Biotransformation of Organophosphorus Insecticides in Mammals

Chapter 3

Biotransformation of Organophosphorus Insecticides in Mammals

Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: March 21, 1991 | doi: 10.1021/bk-1991-0459.ch003

Relationship to Acute Toxicity

Janice E. Chambers1 and Howard W. Chambers2 Departments of Biological Sciences1 and Entomology2, Mississippi State University, Mississippi State, MS 39762 The organophosphorus insecticides or their activated metabolites are potent anticholinesterases which display a wide range of acute toxicity levels in mammals. While target site sensitivity does not effectively explain the acute toxicity levels, the magnitude of various aspects of the parent insecticide's biotransformation, including both bioactivation and detoxication pathways, can predict an insecticide's overall toxicity level. Thus, biotransformation i s a c r i t i c a l factor in determining mammalian sensitivity to the acute lethality of organophosphorus insecticides. The organophosphorus (OP) i n s e c t i c i d e s are a very e f f e c t i v e and widely used group of pesticides of both h i s t o r i c a l and current s i g n i f i c a n c e . Their toxicology i s important because of the l i k e l y accidental exposures to humans and other mammals during t h i s widespread use. This paper describes the common biotransformation routes f o r OP i n s e c t i c i d e s and assesses t h e i r s i g n i f i c a n c e i n determining the acute t o x i c i t y l e v e l displayed by the i n s e c t i c i d e . Organophosphorus I n s e c t i c i d e T o x i c i t y The organophosphorus i n s e c t i c i d e s are potent neurοtoxicants i n vertebrates and invertebrates. The mechanism of acute t o x i c i t y i s c u r r e n t l y accepted to be the i n h i b i t i o n of the enzyme acetylcholinesterase (AChE) i n nervous t i s s u e (1). AChE i s responsible f o r the hydrolysis of the important neurotransmitter acetylcholine which transmits information across c h o l i n e r g i c synapses. Cholinergic synapses i n vertebrates are widely d i s t r i b u t e d , occurring w i t h i n the somatic nervous system which activates s k e l e t a l muscles, w i t h i n the autonomic nervous system (both parasympathetic and sympathetic d i v i s i o n s ) which innervates

(X)97^156/9iy0459-0032$06.00/0 © 1991 American Chemical Society In Pesticide Transformation Products; Somasundaram, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


3.

CHAMBERS AND CHAMBERS

Organophosphorus Insecticides in Mammals

smooth muscles, cardiac muscle and numerous glands, and also a t various locations i n the c e n t r a l nervous system. Therefore a l t e r a t i o n s i n the cholinergic pathways can cause widespread disturbances and diverse symptoms of i n t o x i c a t i o n . This hydrolysis by AChE r a p i d l y inactivates the acetylcholine, leading to the transient e f f e c t of the neurotransmitter during normal nervous system function. When AChE i s i n h i b i t e d , the neurotransmitter accumulates, which r e s u l t s i n h y p e r a c t i v i t y w i t h i n c h o l i n e r g i c pathways and serious imbalances w i t h i n the nervous system. I f doses are high, the main signs of poisoning are parasympathomimetic e f f e c t s such as d i l a t e d p u p i l s , headache and b l u r r e d v i s i o n , and n i c o t i n i c e f f e c t s such as tremors and, i n some cases, convulsions. Respiratory system f a i l u r e i s usually the cause of death i n l e t h a l i n t o x i c a t i o n s i n mammals, r e s u l t i n g from four f a c t o r s : bronchoconstriction, an increase i n bronchiolar secretion of mucus, p a r a l y s i s of the respiratory muscles and an i n h i b i t i o n o f the r e s p i r a t o r y c o n t r o l centers i n the medulla oblongata/pons regions of the b r a i n . Long term e f f e c t s on memory and other cognitive s k i l l s have been reported i n humans as a r e s u l t of s i n g l e high-dose poisonings with an OP i n s e c t i c i d e (2). Thus, numerous target areas e x i s t throughout the c e n t r a l and peripheral nervous systems o f vertebrate non-target organisms, including humans. The threat of l e t h a l or sub-lethal but severe poisonings i s of great concern f o r both humans i n occupational settings and f o r other non-target organisms which might receive inadvertent exposures. I t has been estimated that 500,000 humans are poisoned annually by pesticides throughout the world and that over 20,000 of these are f a t a l . Some 6,000-10,000 poisonings occur i n the United States each year which r e s u l t i n 3,000 farm worker h o s p i t a l i z a t i o n s and 50-60 deaths. OP i n s e c t i c i d e s comprise a large f r a c t i o n of the agents involved i n these poisonings (3). I n addition to the deaths, the long-term sequelae i n survivors of these i n t o x i c a t i o n s are also of continuing concern. Thus, the OP i n s e c t i c i d e s continue to be a health threat from the standpoint of t h e i r acute t o x i c i t y . Over h a l f of the registered OP i n s e c t i c i d e s have r a t o r a l LD 's l e s s than 50 mg/kg p l a c i n g them i n the most t o x i c category i n the U.S. Environmental Protection Agency's c l a s s i f i c a t i o n scheme. A number o f the OP i n s e c t i c i d e s are considerably l e s s t o x i c , however, and thus would constitute a l e s s e r threat of l e t h a l i t y to non-target populations. The acute o r a l LD 's to r a t s range from 3-15 mg/kg f o r such h i g h l y t o x i c i n s e c t i c i d e s as d i s u l f o t o n , parathion and azinphos-methyl to 1,000-13,000 mg/kg f o r such i n s e c t i c i d e s as malathion and temephos (4,5). This wide range of t o x i c i t i e s suggests that there are important differences i n the OP i n s e c t i c i d e s with respect to metabolism and d i s p o s i t i o n and/or target s e n s i t i v i t y which can explain t h i s range. Such differences could be exploited i n the development o f safer i n s e c t i c i d e s which would pose l e s s o f a threat to non-target species. 50

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In Pesticide Transformation Products; Somasundaram, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

34

PESTICIDE TRANSFORMATION PRODUCTS


Xenobiotic Metabolism The reactions of xenobiotic metabolism have been c l a s s i f i e d i n t o two general categories, c a l l e d Phase I and Phase I I (6). The Phase I reactions are involved i n p l a c i n g a more polar and r e a c t i v e group into the xenobiotic to make the compound l e s s l i p o p h i l i c and more l i k e l y to be excreted. The enzymes involved are p r i m a r i l y monooxygenases (both cytochrome Ρ-450-dependent and f l a v i n monooxygenases), reductases and hydrolases, and they catalyze a v a r i e t y of oxidations ( f o r example, hydroxylations, epoxidations or dealkylations) reductions and hydrolyses ( f o r example, ester and amide cleavages). The oxidations and reductions are energyexpensive r e q u i r i n g reducing equivalents, u s u a l l y i n the form o f NADPH, to proceed. Although these reactions are frequently detoxications, they are also the reactions most l i k e l y to form reactive intermediates ( f o r example, epoxides or oxons) and therefore can be a c t i v a t i o n reactions. These b i o a c t i v a t i o n s , then, are l a r g e l y responsible f o r the t o x i c i t y displayed f o l l o w i n g exposure to the parent xenobiotic. However, i t should be borne i n mind that these reactive metabolites w i l l be chemically l a b i l e and would be expected to undergo chemical reactions with numerous nontarget molecules. Therefore, i t should be expected that a r e l a t i v e l y small proportion of the a c t i v a t e d metabolite formed would react with the molecular target and induce t o x i c i t y . The Phase I I reactions are conjugation reactions involved with p l a c i n g a h i g h l y polar or charged group on the xenobiotic/metabolite which renders i t r e a d i l y excretable i n e i t h e r the urine or the b i l e of vertebrates. The group added i s t y p i c a l l y a sugar (glucuronic a c i d ) , s u l f a t e or an amino acid/amino a c i d d e r i v a t i v e . With few known exceptions, the Phase I I reactions are detoxications. Phase I I reactions are also energy-expensive, r e q u i r i n g energy r i c h cofactors such as u r i d i n e diphosphoglucuronic a c i d (UDPGA), phosphoadenosinephosphosulfate (PAPS) or the t r i p e p t i d e glutathione (GSH; glutamic acid-cysteine-glycine) to proceed. The animal can r i d i t s e l f of the conjugates r e s u l t i n g from Phase I I reactions, e s p e c i a l l y small ones, quite r e a d i l y by excretion i n the urine. Excretion of the conjugates from the l i v e r i n t o the b i l e i s also a l i k e l y route, e s p e c i a l l y f o r l a r g e r conjugates. However, these conjugates can also be hydrolyzed by the i n t e s t i n a l m i c r o f l o r a and the aglycone can be reabsorbed v i a the hepatic p o r t a l c i r c u l a t i o n back i n t o the l i v e r . This enterohepatic c i r c u l a t i o n allows some p o t e n t i a l r e c y c l i n g of the compound. I f the compound being recycled i s a t o x i c one, then the animal remains at r i s k . A number of elements w i t h i n the Phase I and Phase I I reactions bear s t r i k i n g s i m i l a r i t y i f not i d e n t i t y to reactions i n v o l v i n g the endogenous s t e r o i d hormones. For example, cytochrome P-450mediated hydroxylations and conjugations to glucuronic a c i d are common reactions f o r many of the s t e r o i d s . Also, the enterohepatic c i r c u l a t i o n r o u t i n e l y reabsorbs the vast majority of b i l e s a l t s so that d a i l y synthesis need be minimal. Therefore, many of the xenobiotic metabolizing reactions share a commonality with metabolism of endogenous compounds. By t h i s token, xenobiotic



3. CHAMBERS AND CHAMBERS

Organophosphorus Insecticides in Mammal

metabolism could compete with normal biochemistry, and has the p o t e n t i a l of i n t e r f e r i n g with the metabolism and d i s p o s i t i o n of endogenous biochemicals. Generally, the highest s p e c i f i c a c t i v i t i e s of the xenobiotic metabolizing enzymes occur i n the l i v e r w i t h considerably lower a c t i v i t i e s i n other tissues. I n the laboratory r a t , the enzymes sometimes display sex differences and developmental differences, with higher a c t i v i t i e s of some enzymes i n male l i v e r s than female l i v e r s , and higher hepatic a c t i v i t i e s i n adults than immatures. Also, several of the enzymes can be induced to higher s p e c i f i c a c t i v i t i e s following i n vivo exposures to selected chemicals such as the barbiturate drug phénobarbital, male sex hormones, p o l y c y c l i c aromatic hydrocarbons ( f o r example, 3-methylcholanthrene or 0-naphthoflavone), or ethanol. Numerous reactions may be a v a i l a b l e f o r any given xenobiotic, and a v a r i e t y of exogenous and endogenous factors may influence the l i k e l i h o o d of those reactions taking place. Understanding the r o l e s of metabolism and d i s p o s i t i o n i n the t o x i c i t y of any p a r t i c u l a r xenobiotic w i l l require concurrent consideration of numerous f a c t o r s . Organophosphorus I n s e c t i c i d e Metabolism Many of the OP i n s e c t i c i d e s are phosphorothionates, which are characterized by one -S and three -OR groups bonded to the c e n t r a l phosphorus atom (Figure 1). The phosphorothionate molecule, such as parathion, i s inherently a weak anticholinesterase, with an I i n the range of 10~ M (7). Thus the phosphorothionate molecule would not be expected to be p a r t i c u l a r l y t o x i c i t s e l f . However, the phosphorothionates can be b i o a c t i v a t e d very e f f e c t i v e l y by the cytochrome Ρ-450-dependent monooxygenases to h i g h l y reactive metabolites, the phosphates or oxons. This b i o a c t i v a t i o n , c a l l e d a desulfuration reaction, i s hypothesized to occur i s a r e s u l t of an attack on the phosphorus by oxygen and the formation of an unstable phosphooxathiiran intermediate, which then undergoes a release of s u l f u r and the formation of the oxon (Figure 1, pathway l a ) (8,9). The s u l f u r released by the desulfuration r e a c t i o n i s h i g h l y reactive and can destroy nearby molecules, such as the cytochrome P-450 which produced i t (8,10). The oxon i s about 3 orders of magnitude more potent as an anticholinesterase than i t s corresponding phosphorothionate (7). I t i n h i b i t s the AChE by phosphorylating the serine hydroxyl present at the active s i t e ; t h i s phosphorylation i s extremely p e r s i s t e n t , with i n h i b i t i o n l a s t i n g hours to days (11). For some organophosphates the persistence i s p a r t i a l l y a r e s u l t of a process known as "aging", a poorly understood d e a l k y l a t i o n which leaves the phosphorylated AChE charged a t p h y s i o l o g i c a l pH. This phosphorylated and aged AChE cannot be reactivated by e i t h e r spontaneous hydrolysis or the a c t i v i t y of therapeutic oximes (such as 2-PAM), and, therefore, i s permanently deactivated. We have found r a t b r a i n AChE I ' s ranging from 1.8 nM f o r c h l o r p y r i f o s methyl-oxon to 89.3 nM f o r methyl paraoxon (12). The oxons are also potent i n h i b i t o r s of other serine esterases which are l e s s c r i t i c a l to s u r v i v a l such as a l i e s t e r a s e s (carboxylesterases) (12), 5 0

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Biotransformation of Organophosphorus Insecticides in Mammals

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