of the production of a protein which has immunoreactivity with anti HSP 72 antibodies. The cells showing evidence of immunoreactivity were within the area of the centrilobular lesion. The level of immunoreactive protein varied directly with the intensity of the lesion. Liver sections from animals treated with phenobarbital alone, phenobarbital plus 10% oxygen, or phenobarbital plus 20% oxygen and 1.0% halothane all were without lesions as well as the immunoreactive protein.
Introduction A family of proteins ~ o l l e c t i v e l y known as stress proteins or heat shock proteins (HSP) have been found to be synthesized and inducible in every species thus far examined. As their name implies HSP's are induced in response to a variety of stress including heat shock, nutrient deprivation, oxygen radicals, and metabolic disruption produced b y s u l f h y d r y l reagents, heavy metals, and glutathione depleting agents, viruses, ischemia and xenobiotics
(l-S) The role of HSP's is presently coming into focus and it is clear they have a function in unstressed as well as stressed cells (6,7). In unstressed cells these proteins play a role in chaperoning other proteins and aid in transmembrane transport of proteins. They are also involved in functions involving facilitating proper folding of proteins as well as preventing improper folding and protein aggregation. In these roles it is almost certain that HSP's are important in protecting cells from the deleterious effects of those treatments, such as heat, which may promote m a l f o l d i n g or protein aggregation (8,9). More recent studies have lead to the observation that stress proteins are important targets of immune response. A number of 4 C o r r e s p o n d i n g author. 2 Abbreviations: HSP 72, Heat shock protein, Mrs72,000 ; PB, phosphate buffer; PBS, phosphate buffered saline. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press plc All rights reserved.
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HSPs in Toxicity of Halothane
Vol. 50, No. 7, 1992
facts have a c c u m u l a t e d to suggest such a role, for example: the abundance of stress proteins, their structure conservation t h r o u g h o u t species and their intrinsic a n t i g e n i c i t y (Reviewed in
lO). The mechanism of h e p a t o t o x i c i t y produced following the a d m i n i s t r a t i o n of h a l o t h a n e either in e x p e r i m e n t a l animals or h u m a n s has not been c o m p l e t e l y resolved. In recent years the focus has been on an immune r e s p o n s e to a h a l o t h a n e m e t a b o l i t e (trifluoro a c e t i c acid) a d d u c t e d p r o t e i n (11,12). We r e p o r t h e r e i n the a p p e a r a n c e of an HSP-72 p r o t e i n in rat livers under c o n d i t i o n s found to p r o d u c e h a l o t h a n e induced h e p a t i c n e c r o s i s in rats. This finding may c o n t r i b u t e to the immunological basis of this toxicity.
Methods T r e a t m e n t of A n i m a l s Male S p r a g u e - D a w l e y rats (approx 250 gm) were used. For p h e n o b a r b i t a l p r e t r e a t m e n t the animals were given p h e n o b a r b i t a l in their d r i n k i n g w a t e r (0.2%) for 5 days and r e t u r n e d to u n t r e a t e d d r i n k i n g w a t e r for 24 hours prior to use. A n i m a l s were divided into groups as follows with each group c o n t a i n i n g 6 or more animals: Group 1. R e c e i v e d p h e n o b a r b i t a l t r e a t m e n t only. G r o u p 2. R e c e i v e d p h e n o b a r b i t a l t r e a t m e n t f o l l o w e d by 10% 02 for 2 hours. Group 3. R e c e i v e d p h e n o b a r b i t a l t r e a t m e n t f o l l o w e d by 2 hours of 10% 02 and 1.0% halothane. Group 4. R e c e i v e d p h e n o b a r b i t a l t r e a t m e n t followed by 2 hours of 20% 02 with 1.0% halothane. F o l l o w i n g the two hour exposures the animals were r e t u r n e d to their cages and given food and water. P r e p a r a t i o n of Liver Slices Liver samples were taken from all rats at 24 hours post e x p o s u r e except for half the animals in group 3 w h i c h were not sampled until 48 hours. Animals were a n e s t h e t i z e d with sodium p e n t o t h a l (20 mg/kg) and the liver and m a j o r v e s s e l s exposed and h e p a r i n injected. The livers were p e r f u s e d t h r o u g h the inferior vena cava w i t h PBS, followed by p e r f u s i o n with p h o s p h a t e buffer c o n t a i n i n g 4% paraformaldehyde. Small cube shaped samples were r e m o v e d and p l a c e d in PBS c o n t a i n i n g paraformaldehyde. Immunocytochemistry The livers were sliced by v i b r a t o m e (50~m) as d e s c r i b e d by Welch et al (13). The slices were treated f l o a t i n g freely i n i t i a l l y in PBS then into PBS c o n t a i n i n g 50mM NH4Cl for 1 hour, w a s h e d in PBS and then into PBS c o n t a i n i n g 0.3% triton X-100 for 1 hour and washed. Normal sheep serum was used to block and 10% sheep serum in PBS was used in all s u b s e q u e n t w a s h e s and a n t i b o d y treatments. M o n o c l o n a l anti HSP-72 (Amersham, A r l i n g t o n Heights, IL) (13) was used at a d i l u t i o n of 1:200 and i n c u b a t e d for 10-12 hours f o l l o w e d by several washes. Endogenous p e r o x i d a s e was b l o c k e d with 3% H202 in PBS with 10% methanol, followed by washing. P e r o x i d a s e c o n j u g a t e d sheep b i o t i n y l a t e d a n t i m o u s e IgG was added for 6-8 hours followed by w a s h i n g overnight. Sections were incubated with s t r e p t a v i d i n "bridge" 1:200 in PBS for 1 hr,
Vol. 50, NO. 7, 1992
HSPs in Toxicity of Halothane
PL-43
followed by biotinylated horseradish peroxidase 1:400 in PBS for 1 hr. Then peroxidase was detected with 1 mg/ml diamine-benzidine, 0.015% in H202 in I00 mM Tris-pH7.6. Results
Sections of livers removed from phenobarbital treated animals exposed to 1.0% halothane and 10% oxygen and stained with hematoxylin-eosin showed many features ordinarily seen following this treatment, such as focal hepatocellular necrosis and fatty cell d e g e n e r a t i o n around the central vein (fig IE). Immunostaining of slices from the same sections with anti-HSP 72 showed strong staining in the same focal areas (fig IC). The immunoreactive protein was evident in areas superimposable on the lesion around the central vein. Liver sections removed from animals exposed to the other treatments showed no evidence of immunoreactivity. Thus all animals in groups 1,2 and 4 (fig IA,B,D) showed no detectable immunoreactivity nor any evidence of liver damage similar to that shown by all animals in group 3 (fig 1 C,E). Only group 3 animals were examined at 24 and 48 hours post exposure. The intensity of immunoreactivity varied with the intensity of the hepatic lesion in the group 3 animals. This variation resulted in immunoreactivity in as few as 2-3 cell rows from the central vein to as many as 12 or more. Because of this individual variation, it was not possible to determine any gradation from 24 to 48 hours. At 48 hours the immunoreactivity was present with the same variation as that seen at 24 hours.
DISCUSSION
The data presented in this communication clearly demonstrate the appearance of a protein in the hepatocytes in the central region of the liver which reacts with anti HSP-72 antibodies. Furthermore the appearance of this protein only occurs as a consequence of those conditions known to promote liver damage by halothane in the rat; i.e. the exposure of the phenobarbital induced animal to low 02 tensions (10% 02) and 1.0% halothane. If any one of the three components of this model is changed or deleted, no histological evidence of toxicity results and as shown here no immunoreactive protein is produced. The m e t a b o l i s m of halothane has been studied extensively and it has been found to undergo either a reductive or oxidative m e t a b o l i s m depending on the oxygen content of the tissue (14,15). Of interest to the present study is the discovery that halothane, when it undergoes reductive metabolism, does so through a free radical which has been implicated in generating free radicals in phospholipids, likely through the unsaturated fatty acid moiety (16,17). Upon reoxygenation this shows evidence of lipid peroxidation, a situation similar to oxidative stress following ischemia/reperfusion. HSP's have been shown to be induced in animal models for ischemia and reperfusion injury (18). Thus, in the present work, the 10% oxygen alone does not produce sufficient hypoxia to result in oxidative stress although halothane may exacerbate the oxidative stress of hypoxia alone by decreasing liver perfusion.
PL-44
HSPs in Toxicity of Halothane
Fig. 1 A-D Immunocytochemistry with anti HSP 72 antibody linked to peroxidase in 5~m slices of liver sections taken from animals with; A, p h e n o b a r b i t a l treatment only; B, p h e n o b a r b i t a l treatment followed by 2 hours of 10% 02; C, phenobarbital treatment followed by 2 hours of 10% 02 and 1.0% halothane; D, and phenobarbital treatment followed by 2 hours of 20% 02 with 1.0% halothane. E, shows an H&E stain of a 5~m slice taken from a liver of an animal treated as in C. Arrows in C and E show the central vein.
Vol. 50, No. 7, 1992
vol. 50, No. 7, 1992
HSPs in Toxicity of Halothane
PL-45
W i t h the current focus of the m e c h a n i s m of h a l o t h a n e induced h e p a t o t o x i c i t y on an immune response there now may be reason to include HSP's in this m e c h a n i s m e s p e c i a l l y since HSP's are believed to play a role in immunity (10,19,20). However it may be p r e m a t u r e to make c o m p a r i s o n s in part because of the a p p a r e n t species d i f f e r e n c e s in conditions required to produce the t o x i c i t y as well as species d i f f e r e n c e s in the incidence of t o x i c i t y (e.g. rat, mouse, guinea pig and human) (21-23). Furthermore the number of HSP's known to exist or to be inducible has been e s t i m a t e d to be as high as 24. Since the present study examines only the p r e s e n c e of a single HSP i.e. HSP-72, it is possible that other HSP's may be induced by p h e n o b a r b i t a l treatment alone or any of the other a s s o c i a t e d treatment. This will be the subject of future studies. Clearly, further e x p e r i m e n t a t i o n is required to define if the HSP's play a role in the toxicity of halothane and w h e t h e r there is any c o n n e c t i o n with the h y p e r s e n s i t i v i t y a s s o c i a t e d with h a l o t h a n e h e p a t i t i s in humans. REFERENCES
1. 2. 3. 4. 5. 6.
S. LINDQUIST, E.A. CRAIG, Annu. Rev. Genet. 22 63-137 (1988). M. SCHLESINGER, J. Cell Biol. 103 321-325 (1986). J.R. NEVINS, Cell 29 913-919 (1982). W. LEVINSON, H. OPPERMAN, J. JACKSON, Biochem. Biophys. Acta. 60 170-180 (1980). J. SCANDRA, J. SUBJECK, C. HUGHES, Proc. Natl. Acad. Sci. USA 8_!1 4843-4847 (1984). W.J. CHIRICO, M.G. WATERS, G. BLOBEL, Nature 332 805-810
(1988). 7.
8.
S.M. HEMMINGSEN, C. WOOLFORD, S.M. VAN DER VIES, K. TILLY, D.T. DENNIS, C.P. GEORGOPOULOS, R.W. HENDRIX, R.J. ELLIS, Nature 333 330-334 (1988). K.T. RIABOWOL, L.A. MIZZEN, W.J. WELCH, Science 242 433-436
(1988). 9. 10. ii. 12. 13. 14. 15. 16.
R.N. JOHNSTON, B.L. KUCEY, Science 242 1551-1554 (1988). R.A. YOUNG, Annu. Rev. Immunol. 8 401-420 (1990). H. TAKAHASHI, T. TAKESHITA, B. MOREIN, S. PUTNEY, R.N. GERMAIN, J.R. BERZOFSKY, Nature 344 873-875 (1990). L.R. POHL, H. SATOH, D.D. CHRIST, J.G. KENNA, Annu. Rev. Pharmacol. Toxicol. 28 367-387 (1988). W.J. WELCH, J.P. SUHAN, J. Cell Biol. 103 2035-2052 (1986). L.A. WIDGER, A.J. GANDOLFI, R.A. VAN DYKE, A n e s t h e s i o l o g y 44 197-201.15 (1976). R.A. VAN DYKE, M.T. BAKER, I. JANNSON, J. SCHENKMAN, Biochem. Pharmacol. 3 7 2357-2361 (1988). R.A. VAN DYKE, A.J. GANDOLFI, Drug Metab. Disp. 4 40-44
(1976). 17. 18. 19. 20. 21.
C.L. WOOD, A.J. GANDOLFI, R.A. VAN DYKE, Drug Metab. Disp. 305-313 (1976). Y.R.A. DONATI, D.C. SLOSMAN, B.S. POLLA, Biochem. Pharmacol. 4-0 2571-2577 (1990). R.A. YOUNG, T.J. ELLIOTT, Cell 5-9 5-8 (1989). P. PARHAM, TIBS 16 357-358 (1991). Summary of the National H a l o t h a n e Study. JAMA 197 775-778
(1966). 22. 23.
R.A. VAN DYKE, Anesth. Analg. 6_!1 812-819 (1982) C. LUMAN, M.J. COUSINS, P. DE LA M. HALL, J. Pharmacol. Ther. 232 801-809 (1985).
Exp.
50, pp.
PL-41 - PL-45
PHARMACOLOGY
Accelerated
Pergamon
LETTERS
Communication
IMMUNOCYTOCHEMICAL DETECTION OF THE 72-kDa HEAT SHOCK PROTEIN IN HALOTHANE INDUCED HEPATOTOXICITY IN RATS R.A. Van Dyke I, S. Mostafapour, Henry Ford Health
Press
H.M. Marsh,
-
Y. Li, M. Chopp
Sciences, 2799 West Grand Boulevard, Detroit, MI 48202
(Submitted November 12, 1991; accepted November 14, 1991; received in final form December 13, 1991) Abstract. Liver sections removed from phenobarbital induced rats 24 to 48 hours after a 2 hour exposure to 1.0% halothane with 10% oxygen and subjected to immunocytochemical treatment showed evidence of centrilobular damage as well as evidence of the production of a protein which has immunoreactivity with anti HSP 72 antibodies. The cells showing evidence of immunoreactivity were within the area of the centrilobular lesion. The level of immunoreactive protein varied directly with the intensity of the lesion. Liver sections from animals treated with phenobarbital alone, phenobarbital plus 10% oxygen, or phenobarbital plus 20% oxygen and 1.0% halothane all were without lesions as well as the immunoreactive protein.
Introduction A family of proteins ~ o l l e c t i v e l y known as stress proteins or heat shock proteins (HSP) have been found to be synthesized and inducible in every species thus far examined. As their name implies HSP's are induced in response to a variety of stress including heat shock, nutrient deprivation, oxygen radicals, and metabolic disruption produced b y s u l f h y d r y l reagents, heavy metals, and glutathione depleting agents, viruses, ischemia and xenobiotics
(l-S) The role of HSP's is presently coming into focus and it is clear they have a function in unstressed as well as stressed cells (6,7). In unstressed cells these proteins play a role in chaperoning other proteins and aid in transmembrane transport of proteins. They are also involved in functions involving facilitating proper folding of proteins as well as preventing improper folding and protein aggregation. In these roles it is almost certain that HSP's are important in protecting cells from the deleterious effects of those treatments, such as heat, which may promote m a l f o l d i n g or protein aggregation (8,9). More recent studies have lead to the observation that stress proteins are important targets of immune response. A number of 4 C o r r e s p o n d i n g author. 2 Abbreviations: HSP 72, Heat shock protein, Mrs72,000 ; PB, phosphate buffer; PBS, phosphate buffered saline. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press plc All rights reserved.
PL-42
HSPs in Toxicity of Halothane
Vol. 50, No. 7, 1992
facts have a c c u m u l a t e d to suggest such a role, for example: the abundance of stress proteins, their structure conservation t h r o u g h o u t species and their intrinsic a n t i g e n i c i t y (Reviewed in
lO). The mechanism of h e p a t o t o x i c i t y produced following the a d m i n i s t r a t i o n of h a l o t h a n e either in e x p e r i m e n t a l animals or h u m a n s has not been c o m p l e t e l y resolved. In recent years the focus has been on an immune r e s p o n s e to a h a l o t h a n e m e t a b o l i t e (trifluoro a c e t i c acid) a d d u c t e d p r o t e i n (11,12). We r e p o r t h e r e i n the a p p e a r a n c e of an HSP-72 p r o t e i n in rat livers under c o n d i t i o n s found to p r o d u c e h a l o t h a n e induced h e p a t i c n e c r o s i s in rats. This finding may c o n t r i b u t e to the immunological basis of this toxicity.
Methods T r e a t m e n t of A n i m a l s Male S p r a g u e - D a w l e y rats (approx 250 gm) were used. For p h e n o b a r b i t a l p r e t r e a t m e n t the animals were given p h e n o b a r b i t a l in their d r i n k i n g w a t e r (0.2%) for 5 days and r e t u r n e d to u n t r e a t e d d r i n k i n g w a t e r for 24 hours prior to use. A n i m a l s were divided into groups as follows with each group c o n t a i n i n g 6 or more animals: Group 1. R e c e i v e d p h e n o b a r b i t a l t r e a t m e n t only. G r o u p 2. R e c e i v e d p h e n o b a r b i t a l t r e a t m e n t f o l l o w e d by 10% 02 for 2 hours. Group 3. R e c e i v e d p h e n o b a r b i t a l t r e a t m e n t f o l l o w e d by 2 hours of 10% 02 and 1.0% halothane. Group 4. R e c e i v e d p h e n o b a r b i t a l t r e a t m e n t followed by 2 hours of 20% 02 with 1.0% halothane. F o l l o w i n g the two hour exposures the animals were r e t u r n e d to their cages and given food and water. P r e p a r a t i o n of Liver Slices Liver samples were taken from all rats at 24 hours post e x p o s u r e except for half the animals in group 3 w h i c h were not sampled until 48 hours. Animals were a n e s t h e t i z e d with sodium p e n t o t h a l (20 mg/kg) and the liver and m a j o r v e s s e l s exposed and h e p a r i n injected. The livers were p e r f u s e d t h r o u g h the inferior vena cava w i t h PBS, followed by p e r f u s i o n with p h o s p h a t e buffer c o n t a i n i n g 4% paraformaldehyde. Small cube shaped samples were r e m o v e d and p l a c e d in PBS c o n t a i n i n g paraformaldehyde. Immunocytochemistry The livers were sliced by v i b r a t o m e (50~m) as d e s c r i b e d by Welch et al (13). The slices were treated f l o a t i n g freely i n i t i a l l y in PBS then into PBS c o n t a i n i n g 50mM NH4Cl for 1 hour, w a s h e d in PBS and then into PBS c o n t a i n i n g 0.3% triton X-100 for 1 hour and washed. Normal sheep serum was used to block and 10% sheep serum in PBS was used in all s u b s e q u e n t w a s h e s and a n t i b o d y treatments. M o n o c l o n a l anti HSP-72 (Amersham, A r l i n g t o n Heights, IL) (13) was used at a d i l u t i o n of 1:200 and i n c u b a t e d for 10-12 hours f o l l o w e d by several washes. Endogenous p e r o x i d a s e was b l o c k e d with 3% H202 in PBS with 10% methanol, followed by washing. P e r o x i d a s e c o n j u g a t e d sheep b i o t i n y l a t e d a n t i m o u s e IgG was added for 6-8 hours followed by w a s h i n g overnight. Sections were incubated with s t r e p t a v i d i n "bridge" 1:200 in PBS for 1 hr,
Vol. 50, NO. 7, 1992
HSPs in Toxicity of Halothane
PL-43
followed by biotinylated horseradish peroxidase 1:400 in PBS for 1 hr. Then peroxidase was detected with 1 mg/ml diamine-benzidine, 0.015% in H202 in I00 mM Tris-pH7.6. Results
Sections of livers removed from phenobarbital treated animals exposed to 1.0% halothane and 10% oxygen and stained with hematoxylin-eosin showed many features ordinarily seen following this treatment, such as focal hepatocellular necrosis and fatty cell d e g e n e r a t i o n around the central vein (fig IE). Immunostaining of slices from the same sections with anti-HSP 72 showed strong staining in the same focal areas (fig IC). The immunoreactive protein was evident in areas superimposable on the lesion around the central vein. Liver sections removed from animals exposed to the other treatments showed no evidence of immunoreactivity. Thus all animals in groups 1,2 and 4 (fig IA,B,D) showed no detectable immunoreactivity nor any evidence of liver damage similar to that shown by all animals in group 3 (fig 1 C,E). Only group 3 animals were examined at 24 and 48 hours post exposure. The intensity of immunoreactivity varied with the intensity of the hepatic lesion in the group 3 animals. This variation resulted in immunoreactivity in as few as 2-3 cell rows from the central vein to as many as 12 or more. Because of this individual variation, it was not possible to determine any gradation from 24 to 48 hours. At 48 hours the immunoreactivity was present with the same variation as that seen at 24 hours.
DISCUSSION
The data presented in this communication clearly demonstrate the appearance of a protein in the hepatocytes in the central region of the liver which reacts with anti HSP-72 antibodies. Furthermore the appearance of this protein only occurs as a consequence of those conditions known to promote liver damage by halothane in the rat; i.e. the exposure of the phenobarbital induced animal to low 02 tensions (10% 02) and 1.0% halothane. If any one of the three components of this model is changed or deleted, no histological evidence of toxicity results and as shown here no immunoreactive protein is produced. The m e t a b o l i s m of halothane has been studied extensively and it has been found to undergo either a reductive or oxidative m e t a b o l i s m depending on the oxygen content of the tissue (14,15). Of interest to the present study is the discovery that halothane, when it undergoes reductive metabolism, does so through a free radical which has been implicated in generating free radicals in phospholipids, likely through the unsaturated fatty acid moiety (16,17). Upon reoxygenation this shows evidence of lipid peroxidation, a situation similar to oxidative stress following ischemia/reperfusion. HSP's have been shown to be induced in animal models for ischemia and reperfusion injury (18). Thus, in the present work, the 10% oxygen alone does not produce sufficient hypoxia to result in oxidative stress although halothane may exacerbate the oxidative stress of hypoxia alone by decreasing liver perfusion.
PL-44
HSPs in Toxicity of Halothane
Fig. 1 A-D Immunocytochemistry with anti HSP 72 antibody linked to peroxidase in 5~m slices of liver sections taken from animals with; A, p h e n o b a r b i t a l treatment only; B, p h e n o b a r b i t a l treatment followed by 2 hours of 10% 02; C, phenobarbital treatment followed by 2 hours of 10% 02 and 1.0% halothane; D, and phenobarbital treatment followed by 2 hours of 20% 02 with 1.0% halothane. E, shows an H&E stain of a 5~m slice taken from a liver of an animal treated as in C. Arrows in C and E show the central vein.
Vol. 50, No. 7, 1992
vol. 50, No. 7, 1992
HSPs in Toxicity of Halothane
PL-45
W i t h the current focus of the m e c h a n i s m of h a l o t h a n e induced h e p a t o t o x i c i t y on an immune response there now may be reason to include HSP's in this m e c h a n i s m e s p e c i a l l y since HSP's are believed to play a role in immunity (10,19,20). However it may be p r e m a t u r e to make c o m p a r i s o n s in part because of the a p p a r e n t species d i f f e r e n c e s in conditions required to produce the t o x i c i t y as well as species d i f f e r e n c e s in the incidence of t o x i c i t y (e.g. rat, mouse, guinea pig and human) (21-23). Furthermore the number of HSP's known to exist or to be inducible has been e s t i m a t e d to be as high as 24. Since the present study examines only the p r e s e n c e of a single HSP i.e. HSP-72, it is possible that other HSP's may be induced by p h e n o b a r b i t a l treatment alone or any of the other a s s o c i a t e d treatment. This will be the subject of future studies. Clearly, further e x p e r i m e n t a t i o n is required to define if the HSP's play a role in the toxicity of halothane and w h e t h e r there is any c o n n e c t i o n with the h y p e r s e n s i t i v i t y a s s o c i a t e d with h a l o t h a n e h e p a t i t i s in humans. REFERENCES
1. 2. 3. 4. 5. 6.
S. LINDQUIST, E.A. CRAIG, Annu. Rev. Genet. 22 63-137 (1988). M. SCHLESINGER, J. Cell Biol. 103 321-325 (1986). J.R. NEVINS, Cell 29 913-919 (1982). W. LEVINSON, H. OPPERMAN, J. JACKSON, Biochem. Biophys. Acta. 60 170-180 (1980). J. SCANDRA, J. SUBJECK, C. HUGHES, Proc. Natl. Acad. Sci. USA 8_!1 4843-4847 (1984). W.J. CHIRICO, M.G. WATERS, G. BLOBEL, Nature 332 805-810
(1988). 7.
8.
S.M. HEMMINGSEN, C. WOOLFORD, S.M. VAN DER VIES, K. TILLY, D.T. DENNIS, C.P. GEORGOPOULOS, R.W. HENDRIX, R.J. ELLIS, Nature 333 330-334 (1988). K.T. RIABOWOL, L.A. MIZZEN, W.J. WELCH, Science 242 433-436
(1988). 9. 10. ii. 12. 13. 14. 15. 16.
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