Biological and nonbiological modifications of organophosphorus compounds*

Bull. Org. mond. Sante 1 Blull. Wld Hlth Org j1971, 44, 133-150 Biological and Nonbiological Modifications of Organophosphorus Compounds* W. C. DAUTERMAN 1 The general types of biological reaction that are most prominent in the modification of organophosphorus compounds involve the mixed-function oxidases, hydrolases, or transferases. In certain cases, more than one of these reactions may be involved at the same site on the pesticide molecule. Examples of various organophosphorus pesticides that are altered by oxidation, hydrolysis, alkyl- or aryl-group transfer, reduction, and conjugation are discussed. The increase or decrease in toxicity of a pesticide that can result from biological modification is emphasized. Non-biological transformations of organophosphorus compounds involve the effect ou the compounds of such factors as light, air, temperature, and solvent. These factors are discussed with special emphasis on desulfuration, rearrangement, and oxidation. Increasing emphasis is being given to research on, and the development of, pesticides that will be effective for the control of insects but that will minimize the " off-target " effects that are characteris- tic of the chlorinated hydrocarbons. This trend involves both the more effective use of currently avail- able nonpersistent pesticides and the development of new compounds. The organophosphorus com- pounds are a chemical group of insecticides that are characterized by their nonpersistence in the environment as well as by their wide spectrum of activity. In order to utilize more effectively the inhibitory power of the phosphate moiety in the development of new compounds, a knowledge of the metabolism and fate of organophosphorus esters in various biological systems is necessary. This information is important for an understanding of the processes of intoxication, detoxification, and resistance. Information on the chemical behaviour and reactions of organophosphorus compounds in a nonliving system, the environment, is essential for the assessment of the potential hazards they present to human health, their longevity in the environment, and their efficacy for the control of insects. The metabolism of organophosphorus compounds by plants and animals, with special emphasis on isolated in vitro systems, is discussed in the first section of this paper. The second section is devoted to environmental factors such as light, temperature, air, and solvent and their effects on nonbiological modifications of organophosphorus compounds. BIOLOGICAL MODIFICATIONS The metabolism of organophosphorus insecticides in plants and animals has been the subject of several recent reviews (O'Brien, 1967; Hodgson, 1968; Casida & Lykken, 1969; Lykken & Casida, 1969; Fukuto & Metcalf, 1969; Menzie, 1969; Menzer & * This study was supported in part by Grant ES-00044 from the US Public Health Service. It is paper No. 3391 of the Journal Series of the North Carolina State University Experiment Station, Raleigh, N.C., USA. 1 Associate Professor, Department of Entomology, North Carolina State University, Raleigh, N.C., USA. Dauterman, 1970; Bull, 1970). This portion of the review summarizes and compares the importance of the various biological processes by which plants and animals modify the chemical structure of insecticidally active organophosphorus compounds. Since the literature is so complex, by virtue of the large number of compounds, specific examples have been chosen to illustrate the various types of reaction. Organophosphorus insecticides are metabolized by four general classes of reaction: (1) reactions 2622 - 133 - 134 W. C. DAUTERMAN Fig. 1 Oxidative desulfuration of parathion s 0 (C2H50)2 P °K/ Noz H (C2 H5 0)2 P 0 NO2 + So402 parathion microsomes paraoxon Dahm (1970). involving the mixed-function oxidases, (2) reactions involving hydrolases, (3) transferase reactions, and (4) miscellaneous reactions. In certain cases, more than one of these reactions may be involved at a common site on the organophosphorus molecule. Therefore the identification of the product is not necessarily indicative of either the type of biological alteration or the route. MIXED-FUNCTION OXIDASES The mixed-function oxidases are a group of enzymes that are important in the metabolism of xenobiotics in mammals (Gillette et al., 1969) and insects (Hodgson & Plapp, 1970). The enzyme systems are associated with the post-mitochrondrial supernatant of plant or animal tissue homogenates and are derived from the endoplasmic reticulum. The particulate enzyme system has an essential requirement for NADPH and molecular oxygen in order to modify a xenobiotic. Oxidative desulfuration Phosphorothioate and phosphorodithioate esters are poor inhibitors of cholinesterases unless the compounds are oxidatively desulfurated. This generally results in an increase in inhibition of the target enzyme as well as in toxicity to the organism (Heath, 1961; O'Brien, 1960). The in vivo activation of parathion to paraoxon has been demonstrated in both insects and mammals (Gage, 1953; Metcalf & March, 1953). Evidence for oxidative desulfu- ration has been demonstrated in both plants and animals for a wide variety of insecticides: parathion- methyl (Hollingworth et al., 1967), malathion (O'Brien, 1957), dimethoate (Dauterman et al., 1960; Brady & Arthur, 1963), fenitrothion (Hollingworth et al., 1967), and Supracide t (Bull, 1970). In vitro t Names against which this symbol appears are identified in the Glossary on pages 445-446. studies with microsomal preparations from cock- roach fat body (Nakatsugawa & Dahm, 1965), rat liver (Neal, 1967a, 1967b; Nakatsugawa & Dahm, 1967), and housefly abdomen (El Bashir & Oppenoorth, 1969) demonstrated that desulfuration of parathion was accomplished by a mixed-function oxidase system in the presence of NADPH and molecular oxygen (Fig. 1). The sulfur atom is removed during the reaction in vitro and bound to the microsomes and the detached sulfur is found in vivo as inorganic sulfate (Nakatsugawa & Dahm, 1967; Nakatsugawa et al., 1969b). It seems reason- able to assume that the mixed-function oxidases are responsible for the in vivo desulfuration of phosphorothioates in most other biological entities, although this reaction has not been demonstrated in vitro in plants. Oxidative N-dealkylation The oxidative N-dealkylation by the mixed- function oxidases of many nitrogen-containing xenobiotics in plants and animals is well documented (Frear et al., 1969; Gillette et al., 1969; Casida & Lykken, 1969; Menzer & Dauterman, 1970). A typical example of this reaction is the N-demethyl- ation of dicrotophos and moncrotophos (Fig. 2). This reaction has been demonstrated to occur in plants, mammals, and insects (Menzer & Casida, 1965; Bull & Lindquist, 1964, 1966; Lindquist & Bull, 1967). Removal of the N-methyl groups pro- ceeds by the formation of relatively stable N-hydroxy- methyl intermediates followed by the elimination of formaldehyde. The removal or modification of the N-substituents can result in an increase, a de- crease, or little change in toxicity. N-demethylation has been reported to occur with schradan (Spencer et al., 1967), dimethoate (Sanderson & Edson, 1964; Lucier & Menzer, 1968, 1970), and famphur t (O'Brien et al., 1965). N-deethylation has been reported to occur with phosphamidon (Bull et al., 1967; Clemmons & Menzer, 1968). With phospha- 135BIOLOGICAL AND NONBIOLOGICAL MODIFICATIONS OF OP COMPOUNDS Fig. 2 Oxidative N-dealkylation of dicrotophos * 0 0 ,. R =(CH30)2 Po c = CH C- CH3 N -h ydroxymethyl dicrotophos dicrotophos CH3 CH 3/ / RH-N R-N CH3 CH20H N-hydroxymethyl monocrotophos monocrotophos CH3/ R-N - H 'H2 OH R N H L D50(mg/kg) mouse P I 50 Fly ChE 18 7.07.2 8 6.8 12 3 6.9 6.5 * Menzer & Casida (1965). midon the N-ethyl groups are sequentially hydroxy- lated on the a-carbon and then eliminated as acetaldehyde. In all cases studied the relative con- centrations of the products formed were usually small but all the metabolites had anticholinesterase properties. Oxidative 0-dealkylation Cleavage of organophosphorus triesters to diesters results in relatively nontoxic metabolites. Since the isolation and identification by Plapp & Casida (1958) of metabolites that were the result of alkyl-phosphate cleavage, this reaction has generally been regarded as being of a hydrolytic nature, catalysed by a phosphatase enzyme. Only recently has in vitro evi- dence been presented to indicate that other mecha- nisms are also responsible for the formation of dealkylated metabolites (see also the sections on hydrolases and transferases, below). Donninger et al. (1967) demonstrated that chlorfenvinphos was oxidatively O-deethylated by liver microsomes in the presence of NADPH and oxygen (Fig. 3). The reaction resulted in the isolation of deethyl chlorfenvinphos and acetaldehyde and it was postu- lated that an unstable 1-hydroxyethyl intermediate was formed, which subsequently broke down. Oxi- dative demethylation has also been demonstrated to occur with Gardona,t the dimethyl analogue Fig. 3 Oxidative O-deethylation of chlorfenvinphos * 1~~~~~~~0 (C2H50)2 P 0 C C V H C Cl L OH CH3CH O 0C \POC acl CH3CH2 HCCI postu l a ted intermediate HO 0 C0 O I'loP0COC CH+C3CF C2H5 0 "c H C C * Donninger et al. (1 967). amide RN H2 _j W. C. DAUTERMAN Fig. 4 Oxidative dearylation of diazinon * I~~~~ s (C2H5 °)2 P 0 CH(CH3)2 CH3 0 (C2H50)2 P 0 N> CH (CH3)2 OH3 *Yang et al. (1971). of chlorfenvinphos (Hutson et al., 1968a). Further work by Hutson et al. (1968b) indicates that oxidative O-dealkylation occurs with dimethyl, diethyl, diiso- propyl, and di-n-butyl analogues of 1-naphthyl phosphate. Lewis (1969) reported that diazoxon (but not diazinon) was deethylated by a microsomal preparation from a resistant strain of the housefly in the presence of NADPH and oxygen. Other studies have failed to demonstrate a role of the mixed-function oxidases in the O-dealkylation of phosphorothioates (Hollingworth, 1969; Naka- tsugawa, Tolman & Dahm, 1969a; Yang et al., 1971a, 1971b). If oxidative O-dealkylation is an important route of detoxification, it may be pri- marily involved with the phosphates rather than the phosphorothioates. Oxidative dearylation It is generally agreed that disruption of the acid- anhydride bond of organophosphorus insecticides is the most important mechanism for in vivo detoxifi- cation. For some time this reaction was regarded as being catalysed exclusively by hydrolases (Al- dridge, 1953; Augustinsson & Heimburger, 1954; O'Brien, 1960). While it is true that these enzymes are important for the degradation of some organo- phosphates, recent in vitro studies have also demon- strated that a NADPH-dependent microsomal oxi- dase system from mammalian liver and housefly abdomens was able to cleave the aryl-phosphate bond (Fukunaga, 1967; Neal, 1967a, 1967b; Naka- tsugawa & Dahm, 1967; El Bashir & Oppenoorth, 1969; Lewis, 1969; Yang et al., 1971a, 1971b). Studies with parathion and diazinon resulted in S NADPH 11 02 (C2H50)2 P 0 H microsomes NADPH ,, o (C2H50)2 P 0H 02 House f y abdomen microsomes the isolation of diethyl phosphorothioic acid and some diethyl phosphoric acid, which is formed by oxidative desulfuration followed by the breakdown of the oxons (Fig. 4). The mixed-function oxidases from rat liver are able oxidatively to degrade diazinon but not diazoxon (Yang et al., 1971a), whereas the microsomal system from houseflies is able oxidatively to dearylate both diazinon and diazoxon, although diazinon is the preferred substrate (Lewis, 1969; Yang et al., 1971b). In studies by Nakatsu- gawa et al. (1968), eight substituted and unsubsti- tuted phosphorothioate analogues containing p- nitrophenol were oxidatively dearylated to p-nitro- phenol by microsomes from rat liver, rabbit liver, and housefly abdomens. From the available in- formation it would appear that the cleavage of an aryl-phosphate bond by oxidative dearylation in mammals and insects is an important degradative reaction, especially if the organophosphate cointains a thiono sulfur atom. There is no evidence that this reaction occurs in plants. Thioether oxidation The thioether oxidation of certain organophos- phates has been demonstrated in vivo in plants, mammals, and insects. This reaction involves the conversion of the thioether moiety to the respective sulfoxide and sulfone and has been demonstrated with demeton (Fukuto et al., 1955; Fukuto et al., 1956), phorate (Bowman & Casida, 1957, Metcalf et al. 1957), disulfoton (Metcalf et al., 1957; Bull, 1965), and fensulfothion (Benjamini et al., 1959a, 1959b). Thioether oxidation is exemplified by the reactions in Fig. 5, which show the oxidation of 136 BIOLOGICAL AND NONBIOLOGICAL MODIFICATIONS OF OP COMPOUNDS Fig. 5 Thioether oxidation of disulfoton * s (C2H5 0)2 P S CH2 CH2 S C2 H 5 I50( x 10-4M) S 0 (C2H50)2 P S CH2 CH2 S C2 H5 150(7 x 10-5M) ,, 0 (C2H50)2 P S CH2 CH2 S C2 H5 I50 (3.5 x 10-6M) Metcalf et al. (1 957). the thioether moiety of disulfoton to the corres- als ponding sulfoxide and sulfone as well as the desul- (19 furation. The I5, values for the various metabolites ph to fly-head ChE are given. In general the initial act oxidation of most thioether-containing organo- in 1 phosphates results in a rapid conversion to the Th sulfoxide and then a slow conversion to the sulfone. int This results in the accumulation of the sulfoxide, Sp( which is probably the principal toxicant. With TC disulfoton, thioether oxidation resulted in an in- fox crease in the '50 value, but this activation was not as atc great as that resulting from oxidative desulfuration mc (Metcalf et al., 1957). sal At present, no in vitro data are available on rea the enzymes or enzyme systems responsible for the acd oxidative reactions with thioether-containing organo- T( phosphates. However, in vitro studies with carba- mates (Tsukamato & Casida, 1967a, 1967b; And- grm rawes et al., 1967) and sulfur compounds (Parke, grc 1968; Lee et al., 1970) indicate that the microsomal N2 oxidases are responsible for the conversion of the It thioether moiety to the sulfoxide and also the sulfone. sib It is reasonable to assume that the mixed-function Th oxidases are also involved in the oxidation of mc thioether-containing organophosphorus insecticides. thi Side-group oxidationt ri The oxidation of aliphatic ring substituents of the certain organophosphorus compounds is probably 0 0 (C2H5 0)2 P S CH2 CH2 S C2H5 150(1.5x 10`6M) - (C2H5 0)2 P S CH2 CH2 S C2H5 150 (6 x 10-7M) o the result of microsomal enzymes. Eto et al. 962), working on the metabolism of tri-o-tolyl osphate (TOCP), obtained evidence that three :ive metabolites were formed in vivo by rats and vitro by rat liver microsomes fortified withNADPH. we structures of the metabolites isolated from rat estines were characterized by chemical tests, IR ectra, and synthesis. The findings indicated that )CP was hydroxylated at the o-methyl group to rm hydroxymethyl TOCP and that this intermedi- was then cyclized (Eto et al., 1967) to form cyclic athylene-o-phenylene o-tolyl phosphate [o-tolyl ligenin phosphate] (Fig. 6, reaction a). This action resulted in an increase in anticholinesterase tivity to a level 12 000 000 times that of the parent )CP. Douch et al. (1968) reported that the 3-methyl)up of fenitrothion was oxidized to a carboxyl)up by mouse liver microsomes fortified withkDP and glucose-6-phosphate (Fig. 6, reaction b). was suggested that this reaction might be respon-ile for the low mammalian toxicity of fenitrothion.ie reported metabolite was identified by indirect ethods and further work is needed to substantiate is identification. Another example of in vivo oxidation of aliphaticig substituents has recently been reported with identification of hydroxy diazinon (Pardue et 1970). This metabolite was isolated from field- 137 W. C. DAUTERMAN Fig. 6 Side-group oxidation * CH3 ° CH3 a. do- p- o- 0 C,~, H3 TOCP b. (CH30)2P OC NO2 CH3 fenitrothion C. S CH3N ( C2H5 0)2 P-OrfI-CH -KN CH, CH3 0 CH3 CH3 o w- P~~~~~~~-0~- 0 O CH2fCH2 OH hydroxymethyl TOCP (CH30)2P 0Q NO2 COOH S CH3 N - (C2 H50 )2P-O0 I- - Oii .N CH3 CH3 CH3 diazinon hydroxy-diazinon CH3 CH3 HO N C HO N C-OH CH 2 CH3 References: reaction a, Eto et al. (1962); reaction b, Douch et al. (1968); reaction c, Pardue et al. (1970); reaction d, Mucke et al. (1 970). treated kale and is the result of oxidation at the tertiary carbon of the isopropyl group (Fig. 6, reaction c). In another in vivo study with diazinon in rats, Mucke et al. (1970) found that the pyrimidine moiety was oxidized at both the primary and the tertiary carbon atoms of the isopropyl group (Fig. 6, reaction d). These metabolites were isolated from rat urine and it was not established whether the pyrimidinol metabolites were formed before or after cleavage of the pyrimidine-phosphate linkage. HYDROLASES Most organophosphorus compounds are degraded to some extent by various hydrolases (Heath, 1961; O'Brien, 1960, 1967). These enzymes may hydro- lytically attack the phosphorus ester bond or the anhydride bond as well as an ester or amide bond in the leaving group. The disruption of any of these bonds to form a diester or a monocarboxylic acid is probably one of the most important mechanisms of inactivation and detoxification. Hydrolases res- ponsible for organophosphate hydrolysis are present in many biological systems and are widely distributed in different organs and tissues, as well as being found in both soluble and subcellular fractions. Triester hydrolysis The first enzymatic hydrolysis of an organophos- phorus compound to be reported was that of DFP t (Mazur, 1946). The findings indicated that DFP and various analogues were hydrolysed at the P-F bond by various rabbit tissue homogenates. Evidence for the hydrolysis of the acid anhydride bond by arylesterases has subsequently been demon- strated both with mammalian and insect tissue homogenates and with partially purified enzymes with DFP (Mounter, 1956; Cohen & Warringa, 1957), tabun t (Augustinsson & Heimburger, 1954), sarin t (Adie, 1956), paraoxon and analogues (Ald- ridge, 1953; Main, 1960a, 1960b; Jarczyk, 1966), and diazinon (Matsumura & Hogendijk, 1964a). With all the compounds mentioned above the hydrolysis occurred at P-O-C, P-CN, or P-F bonds and resulted in relatively nontoxic products (Fig. 7). At present most of the available evidence indicates 138 BIOLOGICAL AND NONBIOLOGICAL MODIFICATIONS OF OP COMPOUNDS Fig. 7 Triester hydrolysis 0 a. (i-C3H70)2 P-F DFP C2H50 0 b. / P - C N (CH3)2 N tabun 0 C. (C2H50)2 P0; N paraoxon CH30 0 d. 'P-OCH = CC C H30 d ichlorvos 0 (i--C3H70 )2 P OH + F C2H5O -0 °_ 0-- zP OH + CN (CH3 )2N 02 - (C2H50)2P OH + O\6_/ CH30 0 '12 - P- OCH = CC 12 H 0 demethyl dichlorvos that the phosphates are probably the preferred substrates for enzymatic hydrolysis rather than the phosphorothioate analogues. O-dealkylation is another possible mechanism by which organophosphorus triesters may be hydro- lysed. Hodgson & Casida (1962) reported that di- chlorvos was O-demethylated by a soluble enzyme fraction from rat liver (Fig. 7, reaction d). Their findings indicated that this reaction was not associ- ated with the microsomal fraction and that NADPH was not required; therefore, it was not the result of oxidative O-dealkylation. However, endogenous glutathione might have been present in the soluble fraction in sufficient quantities to mediate the 0- dealkylation (see glutathione S-alkyl transferase, below). Recently Nolan & O'Brien (1970) reported that 3H-paraoxon was 0-dealkylated in houseflies, resulting in the formation of labelled ethanol and derivatives. Since ethanol was found rather than the aldehyde or S-ethyl glutathione, one may con- clude that this reaction may have been mediated by a hydrolase. In general very little information is available on this reaction and further work is needed to clarify its importance in vivo. Carboxylesterases Carboxylesterases, or carboxylic-ester hydrolases, have been shown to be important in the detoxification of organophosphorus compounds such as malathion and acethion t (O'Brien, 1960). This detoxification reaction involves the hydrolysis of a carboxyester linkage, resulting in a nontoxic ionic product, the mono-acid of malathion (Cook & Yip, 1958) (Fig. 8, reaction a). The enzyme is widely distributed in mammals and has been found in the liver, kidney, serum, lung, spleen, and ileum of the rat (Seume & O'Brien, 1960), the mouse, the guinea-pig, and the dog (Murphy & DuBois, 1957). In each of the insect species studied the activity of the enzyme was low in (or the enzyme was absent from) susceptible insects (Kojima, 1961), partly explaining the selective toxicity of the compound. This hydrolase is present in certain malathion-resistant insects and it is reasonable to assume that resistance to malathion is at least partly due to carboxylesterase activity (Dauterman & Matsumura, 1962; Matsumura & Brown, 1963; Matsumura & Hogendijk, 1964b). Main & Braid (1962) partially purified from rat liver an enzyme that hydrolysed malathion. This 139 W. C. DAUTERMAN Fig. 8 Hydrolases of functional groups s 0 s 0 crboxylesterasea. (CH3O)2 P S CH COC2 H5 " (CH3O)2 P S CH C OH CH2COC2H5 CH2C 0C2H5 0 0 b. malathion a - monoaci d S 0 S 0 11 01 ~~~amidase-JCH3O)2 P S CH2C NHCH3 - (CH30)2 P S CH2 C OH dimethoate dimethoate acid enzyme cleaved only one of the carbethoxy groups and was characterized as a carboxylesterase. Summarizing some of the findings on this enzyme from our laboratory, only one of the carbethoxy groups is hydrolysed in vivo by the rat and by the purified enzyme and the resulting metabolite, as identified by NMR spectroscopy, is the a-mono- acid (Chen et al., 1969) (Fig. 8a). This enzyme is unable to hydrolyse the second carbethoxy group and can attack only un-ionized substrates. Long- chained carbalkoxy compounds are better substrates than carbmethoxy compounds (Dauterman & Main, 1966). Malaoxon is both a substrate and an inhibitor of the carboxylesterase (Main & Dauterman, 1967). The enzyme is unable to hydrolyse the carboxyamide bond of compounds like dimethoate. Amidases Carboxyamidases have been implicated in the metabolism in plants and animals of a number of organophosphorus insecticides containing car- boxyamide groups-e.g., dimethoate, dicrotophos, monocrotophos, and phosphamidon (Menzie, 1969). In each case a carboxylic acid metabolite was identi- fied as an alteration product (Fig. 8b). Since these compounds were either substituted N.N-dimethyl, N-methyl, or N,N-diethyl amides, one must assume that carboxyamidases are able to hydrolyse various substituted N-alkyl groups. Most of the in vitro studies on the amidases have been conducted with mammalian tissues being the source of the enzyme. In mammals the enzyme appears to be concentrated in the liver (Uchida et al., 1964) and is primarily associated with the microso- mal fraction (Uchida & O'Brien, 1967). The latter workers found a correlation between the in vitro degradation of dimethoate by liver homogenates from six vertebrates with the in vivo toxicity for each species. With this in mind we undertook an investigation of some of the properties of the mammalian amidase. Preliminary findings indicated that the enzyme could be rendered soluble by repeated freezing and thawing of sheep liver microsomes. Utilizing rapid-flow hydroxylapatite gel chromatography followed by benzyl-DEAE column chromatography it was possible to obtain a 50-fold purification based on protein. Disc electrophoresis indicated that the enzyme was a single protein band (Chen & Dauter- man, unpublished results). Our results showed that the enzyme has an optimum pH of 9 and a molecular weight of approximately 230 000-250 000. Divalent cations did not stimulate the enzyme at a 10-6M concentration, indicating that it is not an arylamidase since these ions activate arylamidase activity. The various nucleotides, in either the oxidized or the reduced form, did not enhance the activity. Whereas malaoxon is both a substrate and an inhibitor of carboxylesterase, the oxygen analogue of dimethoate is only an inhibitor of the amidase and is not hydrolysed by it. TRANSFERASES Studies on transferases or conjugation reactions involving organophosphorus insecticides have ge- nerally been neglected. Most of the emphasis in organophosphorus metabolism has been on the identification of the hydrolysis products containing the P atom rather than on other portions of the molecule that could be conjugated. With the carba- mates much more information is available on 140 BIOLOGICAL AND NONBIOLOGICAL MODIFICATIONS OF OP COMPOUNDS Fig. 9 Transferase reactions COOH HOKwI OH UDPGA/ / glucuronyl transferase R-OH UDPGglucosyl transferase sulfokinase APS R - OSO3 + UDP CH20H 0 OR OH OH + U DP + PAP conjugates, as has been described by Knaak (1971). However, since organophosphorus compounds can undergo a wide variety of biological modifications that may result in the introduction of sites for conjugation, one must assume that a portion of these metabolites are conjugated by the general transferase reactions. Some important conjugation reactions that may be involved in the in vivo metabolism of organo- phosphates are summarized in Fig. 9. Of those listed, glucuronides are reported to be formed in vertebrates, glucosides in plants and insects, and etheral sulfates in vertebrates. These reactions are some of those most frequently described in the literature. All of these transferase reactions re- quired a high energy donor as well as a suitable enzyme. With glucuronide formation the reaction requires uridine diphosphate glucuronic acid and the glucuronyl transferase enzyme, which is asso- ciated with the microsomal fraction of mammalian livers or kidneys. Glucuronic acid may be transferred to phenolic, hydroxylamino, and alcoholic hydroxyl groups; carboxyl groups; amino and imino groups; and sulfhydryl groups (Smith & Williams, 1966). The formation of glycosidic conjugates requires a uridine diphosphate glucose donor, a glucosyl transferase, and acceptor groups similar to those required for glucuronide formation. Under certain conditions sugars other than glucose may be in- volved as a part of the uridine donor. Sulfate conjugation primarily occurs with aro- matic amino groups, phenolic hydroxyl groups and aliphatic alcoholic hydroxyl groups (Parke, 1968). Sulfate esters are synthesized biochemically by the transfer of sulfate from adenosine-3'-phosphate- 5'-phosphosulfate to the phenol, alcohol, or amine by the sulfate-transferring enzyme aryl sulfotrans- ferase. The accompanying table lists some of the conju- gation products that have been identified for a number of organophosphorus insecticides. All of the conjugated products reported are secondary metabolites in which the phosphorus moiety is lacking. From these data it would appear that con- jugates of the leaving group are synthesized after the cleavage of the acid anhydride bond. None of the other conjugation reactions-such as methylation, peptide conjugation, acetylation, or phosphate conjugation-has been reported to occur with organophosphorus insecticides. Alkyl transferases Glutathione conjugations have been found to be extremely important in the metabolism of organo- 141 J W. C. DAUTERMAN Conjugation products of metabolites of organophosphorus insecticides Compound Organism Product Reference Abate t plant 4,4'-thiodiphenol, 4,4'-sulfinyldiphenol, and 4,4'-sulfonyl- Blinn (1968) diphenol glucosides chlorfenvinphos rat, dog 2,4-dichlorophenylethyl and ethanediol glucuronides Hutson et al. (1967) Colep t rat phenylsulfuric acid Marco & Jaworski plant phenyl a and 6 glucosides (1964) famphur t calf methyl and dimethyl sulfamoylphenyl glucuronides and Gatterdam et al. sulfates (1967) Gardona f rat, dog 2,4,5-trichlorophenylethyl and ethanediol glucuronides Whetstone et al. (1966) parathion cow aminophenol glucuronide Pankaskie et al. (1 952) phosalone plant benzoxazolone glycoside Colinese & Terry (1968) trichlorfon insect trichloroethanol glucuronide Hassan et al. (1965) dichlorvos rat (in vitro) dichloroethanol glucuronide Hodgson & Casida (1 962) phosphates because of the formation of primary metabolites (Fig. 10, reaction a). Studies on the detoxification of parathion-methyl and the dimethyl homologue of paraoxon by mammals and insects demonstrated that 0-demethylation occurred in the presence of a soluble enzyme preparation and reduced glutathione (Fukami & Shishido, 1963, 1966; Shishido & Fukami, 1963). Subsequent in vitro studies on the dimethyl analogue of chlorfenvinphos (Hutson et al., 1968a), mevinphos (Morello et al., 1968), fenitrothion and methyl paraoxon analogues (Hollingworth, 1969), bromophos (Stenersen, 1969), and diazinon (Lewis, 1969) have demonstrated that 0-dealkylation also occurred with other organo- phosphorus compounds. The available information indicates that the alkyl transferase reaction is dependent on the presence of reduced glutathione, that the enzyme is in the soluble fraction and is probably identical to glutathione S-alkyl trans- ferase, and that the substrate specificity favours the dimethyl esters and will 0-dealkylate both phos- phorothioates and phosphates, resulting in S- alkyl glutathione and 0-dealkyl derivatives. The transferase activity is greatest in soluble fractions of mammalian liver and in the midgut and fat body of insects. All available information indicates that only one alkyl group is 0-dealkylated. Stener- sen (1969) reported that both 0-methyl groups Fig. 1 0 S-alkyl and S-aryl transferases S-ALKYL TRANSFERASE 0 ~~~~~~CH30 a. (CH30)2 P O§NO2 + GSH -p NO2 + GSCH3 HO S-ARYL TRANSFERASE b. (C2H50)2 IJ° N02 + GSH - (C2H50)2P OH + GSe N02 WnO I02?9 142 BIOLOGICAL AND NONBIOLOGICAL MODIFICATIONS OF PO COMPOUNDS Fig. 1 1 Miscellaneous reactions * Is Reduction a. (C2 H50)2 P O°\) NO2 > parath ion LD50 4mg/kg rat COOH /al b. (CH3°)2 P S CH2CN CH3 '_ /CH20H / red -N formothion CH3 LD50 375-535 mg/kg rat Postulated Intermediate (C2H5 )2 P 0/3NH2 amino- para th ion LD50450 mg / kg rat ( I(CH30)2 P 0 H S CH2 C N \CH3 dim et hoa te LD50 215mg/kg rat * References: reaction a, Ahmed et al. (1958); reaction b, Laroche et al. (1970). of bromophos were removed; however, he subse- quently indicated that bromophos is only mono- demethylated. Aryl transferases Preliminary data also indicate that a glutathione transferase reaction might be responsible for the transfer of aryl groups (Fig. 10, reaction b). Dahm (1970) reported some evidence that the P-O-aryl bond of parathion was cleaved by a non-oxidative soluble enzyme requiring glutathione. Studying the in vitro metabolism of diazinon in the housefly, we found that diethyl phosphoric acid and diethyl phosphorothioic acid were produced in the presence of GSH and the soluble enzyme fraction, thus indi- cating a possible aryl transfer (Yang et al., 1971b). However, we have not actually identified an S-aryl glutathione metabolite. MISCELLANEOUS REACTIONS There are reports of reductive reactions involved in the metabolism of organophosphates. The in vivo reduction of parathion and paraoxon to the amino derivatives is an important detoxification mechanism in ruminants, but is of minor importance in other animals (Ahmed et al., 1958). The toxicity of the amino derivatives is much lower than that of the parent compound (Fig. 11, reaction a). Re- cent in vitro studies (Hitchcock & Murphy, 1967) on the reduction of nitrophenyl-containing insecti- cides indicated that reductase activity was uniformly distributed between various cell fractions and that the enzyme required NADPH for activity. Reduc- tase activity was greatest in liver and kidney but was also found in several other tissues. Evidence that formothion is metabolized in vivo to dimethoate was recently obtained by Laroche et al. (1970). It is possible to postulate two routes of metabolism (Fig. 11, reaction b), one involving the oxidation of the aldehyde to the acid followed by decarboxylation and the other involving the reduc- tion of the aldehyde to the N-hydroxymethyl group. This reaction can be considered an intoxication reaction since dimethoate is more toxic than for- mothion. 11 143 W. C. DAUTERMAN NONBIOLOGICAL MODIFICATIONS Considerable research has been conducted on the "biological modifications " of organophosphorus compounds, but less attention has been devoted to the physical factors that may modify these compounds (Crosby, 1969). Some of the physical factors that may alter or modify the structure of organophosphorus compounds are light, temperature, air, and solvent. When a pesticide is used for insect control it is exposed to various environmental factors, which may alter it in the absence of a living organism. Since many of the alteration pro- ducts that have been isolated and identified are quite similar to those obtained from biological systems, these reactions may also be important for the intoxication and detoxification of organophos- phorus compounds, and may present a health hazard in the environment. Light Of all the physical factors that are responsible for chemical change, light is probably one of the most important. Most photochemical reactions appear to be mediated by the short-wavelength or the ultraviolet component of sunlight. Parathion was one of the first organophosphorus compounds whose anticholinesterase activity was shown experimentally to increase during exposure to UV light and sunlight (Payton, 1953). Subsequent work by Cook (1955) and Cook & Pugh (1957) indicated that the toxicity of parathion decreased under UV light, but the in vitro anticholinesterase activity increased as the result of the formation of more polar products. Frawley et al. (1958) found that the exposure of parathion to UV light resulted in a mixture of compounds with greater in vitro anticholinesterase activity than parathion. A study by Mitchell (1961) indicated that most organo- phosphorus compounds break down to form a wide variety of new compounds on irradiation, but no attempt was made to characterize them. Studies by Koivistoinen & Meriliiinen (1963) demonstrated that both UV light and sunlight changed parathion to several cholinesterase inhibitors. On the basis of chromatographic behaviour, the metabolites were identified as paraoxon and the S-ethyl and S- phenyl isomers of parathion, together with unknown products (Fig. J 2). This study showed that UV light is able to oxidize as well as isomerize parathion. When Fig. 12 Effect of light on some organophosphorus insecticides S 254 nm (C2H50)2 P ° \) 2 sunlight _ (C2H50)2 P S N02 S-phenyl isomer (C2 H50)2 P0 NO2 paraoxon C2H5 S° of C2H50 \/ 2 + Unknown Products S-ethyl isomer S S 0 s 0254nm 4 b. (C2H50)2 P S CH2 S CH5 idt- - (C2H50)2 P S CH2 S C2H5 + (_2H50)2 P S OH2 S 02H5 0 phorate sulfox ide sulfone 0 0 (C2H50)2P 0 C(CH3) = CHCOOC2H5-w.(C2H50)2P-OC = CH OH3 COOC2H5 0 COOC2H5 Il + (C2H50)2P-OuO= OH CH3 carbethoxy analog of mevinphos 30% -cis 70% a. C. 144 30% - cis trans BIOLOGICAL AND NONBIOLOGICAL MODIFICATIONS OF OP COMPOUNDS parathion-methyl was given the same UV treatment only the methyl homologue of paraoxon was found. A similar study in which EPN t was exposed to UV light resulted in the identification of the oxygen analogue of EPN and p-nitrophenol, together with unidentified resins (Okada & Uchida, 1964), also indicating cleavage of the P-O-aryl bond. Studies with seven organophosphorus pesticides containing sulfur in a thioether group indicated that exposure to UV light (254 nm) resulted in a variety of oxidation products (Mitchell et al., 1968). With phorate, disulfoton, and thiometon the corresponding sulfoxides and sulfones were identified as products of UV irradiation. With thiometon evidence of oxi- dation of the thiono sulfur was also obtained. In all cases the oxidation products were more toxic than the parent compound (see Thioether oxidation, above). Ultraviolet irradiation of a carbethoxy analogue of mevinphos results in another type ofphotoisomeri- zation (Fig. 12) (Casida, 1955). Starting with either the cis or the trans isomer or a mixture of the isomers and exposing the compounds to UV light resulted in a mixture of approximately 30% of the cis and 70% of the trans isomer. In all cases the trans isomer was predominant. When Dursban t is exposed to UV light or sun- light it undergoes hydrolysis in the presence of water to liberate 3,5,6-trichloro-2-pyridinol (Smith, 1968), which then undergoes complete photo- dechlorination with the formation of diols, triols, and tetraols. The effect of light, therefore, can be responsible for oxidation of thiono sulfur as well as thioether groups, isomerization of the thiono sulfur and iso- merization across double bonds, as well as hydro- lysis and dehalogenation. Temperature The first report of thermal isomerization of an alkyl phosphorothioate was published by Emmett & Jones (1911), who reported that O,O,O-trimethyl phosphorothioate, when heated in a sealed tube at 1 50°C, isomerized to O, O,S-trimethyl phosphoro- thioate. Subsequent studies with insecticidal organo- phosphates also indicated that heat modifies the biological activity. Metcalf & March (1953) found that when para- thion was heated at 150°C for 24 hours, 8 breakdown products were formed. Five of the compounds were identified: parathion, paraoxon, p-nitrophenol, bis(p-nitrophenyl) thionophosphate, and-the prin- cipal product S-ethyl parathion (Fig. 13, reaction a). Thermal isomerization has also been demonstra- ted for malathion, parathion-methyl, the diisopropyl homologue of parathion, EPN t (Metcalf & March 1953); Chlorthion,f diazinon (Augustinsson & Jonsson, 1957); and fensulfothion (Benjamini et al., 1959a). In each case the S-alkyl isomer showed greater in vitro anticholinesterase activity than the Fig. 13 Thermal isomerization S 0 ~~A ____H111 / ~ 0(C2H50)2 P 0F NO02 C25O~,p NO irath ion S-ethyl parathion S 0 .,0)P0CCHS2H - -(2H5A)2 2CH2 SC2 H5(C2H50)2- P CH2CH2 SC 5 (C2H50)2 P S CH22 H5 demetond e m e t o n - 0 S 0 A (C2H50)2 P 0 CH2 CH2 N (02 H5)2 -b.- (C2H5o)2 p S CH2CH2 N ( 02 H5)2 a. pa b. C. 145 t h iono - 1 et1 ra m tetram W. C. DAUTERMAN parent compound. Heating insecticidal organo- phosphates above 200°C also results in decomposi- tion. With parathion-methyl, isomerization to the S-methyl isomer is followed by the generation of dimethyl sulfide and sulfur dioxide and a mix- ture of poly(aryl metaphosphates), which decompose to a carbonaceous residue that is explosive (McPher- son & Johnson, 1956). Parathion-methyl, malathion, Chlorthion,t and dicapthon t may also decompose at temperatures between 65°C and 115°C. However, the time for decomposition is measured in days rather than minutes. A second type of thermal isomerization involves compounds containing sulfur or nitrogen in the side chain. O-dialkylaminoethyl and O-alkylthio- ethyl dialkyl phosphorothioates isomerize readily to yield the S-2-ethylthioethyl and S-2-dialkyl- aminoethyl isomers (Fukuto & Metcalf, 1954; Fukuto & Stafford, 1957; Tammelin, 1957) (Fig. 13, reactions b and c). No evidence was found for S-ethyl or S-methyl isomerization. It is generally accepted that this type of isomerization probably proceeds via a cyclic intermediate and results in an increase in toxicity as well as an increase in the anticholine- sterase properties. Simple alkyl and aryl phosphates also decompose under the influence of heat. A temperature of 100-200°C is required before the extent of decompo- sition becomes significant. Two types of reaction are shown in Fig. 14. In the first reaction, heating a diaryl alkyl phosphate results in the formation of an unsaturated aliphatic hydrocarbon and a diaryl phosphate. For this type of thermal decom- position, it is postulated that an available hydrogen is necessary on carbon number 2 for the formation of a 1-alkene (Gamrath et al., 1954). For the second type of reaction, compounds containing a pyro- phosphate bond tend to undergo dissociation and rearrangement on heating. This reaction is catalysed by acids, which are readily formed by hydrolysis if a trace of water is present, and is known to occur with TEPP. Heat may be extremely important in the manu- facture, purification, and storage of organophos- phorus insecticides. This effect of temperature may increase-or, with some compounds, decrease-the toxicity. From this limited discussion it is obvious that the effect of temperature on organophosphorus compounds is extremely complex-both chemically and biologically. Air It is generally recognized that a component of air, oxygen, is required for many biological reac- tions catalysed by the mixed-function oxidases. Generally, it has been assumed that the conversion of P = S to P=0 compounds was strictly a biological reaction. However, when dimethoate was exposed to air in the absence of UV light, dimethoxon was rapidly formed. This reaction occurred on leaves of cotton, potato, and maize (corn) and on glass plates (Dauterman et al., 1960). Koivistoinen & Merilainen (1963) also showed that when parathion was exposed as a thin film, trace amounts of para- oxon were formed in the absence of light. Solvents Various types of solvent may have a marked effect on the stability as well as the toxicity of organophosphorus compounds. It is generally recognized that in aqueous solution most organo- phosphorus esters are readily hydrolysed. Under acid or neutral conditions the alkyl-oxygen bond is attacked, while in alkaline conditions the phos- phorus-oxygen bond is ruptured. The hydrolysis products are generally less susceptible to further Fig. 14 Effect of heat on organophosphorus compounds 0 2 0 a. (RO )2 P CH2 CH2 R- - (RO)2 P OH + RCH = CH2 R'= aryl R= alkyl 00 0 to *e Zsb. (C2H50)2 P o P(OC2H5)2- - (C2H50)3 P + /n (c2H5o PO2)n 146 BIOLOGICAL AND NONBIOLOGICAL MODIFICATIONS OF OP COMPOUNDS Fig. 1 5 Transalkylation * Of H20 0 +,C2H5 CH3 0'1 0 2 (CH30)2 P S CH2CH2 S C2H5 (CH3O)2P S CH2CH2 Ss + P S CH2CH2 S C2H5 + OH CH3 HO demeton- methyl sulfonium compound *H60 1025c . Heath sVandekar (1957). The intravenous LDso0for rats is 65mg/kg of demeton-methyl and 0.06 mg/kg of the sulfonium compound. degradation and are markedly less toxic to insects and mammals (O'Brien, 1960). A number of agents such as amino acids, hydroxylammonium derivatives, and metal ions such as Cu++ catalyse the hydrolysis of the phosphorus esters. Solvents are also used in formulating organo- phosphates to obtain properties that will increase the chances of contact between the insecticide and the pest. Casida & Sanderson (1963) found that dimethoate increases in toxicity on storage in certain hydroxylic solvents, particularly 2-alkoxy- ethanols. The oral LD50 for the rat decreased on storage from 150-250 mg/kg to 30-40 mg/kg after 7 months at normal temperatures. Studies indi- cated that 14 phosphorus-containing metabolites were formed in the presence of methyl Cellosolve. The degradation involved hydrolysis of the amide bond, hydrolysis of all the ester groups, and loss of the thiono sulfur. The most toxic fraction was identified as an O,O-dialkyl S-(N-methylcarbamoyl- methyl) phosphorothioate with probably one, but possibly both, of the methyl groups replaced by 2-methoxyethyl groups. No evidence was obtained for the formation of pyrophosphates. The toxicity of a few other phosphorothioate insecticides also increased in the presence of 2-methoxyethanol. Another type of reaction occurs when organo- phosphorus compounds containing a secondary sulfur are stored undiluted or in an aqueous solu- tion. This reaction involves one molecule of the compound alkylating another (Fig. 15). Heath & Vandekar (1957) observed that a 1% solution of demeton-methyl increased in toxicity spontaneously at 35°C during the course of one day. This increase was due to the formation of the sulfonium ion, whose toxicity is more than 1 000 times that of the parent compound. A similar reaction has also been shown to take place with demeton-O. Samples of demeton-methyl that have been stored for a few months may contain up to 4% of the sulfonium compound. The transalkylation is extremely rapid with demeton-methyl and slower with demeton. CONCLUSIONS From this brief review it is obvious that our knowledge of the different reactions involved in the biological and nonbiological modification of organo- phosphorus compounds is advancing but is far from complete. Recent in vitro studies, especially those involving the mixed-function oxidases, have helped to elucidate some of these reactions. How- ever, a number of different enzymatic reactions can produce the same metabolites in vitro (i.e., oxidative dearylation, hydrolysis, and aryl transfer). There- fore, it is difficult to assess the importance of the reaction in vivo. Further work is needed to evaluate these reactions and determine their relative impor- tance in vivo in both target and nontarget organisms. The metabolic fate of many of the leaving groups of organophosphorus compounds has only been partially investigated. Since in many instances the basic anticholinesterase activity has been destroyed by both environmental and biological reactions, it has generally been assumed that the heterocyclic, aryl, and alkyl leaving groups are non- toxic in the environment. Further research is needed to clarify the importance of these chemical break- down products as possible hazards in the environ- ment and the extent to which they may accumulate therein. 147 148 W. C. DAUTERMAN REFERENCES Adie, P. A. (1956) Canad. J. Biochem., 34, 1091-1094 Ahmed, M. K., Casida, J. E. & Nichols, R. E. (1958) J. agric. Food Chem., 6, 740-746 Aldridge, W. N. (1953) Biochem. J., 53, 117-124 Andrawes, N. R., Dorough, H. W. & Lindquist, D. A. (1967) J. econ. Ent., 60, 979-987 Augustinsson, K. B. & Heimburger, G. (1954) Acta chem. scand., 8, 1533-1541 Augustinsson, K. B. & Jonsson, G. (1957) Experientia (Basel), 13, 438-440 Benjamini, E., Metcalf, R. L. & Fukuto, T. R. (1959a) J. econ. Ent., 52, 94-98 Benjamini, E., Metcalf, R. 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