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N-acetyltransferase — An important polymorphism in drug-induced immunotoxicity
Abstracts
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MODULATION OF FcyRECEPTOR EXPRESSION AND FUNCTION IN MURINE PERITONEAL MACROPHAGES BY AMMONIUN METAVANADATE. K. Vaddi and C. I. Wei, Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611-0163. Female B~CRFI mice were qiven intraperitoneal injection of ammoniummetavanadate (2.5 or 10 mg V/kg), a/hm~nlumchloride, or sodium phosphate buffer (0.1M pH 7.2) every 3d for 6 wks. Resident peritoneal macrophages (M¢) were harvested and subjected to flow cytometric analysis of Fcy2a and Fcy2b receptor expression, and photometric microassay to measure receptor mediated binding and phagocytosis of sheep red blood cells. The NH4CI and IOVgroups showed 21.7 and 17.2% lower mean fluorescence channel (MFC) values and 7.1 and 5.9% lower values in % fluorescence positive cells than phosphate buffer control with respect to Fcy2aR expression. For Fc~2bR expression, the IOV group showed significant (P< 0.05) differences of 31.2% lower MFC and 15.7% lower % positive cells than phosphate buffer control. Similar cell sizes as expressed in the plots of fonvard scatter (FSC) vs. side scatter (SSC) were observed among a l l the four groups. Results of the photometric microassay indicated insignificant differences among the treatments in Fcy2aR mediated binding and phagocytosis. However, the IOV group showed significantly lower Fcy2bR mediated binding and phagocytosis. Thus, the reduction in Fcy2bR expression and function could contribute toward the depression in phagocytosis, NADPH-oxidase and superoxide generation in vanadate treated animals previously observed in this laboratory.
122. IMMUNOTOXICOLOGICAL S T A T U S OF Z I N C IN S E L E C T I V E I N H I B I T I O N BY C A D M I U M OF M I T O G E N INDUCED T CELL PROLIFERATION Fuminori OTSUKA and Motoyasu OHSAWA, F a c u l t y of P h a r m a c e u t i c a l Sciences, Teikyo University, S a g a m i k o , K a n a g a w a 199-01, J a p a n In m o u s e s p l e e n cell c u l t u r e s , c a d m i u m at 1 0 p M or less i n h i b i t s c o n c a n a v a l i n Ai n d u c e d T cell p r o l i f e r a t i o n but n o t l i p o p o l y s a c c h a r i d e - i n d u c e d B cell p r o l i f e r a tion. C o u n t e r e f f e c t of zinc r e q u i r e d for T cell p r o l i f e r a t i o n w a s f u r t h e r i n v e s t i g a t e d on the s e l e c t i v e i n h i b i t i o n of T cell p r o l i f e r a t i o n by c a d m i u m . The selec t i v e i n h i b i t i o n was s p e c i f i c to c a d m i u m , as not f o u n d on v a r i o u s h e a v y m e t a l s tested. T h e i n h i b i t o r y e f f e c t of c a d m i u m was e x e r t e d e f f i c i e n t l y by a d d i t i o n of c a d m i u m w i t h i n 16h a f t e r the m i t o g e n - s t i m u l a t i o n , and d i s a p p e a r e d a l m o s t c o m p l e t e l y u n d e r the p r e s e n c e of 30)aM zinc. T h e c o u n t e r e f f e c t to c a d m i u m was not p r o d u c e d w i t h f e r r o u s iron, n i c k e l a n d copper. The e f f e c t r e q u i r e d a d d i t i o n of zinc w i t h i n 16h a f t e r the m i t o g e n - s t i m u l a t i o n , w e l l c o n s i s t e n t w i t h the k i n e t i c s of c a d m i u m - i n h i b i t i o n of T cell p r o l i f e r a t i o n . Neither cellular cadmium content nor cadmium-induced metallothionein level w e r e c h a n g e d by a d d i t i o n of zinc. All t a k e n t o g e t h e r , it is s u g g e s t e d that c a d m i u m i n h i b i t s some z i n c - d e p e n d e n t proce s s e s n e c e s s a r y for T cell p r o l i f e r a t i o n . ( S u p p o r t e d in part by G r a n t - i n - A i d f r o m M i n i s t r y of E d u c a t i o n , S c i e n c e s a n d C u l t u r e , Japan)
123. N acetyltransferase - an i m p o r t a n t p o l y m o r p h i s m in d r u g - i n d u c e d immunotoxicity. D Hickman, S Kelly, E C o r o n e o s , J W o o d a n d E S i m D e p t of P h a r m a c o l o g y , U n i v e r s i t y of Oxford, S o u t h P a r k s Road, O x f o r d OXl 3QT, U . K Immunotoxicity resembling systemic lupus erythematosus occurs in certain individuals during therapy with hydralazine, a hydrazine used in hypertension, and procainamide, an arylamine anti-arrhythmic agent. Hydralazine and procainamide are metabolised by polymorphic N-acetyltransferase (NAT). A major factor predisposing to development of immunotoxlc side effects is the slow acetylator phenotype. Determination of acetylator phenotype has relied on the detection of acetylated substrate in urine. We describe a simple DNA based method for NAT genotyping using PCR with oligonucleotide primers specific for polymorphic NAT. The 'fast' NAT allele and the three 'slow' alleles can be distinguished either by restriction enzyme digestion after amplification or by using allele-specific oligonucleotide primers. Heterozygotes can easily be identified. The allele distribution differs from that reported after Southern blot analysis of a Japanese population and may be the basis of the inter ethnic variation in acetylator phenotype frequencies which has been described I. Deguchl, T. et al., (1990) J.Biol. Chem. 265, 12757-12760
769
121.
MODULATION OF FcyRECEPTOR EXPRESSION AND FUNCTION IN MURINE PERITONEAL MACROPHAGES BY AMMONIUN METAVANADATE. K. Vaddi and C. I. Wei, Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611-0163. Female B~CRFI mice were qiven intraperitoneal injection of ammoniummetavanadate (2.5 or 10 mg V/kg), a/hm~nlumchloride, or sodium phosphate buffer (0.1M pH 7.2) every 3d for 6 wks. Resident peritoneal macrophages (M¢) were harvested and subjected to flow cytometric analysis of Fcy2a and Fcy2b receptor expression, and photometric microassay to measure receptor mediated binding and phagocytosis of sheep red blood cells. The NH4CI and IOVgroups showed 21.7 and 17.2% lower mean fluorescence channel (MFC) values and 7.1 and 5.9% lower values in % fluorescence positive cells than phosphate buffer control with respect to Fcy2aR expression. For Fc~2bR expression, the IOV group showed significant (P< 0.05) differences of 31.2% lower MFC and 15.7% lower % positive cells than phosphate buffer control. Similar cell sizes as expressed in the plots of fonvard scatter (FSC) vs. side scatter (SSC) were observed among a l l the four groups. Results of the photometric microassay indicated insignificant differences among the treatments in Fcy2aR mediated binding and phagocytosis. However, the IOV group showed significantly lower Fcy2bR mediated binding and phagocytosis. Thus, the reduction in Fcy2bR expression and function could contribute toward the depression in phagocytosis, NADPH-oxidase and superoxide generation in vanadate treated animals previously observed in this laboratory.
122. IMMUNOTOXICOLOGICAL S T A T U S OF Z I N C IN S E L E C T I V E I N H I B I T I O N BY C A D M I U M OF M I T O G E N INDUCED T CELL PROLIFERATION Fuminori OTSUKA and Motoyasu OHSAWA, F a c u l t y of P h a r m a c e u t i c a l Sciences, Teikyo University, S a g a m i k o , K a n a g a w a 199-01, J a p a n In m o u s e s p l e e n cell c u l t u r e s , c a d m i u m at 1 0 p M or less i n h i b i t s c o n c a n a v a l i n Ai n d u c e d T cell p r o l i f e r a t i o n but n o t l i p o p o l y s a c c h a r i d e - i n d u c e d B cell p r o l i f e r a tion. C o u n t e r e f f e c t of zinc r e q u i r e d for T cell p r o l i f e r a t i o n w a s f u r t h e r i n v e s t i g a t e d on the s e l e c t i v e i n h i b i t i o n of T cell p r o l i f e r a t i o n by c a d m i u m . The selec t i v e i n h i b i t i o n was s p e c i f i c to c a d m i u m , as not f o u n d on v a r i o u s h e a v y m e t a l s tested. T h e i n h i b i t o r y e f f e c t of c a d m i u m was e x e r t e d e f f i c i e n t l y by a d d i t i o n of c a d m i u m w i t h i n 16h a f t e r the m i t o g e n - s t i m u l a t i o n , and d i s a p p e a r e d a l m o s t c o m p l e t e l y u n d e r the p r e s e n c e of 30)aM zinc. T h e c o u n t e r e f f e c t to c a d m i u m was not p r o d u c e d w i t h f e r r o u s iron, n i c k e l a n d copper. The e f f e c t r e q u i r e d a d d i t i o n of zinc w i t h i n 16h a f t e r the m i t o g e n - s t i m u l a t i o n , w e l l c o n s i s t e n t w i t h the k i n e t i c s of c a d m i u m - i n h i b i t i o n of T cell p r o l i f e r a t i o n . Neither cellular cadmium content nor cadmium-induced metallothionein level w e r e c h a n g e d by a d d i t i o n of zinc. All t a k e n t o g e t h e r , it is s u g g e s t e d that c a d m i u m i n h i b i t s some z i n c - d e p e n d e n t proce s s e s n e c e s s a r y for T cell p r o l i f e r a t i o n . ( S u p p o r t e d in part by G r a n t - i n - A i d f r o m M i n i s t r y of E d u c a t i o n , S c i e n c e s a n d C u l t u r e , Japan)
123. N acetyltransferase - an i m p o r t a n t p o l y m o r p h i s m in d r u g - i n d u c e d immunotoxicity. D Hickman, S Kelly, E C o r o n e o s , J W o o d a n d E S i m D e p t of P h a r m a c o l o g y , U n i v e r s i t y of Oxford, S o u t h P a r k s Road, O x f o r d OXl 3QT, U . K Immunotoxicity resembling systemic lupus erythematosus occurs in certain individuals during therapy with hydralazine, a hydrazine used in hypertension, and procainamide, an arylamine anti-arrhythmic agent. Hydralazine and procainamide are metabolised by polymorphic N-acetyltransferase (NAT). A major factor predisposing to development of immunotoxlc side effects is the slow acetylator phenotype. Determination of acetylator phenotype has relied on the detection of acetylated substrate in urine. We describe a simple DNA based method for NAT genotyping using PCR with oligonucleotide primers specific for polymorphic NAT. The 'fast' NAT allele and the three 'slow' alleles can be distinguished either by restriction enzyme digestion after amplification or by using allele-specific oligonucleotide primers. Heterozygotes can easily be identified. The allele distribution differs from that reported after Southern blot analysis of a Japanese population and may be the basis of the inter ethnic variation in acetylator phenotype frequencies which has been described I. Deguchl, T. et al., (1990) J.Biol. Chem. 265, 12757-12760