Chapter 13
Chronic Organophosphorus Exposure Biomarkers in the Detection of Immune Dysfunction and the Development of Lymphomas 1,2,4
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David S. Newcombe , Ali M. Saboori , and Ahmed H. Esa 1
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Department of Environmental Health Sciences, Department of Medicine, and Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD 21205 3
The health effects of chronic organophosphate (OP) exposures are incompletely understood, but evidence in animals and man suggests an association between organophosphate exposure, immune dysfunction, and the occurrence of hematopoietic tumors, especially lymphomas. Epidemiological studies have shown an excess of these tumors in populations exposed to OPs, and immune dysfunctions have also been associated with the development of lymphomas in animals and man. Despite the predisposition to lymphomas in humans with immune dysfunction and the increased risk of lymphoma in OP-exposed populations, links between organophosphates and the immune system have not been defined. Evaluation of in vitro human natural killer (NK) cell and cytotoxic Τ lymphocyte (CTL) activities in the presence of OPs shows that the cytotoxic functions of these cells are significantly inhibited by some organophosphate compounds. Such functional OPinduced changes may represent sensitive biomarkers of chronic organophosphate exposure and effect when integrated with duration and intensity of exposure. In turn, possible linkages to the development of hematopoietic tumors in some human hosts through the suppression of immune surveillance mechanisms may be uncovered in this process.
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Current address: Bedford VA Hospital, 200 Springs Road, Bedford, MA 01730 Current address: Department of Immunology, Pasteur Institute, 25 rue du docteur Roux, 75724 Paris, France
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0097-£156/94/0542-0197$06.00/0 © 1994 American Chemical Society
In Biomarkers of Human Exposure to Pesticides; Saleh, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
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B I O M A R K E R S OF H U M A N E X P O S U R E TO PESTICIDES
A c u t e o r g a n o p h o s p h o r u s e x p o s u r e s and t h e i r h e a l t h e f f e c t s have b e e n e x t e n s i v e l y i n v e s t i g a t e d and c h a r a c t e r i z e d ( 1 ) , b u t c h r o n i c e x p o s u r e s t o t h i s same c l a s s o f c h e m i c a l s h a v e been i n c o m p l e t e l y e v a l u a t e d even though t h e r e i s e v i d e n c e r e l a t i n g OP e x p o s u r e s t o t h e d e v e l o p m e n t o f h e m a t o p o i e t i c tumors. Since organophosphates are not generally i d e n t i f i e d a s d e f i n i t e human c a r c i n o g e n s , t h e a s s o c i a t i o n between OP e x p o s u r e and t u m o r i g e n e s i s may r e p r e s e n t e i t h e r a y e t t o b e d e f i n e d c a r c i n o g e n i c e f f e c t o r may o c c u r b y a n i n d i r e c t mechanism t h a t p l a c e s t h e h o s t a t r i s k f o r t h e development o f cancer. In t h i s paper, the evidence supporting the p o t e n t i a l for c h r o n i c OP e x p o s u r e t o promote t h e d e v e l o p m e n t o f s e r i o u s human h e a l t h e f f e c t s s u c h a s lymphomas i s f i r s t reviewed. T h e n , t h e mechanisms b y w h i c h t h i s c l a s s o f chemicals causes suppression o f t h e human immune s u r v e i l l a n c e system i s d e s c r i b e d . Biomarkers o f exposure and e f f e c t a r e d e f i n e d f o r t h e r e c o g n i t i o n a n d p o s s i b l e p r e v e n t i o n o f immune d y s f u n c t i o n s and t h e i r s e r i o u s h e a l t h e f f e c t s i n humans e x p o s e d t o o r g a n o p h o s p h a t e s . Epidemiological Populations
Studies
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Organophosphate
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Lymphomas i n Farmers. The i n i t i a l impetus for our l a b o r a t o r y t o examine t h e mechanisms b y w h i c h s i g n i f i c a n t health effects might be associated with chronic organophosphorus exposures arose from published e p i d e m i o l o g i c a l s t u d i e s (2-6). A l a r g e number o f s t u d i e s determined that farmers succumb more frequently t o lymphomas o r h e m a t o p o i e t i c tumors t h a n w o u l d b e e x p e c t e d i n h e a l t h y non-farming p o p u l a t i o n s (2-6). S u c h d a t a have b e e n documented n o t o n l y f o r f a r m e r s r e s i d i n g i n t h e u n i t e d S t a t e s , but a l s o f o r those i n other c o u n t r i e s (3,6). An e x c e s s o f lymphomas h a s been most r e c e n t l y r e p o r t e d i n I t a l i a n f a r m e r s (7). T h e r e have b e e n no e p i d e m i o l o g i c a l d a t a s u p p o r t i n g a s p e c i f i c c h e m i c a l compound o r g r o u p o f compounds a s c o n t r i b u t o r s t o t h i s i n c r e m e n t i n l y m p h a t i c c a n c e r s i n f a r m i n g p o p u l a t i o n s , b u t p e s t i c i d e s have b e e n prominently suggested as p o s s i b l e e t i o l o g i c f a c t o r s . The r e a s o n s f o r t h e e x c e s s o f lymphomas o b s e r v e d i n some farming p o p u l a t i o n s and not o t h e r s has remained e l u s i v e . To d a t e i t i s n o t known w h e t h e r s u c h f a c t o r s r e p r e s e n t d i f f e r e n c e s i n host responses t o chemicals, theuse of d i f f e r e n t chemicals, o r the h a b i t s o f farming groups i n t h e i r u s e and a p p l i c a t i o n o f c h e m i c a l s . Lymphomas i n G r a i n M i l l W o r k e r s . Investigations of grain m i l l w o r k e r s who a l s o have been d e t e r m i n e d t o have more lymphomas t h a n w o u l d be e x p e c t e d i n n o r m a l p o p u l a t i o n s h a v e
In Biomarkers of Human Exposure to Pesticides; Saleh, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
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NEWCOMBE ET AL.
Chronic Organophosphorus Exposure
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p r o v i d e d a d d i t i o n a l d a t a as t o t h e p o s s i b l e causative a g e n t s f o r t h e e x c e s s tumors i n t h e s e w o r k e r s . Studies p e r f o r m e d by e p i d e m i o l o g i s t s from the N a t i o n a l Cancer I n s t i t u t e have r e p o r t e d t h a t t h e w o r k e r s who a c q u i r e t h e s e t u m o r s i n e x c e s s have b e e n e x p o s e d t o p e s t i c i d e s (8), and t h e l a t t e r s u b s t a n c e s have been p o s t u l a t e d t o be p r i m e c a n d i d a t e s as t h e cause f o r t h e development o f such tumors i n exposed h o s t s . Further, only j a n i t o r s c l e a n i n g around g r a i n s t o r a g e s i l o s and t h o s e w o r k i n g d i r e c t l y i n t h e s i l o s t h a t have b e e n t r e a t e d w i t h p e s t i c i d e s t o p r e v e n t r o d e n t s a n d o t h e r a n i m a l s f r o m damaging t h e g r a i n s t o r e s show an increased risk f o r t h e development o f lymphomas ( 9 ). M a l a t h i o n , an o r g a n o p h o s p h o r u s p e s t i c i d e , was one o f t h e several chemicals identified by workers exposed to pesticide-treated grain (9). Lymphomas i n R e s i n W o r k e r s and A u t o m o b i l e M a n u f a c t u r e r s . F i n a l l y , two a d d i t i o n a l p o p u l a t i o n g r o u p s , r e s i n w o r k e r s a n d a u t o m o b i l e m a n u f a c t u r e r s , have a l s o b e e n shown t o h a v e an e x c e s s o f lymphomas ( 1 0 ) . I t i s clear that resin w o r k e r s i n t h e c o u r s e o f t h e i r work may be e x p o s e d t o organophosphorus c h e m i c a l s used e i t h e r as flame r e t a r d a n t s , s t a b i l i z e r s , or p l a s t i c i s e r s . However, s u c h w o r k e r s h a v e not been evaluated for their exposures to specific chemicals that may be lymphomagenic. Automobile m a n u f a c t u r e r s may a l s o be e x p o s e d t o p l a s t i c components i n addition t o a number o f o t h e r substances that could c o n t r i b u t e t o t h e i n c r e a s e d e x p r e s s i o n o f lymphomas i n t h i s group. N o n e t h e l e s s , one s h o u l d r e c o g n i z e t h a t a b o u t 30 t o 40 p e r c e n t o f t h e components i n an a u t o m o b i l e a r e made o f p l a s t i c , and some a u t o m o b i l e w o r k e r s may be i n v o l v e d i n t h e molding of these p l a s t i c parts. S u c h a g r o u p c o u l d w e l l be at risk from exposure t o molten p l a s t i c s containing organophosphorus agents. O r g a n o p h o s p h o r u s - i n d u c e d Immune D y s f u n c t i o n The determination that organophosphorus compounds are p o t e n t i n h i b i t o r s o f i n v i v o and i n v i t r o monocyte e s t e r a s e activity ( 1 1 - 1 6 ) , an enzyme known t o be an essential component o f t h e c y t o t o x i c e f f e c t s o f m o n o c y t e s a n d o t h e r immune c e l l s , was a key l i n k between impaired immune f u n c t i o n s and o r g a n o p h o s p h o r u s e x p o s u r e s . Perhaps the e a r l i e s t c l u e t o an o r g a n o p h o s p h o r u s - i n d u c e d d e f e c t i n m o n o c y t e e s t e r a s e a c t i v i t y comes f r o m t h e work o f L e e and Waters ( 1 5 ) . These i n v e s t i g a t o r s determined t h a t a v a r i e t y o f o r g a n o p h o s p h o r u s p e s t i c i d e s and c a r b a m a t e i n s e c t i c i d e s i n h i b i t e d , t o a s i g n i f i c a n t d e g r e e , human p e r i p h e r a l b l o o d monocyte e s t e r a s e a c t i v i t y . Such i n h i b i t i o n o c c u r r e d i n t h e absence o f d i r e c t c o n t a c t w i t h S h e l l p e s t s t r i p s i n w h i c h t h e c h e m i c a l s were imbedded s i n c e t h e s e p e s t i c i d e s were v o l a t i l e . The i n h i b i t o r c o n c e n t r a t i o n s r e s u l t i n g i n 50 p e r c e n t i n h i b i t i o n o f t h e t a r g e t enzyme ( I C ) f o r t h e v a r i o u s p e s t i c i d e s t e s t e d by L e e and W a t e r s h a v e been 5 0
In Biomarkers of Human Exposure to Pesticides; Saleh, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
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c a l c u l a t e d f r o m t h e i r p u b l i s h e d d a t a a n d a r e shown i n T a b l e I ( 1 5 ) along with t h e LD v a l u e s g i v e n i n t h e same p a p e r . As c a n be s e e n f r o m t h e s e d a t a , t h e organophosphorus insecticides i n v e s t i g a t e d by t h e s e w o r k e r s are f a i r l y p o t e n t i n h i b i t o r s o f human monocyte e s t e r a s e a c t i v i t y . T h e key t o t h e s i g n i f i c a n c e o f t h i s work came l a t e r when O e r t e l and h i s c o l l e a g u e s f i r s t showed t h a t i n h i b i t i n g m o n o c y t e esterase with b i s nitrophenyl phosphate r e s u l t e d i n a s i g n i f i c a n t suppression of the capacity of t h i s c e l l t o form conjugates with a target cell, the K562 erythroleukemic c e l l ( 1 2 ) . C o n s e q u e n t l y , t h e monocyte f a i l e d t o l y s e t h e s e same t a r g e t c e l l s ( 1 2 ) . Although the d o s e s o f t h e b i s n i t r o p h e n y l p h o s p h a t e i n t h i s s t u d y were r e l a t i v e l y h i g h and t h e decrement i n c o n j u g a t e formation and l y s i s was n o t s t r i k i n g , t h e s e d a t a p r o v i d e d t h e f i r s t e v i d e n c e t h a t o r g a n o p h o s p h o r u s compounds c o u l d i m p a i r t h e monocyte's a b i l i t y t o c a r r y o u t c y t o t o x i c r e a c t i o n s a g a i n s t t h e tumor c e l l l i n e , K 5 6 2 .
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O r g a n o p h o s p h o r u s S u p p r e s s i o n o f NK C e l l A c t i v i t y . Both cytotoxic Τ lymphocytes and n a t u r a l killer cells are essential f o r cell-mediated immunity, t h e e l i m i n a t i o n o f v i r u s - i n f e c t e d c e l l s , a n d p r o v i d e immune s u r v e i l l a n c e f o r premalignant c e l l s . We p o s t u l a t e d t h a t o r g a n o p h o s p h a t e i n d u c e d d e f e c t s i n human immune s u r v e i l l a n c e s y s t e m s m i g h t provide a possible link between immune dysfunction, l y m p h o p r o l i f e r a t i o n , and lymphomagenesis. I n i t i a l studies by o u r g r o u p showed t h a t n o n - p e s t i c i d e organophosphates used i n t h e manufacture o f p l a s t i c s r e s u l t e d i n both i n v i v o and i n v i t r o i n h i b i t i o n o f monocyte e s t e r a s e s ( 1 7 ) . Subsequently, we showed that some organophosphorus compounds s u p p r e s s e d N K c e l l a c t i v i t y ( F i g . 1 ) . In these studies, p e r i p h e r a l blood l y m p h o c y t e s were i s o l a t e d b y s t a n d a r d p r o c e d u r e s and i n c u b a t e d f o r one h o u r i n t h e p r e s e n c e o r a b s e n c e o f o r g a n o p h o s p h o r u s compounds. K562 c e l l s were l a b e l e d f o r 1 h o u r w i t h C r and graded doses o f organophosphorus-treated or control peripheral blood l y m p h o c y t e s were e x p o s e d t o t a r g e t c e l l s f o r f o u r h o u r s . Target c e l l l y s i s was t h e n measure b y q u a n t i f y i n g t h e r e l e a s e o f r a d i o l a b e l e d chromium a f t e r c o r r e c t i o n s were made f o r n o n s p e c i f i c r e l e a s e . A s c a n be s e e n i n t h i s f i g u r e , t e t r a - o - c r e s y l p i p e r a z i n y l d i p h o s p h a m i d a t e (TCPD), t r i p h e n y l phosphate (TPP), and t e r t i a r y b u t y l t r i p h e n y l p h o s p h a t e (TBTPP) a t 2 0 uM c o n c e n t r a t i o n s significantly suppressed N K c e l l a c t i v i t y when compared t o c o n t r o l s containing no organophosphates (p
