Archives of
TOXICOLOGY
Arch. Toxicol. 42, 1--18 (1979)
9 Springer-Verlag 1979
Original Investigations The Chemical and Biochemical Reactivity of Dichlorvos A. S. Wright, D. H. Hutson, and M. F. Wooder Shell Research Ltd., Shell ToxicologyLaboratory (Tunstall), Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG, Great Britain Abstract. The chemical structure, reactivity and metabolic fate of the insecticide dichlorvos (2,2-dichlorovinyl dimethyl phosphate) are discussed in relation to the possible genotoxicity of this and other methyl phosphate triesters. Recent attempts to demonstrate the methylation of DNA following exposure of bacteria and animals to dichlorvos are reviewed. On the basis of comparative data relating mutagenesis to methylation reactions, it seems entirely appropriate to conclude that the mutagenicity of dichlorvos to bacteria is due solely to methylation of the bacterial DNA under the conditions of these tests. However, the methylation of mammalian DNA could not be demonstrated under realistic exposure conditions (when the alkylating mutagen methyl methanesulphonate afforded clearly measurable methylation). The failure to detect methylation by dichlorvos in vivo is attributed to the operation of highly efficient enzyme-catalysed biotransformations which rely largely on the phosphorylating reactivity of dichlorvos. The biotransformation pathways, characterised mostly in the rat, appear to be common also to pig, mouse, hamster, and man.
Key words: 2,2-Dichlorovinyl dimethyl phosphate - Dichlorvos -- Rat genotoxicity -- Metabolism - Mouse -- Hamster - Pig - Man -- Methylation DNA -- 7-Methylguanine. Introduction
During the last 10 years the insecticide and anthelmintic dichlorvos (Fig. 1, 2,2dichlorovinyl dimethyl phosphate) has been the subject of rigorous toxicological investigations orientated towards the assessment of genotoxicity. These studies were prompted by physicochemical considerations that resulted in the demonstration that dichlorvos possesses weak methylating activity (L6froth et al., 1969; L6froth, 1970). By analogy with the biological effects of powerful alkylating agents, this finding led to the speculation that dichlorvos might be a mammalian mutagen and possible carcinogen. However, although dichlorvos can induce mutation in bacteria and lower eukaryotes (yeasts) (for a comprehensive review see Wild, 1975) there is no
0340-5761/79/0042/0001/$ 03.60
2
A.S. Wright et al.
cH~-o,,,,p#o CH3--O' / ~O-CH-----CCI 2
Fig. 1. The structure of dichlorvos
evidence that this compound produces genotoxic effects in mammals. Thus dichlorvos has been extensively evaluated for mutagenicity and carcinogenicity in mammalian test systems with entirely negative results (Bootsma et al., 1971; Witherup et al., 1971; Buselmaier et al., 1972; Dean, 1972; Dean and Thorpe, 1972a, b; Dean et al., 1972; Epstein et al., 1972; Voogd et al., 1972; Blair et al., 1976; Dean and Blair, 1976; NCI report, 1977; Molina et al., 1978). The results of recent microbial and biochemical investigations have provided an explanation as to why dichlorvos induces mutations in micro-organisms but fails to produce genotoxic effects in mammals. It is, therefore, apposite to review the biochemistry of this compound, particularly those aspects of possible relevance to genotoxicity.
Determinants of Toxicity Five factors are recognized as governing the nature and magnitude of toxic effects resulting from the exposure of an organism to a foreign compound: 1. The chemical structure and reactivity of the compound in the microenvironment of the target. 2. The topography, specificities and activities of enzymes catalysing the metabolism of the compound. 3. The physico-chemical properties of the target. 4. The target dose, defined as the concentration of the compound or its toxic metabolites at the target locus and the duration of such exposure (determined by the route of exposure, dose level, exposure time and also by the efficiencies and capacities of protective barriers such as cell membranes and detoxifying enzyme systems). 5. The efticiencies, capacities and fidelity of operation of repair systems and modulating factors. Leaving aside rate and route of exposure, the four latter factors are largely determined by genetic structure, cell phenotype and cellular organisation and they are responsible for differences in response among tissues, individuals and species. In those instances where the target locus has been identified, it is usually possible to obtain pertinent information concerning all of these determinants of toxicity. In other instances, where the critical target loci have not been established, e.g., chemical carcinogenesis, the information is, of necessity, less precise.
Target Locus for Chemical Mutagens The genetic material, DNA, is established as the ultimate target of chemical mutagens. In many instances, DNA is also a primary target. For example, the primary mutagenic lesions induced by the powerful alkylating agents are ascribed to direct
The Chemicaland BiochemicalReactivityof Dichlorvos interactions between the electrophilic centre(s) in such compounds and nucleophilic centres in DNA. These initial structural changes in DNA are realised as mutations either by direct miscoding during DNA replication or indirectly as a consequence of misrepair or failure to repair. During the last l0 years it has been recognised that many chemicals are converted into electrophilic reactants by biotransformation of the parent compounds during metabolism within exposed animals. Such metabolites may be capable of reacting with DNA. Consequently, in any mechanistic consideration of chemical mutagenesis, attention must be focussed not only upon the parent compound but also upon the intermediates and products generated during the metabolism of these compounds. It is clear that electrophilic reactivity per se does not automatically confer mutagenic or other overt toxic properties upon a substance (for a review see Miller and Miller, 1974). Thus, the chemistry of the individual electrophiles and their concentrations at target sites are critical determinants of the nature and magnitude of any primary toxic effects. Furthermore, studies of the relationships between methylation reactions and mutagenesis suggest that not all of the primary structural modifications introduced into DNA by reaction with electrophiles are pro-mutagenic. For example, largely because of its quantitative significance it was generally presumed that methylation at N 7 of guanine moieties of DNA was causally related to mutations induced by methylating agents. However, while genetic damage resulting from scissions of 7-methylguanine moieties and subsequent misrepair or failure to repair cannot be discounted, in vitro experiments with synthetic polymers containing varying proportions of 7-methylguanine have demonstrated that the presence of this base does not give rise to errors at the level of transcription (Ludlum, 1970) or translation (Wilhelm and Ludlum, 1966). In addition, methylation of N 7 of guanine moieties in DNA correlates poorly with carcinogenic effects (Kleihues and Magee, 1973). On the other hand, methylations at O6 of guanine and certain other centres, although quantitatively less important than methylation at N 7 of guanine, distort base pairing, affect the secondary structure of DNA and are generally regarded as pro-mutagenic (Lawley, 1974). Current evidence indicates that a good correlation exists between the persistence of these latter molecular lesions and the susceptibility of tissues to tumour formation (Margison and Kleihues, 1975). The Chemistry of Diehlorvos As a mixed triester of phosphoric acid, dichlorvos possesses twe. methyl groups and an electron-withdrawing dichlorovinyl group. The electron-withdrawing capacity of the attached oxygen atoms and of the dichlorovinyl group result in a marked residual positive charge at phosphorus.
The Chemistry of the Phosphoryl Centre The reactivity of nucleophiles for electrophiles varies markedly according to the structures of the reactants. Thus, the electrophilic phosphorus atom is particularly susceptible to attack by those nucleophiles classified as "hard" (Pearson and Song-
4
A.S. Wright et al.
stad, 1967). The general mechanism of nucleophilic substitution reactions at the phosphoryl centre is illustrated in Figure 2. In addition to contributing to the withdrawal of electrons from the phosphorus atom, thus activating this centre towards nucleophilic attack, the diehlorovinyloxy group is a better leaving group than methoxy. Consequently, nucleophilic substitution reactions at phosphorus lead to scission of the P - O (dichlorovinyl) bond and to the dimethylphosphorylation of the attacking nucleophile. This reaction mechanism (Fig. 2) is fundamental to both the insecticidal action of dichlorvos and the detoxification of dichlorvos by mammals. Thus, dichlorvos exerts its acute toxic action by inhibiting the enzyme acetylcholinesterase (Braid and Nix, 1969). This is a general reaction of the insecticidal dialkyl phosphoric acid triesters and is thought to be achieved by attack on phosphorus by the serine hydroxyl (or corresponding oxyanion) located at the active centre of the enzyme. This affords, in the ease of dichlorvos, dimethylphosphorylation of the active site and the loss of enzyme activity (Fig. 3) (for reviews see Aldridge and Riener, 1972; Matsumura, 1975). This type of reaction mechanism also applies when the attacking nucleophile is water (Fig. 4). Although the most abundant nucleophile in living organisms, water is also one of the slowest reacting of all nucleophiles. Thus, in order to be effective in affording protection to other nucleophiles, e.g., the dimethylphosphorylation of serinyl moieties in important proteins, the reaction of dichlorvos with water must be accelerated. In mammals, this acceleration is achieved by the catalytic action of esterases. These esterases (arylesterases) are of a different type to those inhibited by dichlorvos and do not contain serine at the active centre (Augustinsson, 1964). Such enzymes are present in all mammalian tissues, including blood. Consequently, the dimethylphosphorylation of water proceeds extremely efficiently and constitutes the predominant CH3-O (~- 0 CH3-O
CH3-O ~ ~0
" 10 mg/kg body weight) (Wennerberg, 1973).
Conclusions
In vitro bacterial tests that have been employed to assess the mutagenicity of dichlorvos take account of all spontaneous reactions that are dependent upon the presence of the parent compound, e.g., methylation reactions, dimethylphosphorylation reactions with attendant generation of dichloroacetaldehyde together with any theoretical reaction such as transfer of the dichloroalkene moiety. Comparisons of quantitative data relating mutagenesis and methylation reactions indicate that the mutagenicity of dichlorvos in simple bacterial systems is due solely to methylation of bacterial DNA. 9 Methylation of DNA could not be demonstrated in mammals under realistic exposure conditions or after exposure to high doses of this compound. The failure of dichlorvos to induce genotoxic effects in mammals is attributed to the very efficient metabolism of dichlorvos in mammals which results in the destruction of the methylating potential of this compound.
References
Aldridge, W. N., Reiner, E.: In: Enzymesubstrates as inhibitors. Interactionsof esterases with esters of organophosphorus and carbamic acids, Chapt. 11, pp. 170-175. Amsterdam: North Holland Publishing Comp. 1972 Augustinsson, K. B.: Arylesterases. J. Histochem. Cytoehem. 12, 744-747 (1964) Bedford, C. T., Robinson,J.: The alkylatingpropertiesof organophosphates. Xenobiotica2, 307-337 (1972)
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Blair, D., Hoadley, E. C., Hutson, D. H.: The distribution of dichlorvos in the tissues of mammals after inhalation and intravenous administration of dichlorvos. Toxicol. Appl. Pharmacol. 31, 243-253 (1975) Blair, D., Dix, K. M., Hunt, P. F., Thorpe, E., Stevenson, D. E.: Dichlorvos - a two-year inhalation carcinogenesis study in rats. Arch. Toxicol. (Bed.) 35, 281-294 (1976) Bootsma, D., Herring, H., Kleijar, W., Budke, L., de Jong, L. O. A., Berends, F.: The effects of dichlorvos on human cells in tissue culture. Med. Biol. Lab. RVO-TNO. MBL 5 (1971) Braid, P. E., Nix, M.: The kinetic constants for the inhibition of acetylcholinesterase by phosdrin, Sumioxon, DDVP and phosphamidon. Can. J. Biochem. 47, 1-6 (1969) Bridges, B. A., Mottershead, R. P., Green, M. H. L., Gray, W. J. H.: Mutagenicity of dichlorvos and methyl methanesulphonate for Escherichia coli WP2 and some derivatives deficient in DNA repair. Mutat. Res. 19, 295-303 (1973) Buselmaier, W., Rohrborn, G., Propping, P.: Mutagenit/itsuntersuchungen mit Pestiziden im hostmediated Assay und mit dem dominanten Letaltest an der Maus. Biol. Zbl. 91, 311-325 (1972) Casida, J. E., McBride, L., Niedermeier, R. P.: Metabolism of 2,2-dichlorovinyl dimethyl phosphate in relation to residues in milk and mammalian tissues. J. Agric. Food Chem. 10, 370-377 (1962) Chasseaud, L. F.: The nature and distribution of enzymes catalysing the conjugation of glutathione with foreign compounds. Drug Metab. Rev. 2, 185-220 (1974) Chu, B. C. F., Lawley, P. D.: Increased urinary excretion of pyrimidine and nicotinamide derivatives in rats treated with MMS. Chem.-Biol. Interact. 8, 65-73 (1974) Chu, B. C. F., Lawley, P. D.: Increased urinary excretion of nucleic acid and nicotinamide derivatives by rats after treatment with alkylating agents. Chem.-Biol. Interact. 10, 333-338 (1975a) Chu, B. C. F., Lawley, P. D.: Increased urinary excretion of labelled deoxyribonucleosides in rats with stable pre-labelled DNA treated with methyl metbanesulphonate or nitrogen mustard. Chem.-Biol. Interact. 10, 407-412 (1975b) Cohen, S. D., Ehrich, M.: Cholinesterase and carboxylesterase inhibition by dichlorvos and interactions with malathion and triorthotolyl phosphate. Toxicol. Appl. Pharmacol. 37, 39--48 (1976) Cohen, S. D., Murphy, S. D.: Inactivation of malaoxon by mouse liver. Proc. Soc. Exp. Biol. Med. 139, 1385-1389 (1972) Craddock, V. M., Magee, P. N.: Effect of administration of the carcinogen DMN on urinary 7methylguanine. Biochem. J. 104, 435-440 (1967) Dean, B. J.: The effects of dichlorvos on cultured human lymphocytes. Arch. Toxicol. (Bed.) 30, 75--85 (1972) Dean, B. J., Blair, D.: Dominant lethal assay in female mice after oral dosing with dichlorvos or exposure to atmospheres containing dichlorvos. Mutat. Res. 40, 67-72 (1976) Dean, B. J., Doak, S. M. A., Funnel, J.: Genetic studies with dichlorvos in the host-mediated assay in liquid medium using Saccharomyces cerevisiae. Arch. Toxicol. (Berl.) 30, 61-66 (1972) Dean, B. J., Thorpe, E.: Cytogenetic studies with dichlorvos in mice and Chinese hamsters. Arch. Toxicol. (Berl.) 30, 39--49 (1972a) Dean, B. J., Thorpe, E.: Studies with dichlorvos vapour in dominant lethal mutation tests on mice. Arch. Toxicol. (Berl.) 30, 51-59 (1972b) Dicowsky, L., Morello, A.: Glutathione-dependent degradation of 2,2-dichlorovinyl dimethyl phosphate (DDVP) by the rat. Life Sci. 10, 1031-1037 (1971) Donninger, C., Hutson, D. H., Picketing, B. A." Oxidative cleavage of phosphoric acid triesters to diesters. Biochem. J. 102, 26P (1967) Donninger, C., Hutson, D. H., Picketing, B. A.: The oxidative dealkylation of insecticidal phosphoric acid triesters by mammalian liver enzymes. Biochem. J. 126, 701-707 (1972) Donninger, C., Nobbs, B. T., Wilson, K.: An enzyme catalysing the hydrolysis of phosphoric acid diesters in rat liver. Biochem. J. 122, 51P (1971) Ehrieh, M., Cohen, S. D.: Effect of dichlorvos (DDVP) on mouse liver glutathione levels and lack of potentiation by methyl iodide and TOTP. Biochem. Pharmaeol. 26, 997--1000 (1977) Elgar, K., Steer, B. D.: Dichlorvos concentrations in the air of houses arising from the use of dichlorvos PVC strips. Pestic. Sci. 3, 591--600 (1972)
The Chemical and Biochemical Reactivity of Dichlorvos
17
Epstein, S. S., Arnold, E., Andrea, J., Bass, W., Bishop, Y.: Detection of chemical mutagens by the dominant lethal assay in the mouse. Toxicol. Appl. Pharmacol. 23, 288-325 (1972) Fischer, G. W., Schneider, P., Schleufler, H.: Zur Mutagenit~it yon Dichloroacetaldehyde und 2,2Dichlor-l,l-dilaydroxy-~ithanphosphods~iure-methylester, m6glichen Metaboliten des phosphororganischen Pesticides Triehlorphon. Chem.-Biol. Interact. 19, 205-213 (1977) Fukami, J., Shishido, T.: Nature of a soluble, glutathione-dependent enzyme system active in cleavage of methyl parathion to desmethyl parathion. J. Econ. Entomol. 59, 1338-1346 (1966) Hilgetag, G., Teichmann, H.: The alkylating properties of alkyl thiophosphates. Angew. Chem. [Engl.] 4, 914-922 (1965) Hodgson, E., Casida, J. E.: Mammalian enzymes involved in the degradation of 2,2-dichlorovinyl dimethyl phosphate. J. Agric. Food Chem. 10, 208-214 (1962) Hollingworth, R. M.: The dealkylation of organophosphorus esters by mouse liver enzymes in vitro and in vivo. J. Agric. Food Chem. 17, 987-996 (1969) Hollingworth, R. M.: The dealkylation of organophosphorus triesters by liver enzymes. In: Biochemical toxicology of insecticides. O'Brien, R. D., Yamamoto, I. (eds.), pp. 75--92. New York: Academic Press 1970 Hutson, D. H.: Some observations on the chemical and stereochemical specificity of the de-alkylation of organophosphorus esters by a hepatic glutathione transferase. Chem.-Biol. Interact. 16, 315--323 (1977) Hutson, D. H., Hoadley, E. C.: The metabolism of [14C-methyl]dichlorvos in the rat and the mouse. Xenobiotica 2, 107--116 (1972a) Hutson, D. H., Hoadley, E. C.: The comparative metabolism of [14C-vinyl]dichlorvos in animals and man. Arch. Toxikol. (Berl.) 30, 9-18 (1972b) Hutson, D. H., Hoadley, E. C., Pickering, B. A.: The metabolic fate of [vinyl-l-14C]dichlorvos in the rat after oral and inhalation exposure. Xenobiotica 1, 593-611 (1971) Hutson, D. H., Pickering, B. A., Donninger, C.: Phosphoric acid triester-glutathione alkyltransferase. A mechanism for the detoxication of dimethyl phosphate triesters. Biochem. J. 127, 285--293 (1972) Kleihues, P., Mageee, P. N.: Alkylation of rat brain nucleic acids by N-methyl-N-nitrosourea and methyl methanesulphonate. J. Neurochem. 20, 595-606 (1973) Kleihues, P., Patzschke, K., Margison, G. P., Wagner, L. A., Mende, C.: Reaction of methyl methanesulphonate with nucleic acids of fetal and newborn rats in vivo. Z. Krebsforsch. 81, 273-283 (1974) Lawley, P. D.: Some chemical aspects of dose-response relationships in alkylation mutagenesis. Mutat. Res. 23, 283-295 (1974) Lawley, P. D., Brookes, P.: Cytotoxicity of alkylating agents towards sensitive and resistant strains of Escherichia coli in relation to extent and mode of alkylation of cellular macromolecules and repair of alkylation lesions in deoxyribonucleic acids. Biochem. J. 109, 433--447 (1968) Lawley, P. D., Shah, S. A.: Reaction of alkylating mutagens and carcinogens with nucleic acids: detection and estimation of a small extent of methylation at 0--6 of guanine in DNA by methyl methanesulphonate in vitro. Chem.-Biol. Interact. 5, 286-288 (1972) Lawley, P. D., Shah, S. A., Orr, D. J.: Methylation of nucleic acids by 2,2-dichlorovinyl dimethyl phosphate (dichlorvos, DDVP). Chem.-Biol. Interact. 8, 171-182 (1974) Loeffler, J. E., Potter, J. C., Scordelis, S. L., Hendrickson, H. R., Huston, C. K., Page, A. C.: Longterm exposure of swine to a [14C]dichlorvos atmosphere. J. Agric. Food Chem. 24, 367-371 (1976) L/Sfroth, G.: Alkylation of DNA by dichlorvos. Naturwissenschaften 57, 393-394 (1970) l.,/Sfroth, G., Kim, C. H., Hussaln, S.: Alkylating property of 2,2-dichlorovinyl dimethyl phosphate: a disregarded hazard. Environ. Mutat. Soc. New. Lett. 2, 21-26 (1969) L6froth, G., Wennerberg, R.: Methylation of purines and nicotinamide in the rat by dichlorvos. Z. Naturforsch. [C] 29, 651 (1974) Ludlum, D. B.: Alkylated polycytidylic acid templates for RNA polymease. Biochim. Biophys. Acta 213, 142-148 (1970) Margison, G. P., Kleihues, P.: Chemical carcinogenesis in the nervous system - Preferential accumulation of 0~-methylguanine in rat brain deoxyribosnucleic acid during repetitive administration of Nmethyl-N-nitrosourea. Biochem. J. 148, 521--525 (1975)
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Matsumura, F.: Mode of action of insecticides. In: Toxicology of insecticides, p. 142. New York: Plenum Press 1975 Miller, J. A., Miller, E. C.: Mechanisms of chemical carcinogenesis. In: The molecular biology of cancer. Busch, H. (ed.), pp. 377-402. New York, London: Academic Press 1974 Miyata, T., Matsumura, F.: Organophosphate degrading enzymes in the crude supernatant fraction from the rat liver. J. Agric. Food Chem. 20, 30-32 (1972) Mohn, G.: 5-Methyltryptophan resistance mutations in Escherichia coli K12. Mutagenic activity of monofunctional alkylating agents including organophosphorus insecticides. Mutat. Res. 20, 7-15 (1973) Molina, L., Rincus, S., Legator, M.: Evaluation of the micronucleus test over a two-year period. Paper presented at the Eighth Annual Meeting, Environmental Mutagen Society, 13-17th February, 1977, Colorado Springs, USA. Abstract No. 105. Mutat. Res. 53, 125 (1978) NCI Report: Bioassay of dichlorvos for possible carcinogenicity (1977) Page, A. C., Loeftler, J. E., Hendrickson, H. R., Huston, C. K., De Vries, D. M.: Metabolic fate of dichlorvos in swine. Arch. Toxicol. (Bed.) 30, 19-27 (1972) Pearson, R. G., Songstad, J.: Application of the principle of hard and soft acids and bases to organic chemistry. J. Am. Chem. Soc. 89, 1827--1836 (1967) Potter, J. C., Boyer, A. C., Marxmiller, R. L., Young, R., Loeffler, J. E.: Radioisotope residues and residues of dichlorvos and its metabolites in pregnant sows and their progeny dosed with dichlorvos-14C or dichlorvos-36Cl. J. Agric. Food Chem. 21, 734-738 (1973) Sakai, K., Matsumura, F.: Degradation of certain organophosphate and earbamate insecticides by human brain esterases. Toxicol. Appl. Pharmacol. 19, 660--666 (1971) Singer, B.: The chemical effects of nucleic acid alkylation and their relationship to mutagenesis and carcinogenesis. Prog. Nucleic Acid Res. Mol. Biol. 15, 219-284, 330-332 (1975) Traey, R. L., Woodcock, J. G., Chodroff, S.: Toxicological aspects of 2,2-diehlorovinyl dimethyl phosphate (DDVP) in cows, horses, and white rats. J. Econ. Entomol. 53, 593--601 (1960) Voogd, C. E., Jacobs, J. J. A. A., van der Stel, J. J.: On the mutagenic action of dichlorvos. Mutat. Res. 16, 413-416 (1972) Wennerberg, R.: The methylation products after tratment with diehlorvos and dimethylsulphate in vivo. Ph.D. Dissertation, University of Stockholm, Sweden (1973) Wennerberg, R., Lrfroth, G.: Formation of 7-methylguanine by dichlorvos in bacteria and mice. Chem.-Biol. Interact. 8, 339--348 (1974) Wild, D.: Chemical induction of streptomycin-resistant mutations in Escherichia coll. Dose and mutagenie effects of dichlorvos and methyl methanesulphonate. Mutat. Res. 19, 33-41 (1973) Wild, D.: Mutagenieity studies on organophosphorus pesticides. Mutat. Res. 32, 133-150 (1975) Wilhelm, R. C., Ludlum, D. B.: Coding properties of 7-methylguanine. Science 153, 1403-1405 (1966) Witherup, S., Jolley, W. S., Stemmer, K., Pfitzer, A. E.: Chronic toxicity studies with 2,2-dichlorovinyl dimethylphosphate (DDVP) in dogs and rats including observations on rat reproduction. Toxicol. Appl. Pharmacol. 19, 377 (1971) Wooder, M. F., Wright, A. S., King, L. J.: In vivo alkylation studies with dichlorvos at practical use concentrations. Chem.-Biol. Interact. 19, 25--46 (1977) Zaika, A. P.: Detoxication of dimethyl dichlorovinyl phosphate (DDVP) in rabbits. Fiziol. Aktiv. Veshchestva, p. 24 (1972); CA 79, 28098 (1972) Received June 13, 1978
TOXICOLOGY
Arch. Toxicol. 42, 1--18 (1979)
9 Springer-Verlag 1979
Original Investigations The Chemical and Biochemical Reactivity of Dichlorvos A. S. Wright, D. H. Hutson, and M. F. Wooder Shell Research Ltd., Shell ToxicologyLaboratory (Tunstall), Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG, Great Britain Abstract. The chemical structure, reactivity and metabolic fate of the insecticide dichlorvos (2,2-dichlorovinyl dimethyl phosphate) are discussed in relation to the possible genotoxicity of this and other methyl phosphate triesters. Recent attempts to demonstrate the methylation of DNA following exposure of bacteria and animals to dichlorvos are reviewed. On the basis of comparative data relating mutagenesis to methylation reactions, it seems entirely appropriate to conclude that the mutagenicity of dichlorvos to bacteria is due solely to methylation of the bacterial DNA under the conditions of these tests. However, the methylation of mammalian DNA could not be demonstrated under realistic exposure conditions (when the alkylating mutagen methyl methanesulphonate afforded clearly measurable methylation). The failure to detect methylation by dichlorvos in vivo is attributed to the operation of highly efficient enzyme-catalysed biotransformations which rely largely on the phosphorylating reactivity of dichlorvos. The biotransformation pathways, characterised mostly in the rat, appear to be common also to pig, mouse, hamster, and man.
Key words: 2,2-Dichlorovinyl dimethyl phosphate - Dichlorvos -- Rat genotoxicity -- Metabolism - Mouse -- Hamster - Pig - Man -- Methylation DNA -- 7-Methylguanine. Introduction
During the last 10 years the insecticide and anthelmintic dichlorvos (Fig. 1, 2,2dichlorovinyl dimethyl phosphate) has been the subject of rigorous toxicological investigations orientated towards the assessment of genotoxicity. These studies were prompted by physicochemical considerations that resulted in the demonstration that dichlorvos possesses weak methylating activity (L6froth et al., 1969; L6froth, 1970). By analogy with the biological effects of powerful alkylating agents, this finding led to the speculation that dichlorvos might be a mammalian mutagen and possible carcinogen. However, although dichlorvos can induce mutation in bacteria and lower eukaryotes (yeasts) (for a comprehensive review see Wild, 1975) there is no
0340-5761/79/0042/0001/$ 03.60
2
A.S. Wright et al.
cH~-o,,,,p#o CH3--O' / ~O-CH-----CCI 2
Fig. 1. The structure of dichlorvos
evidence that this compound produces genotoxic effects in mammals. Thus dichlorvos has been extensively evaluated for mutagenicity and carcinogenicity in mammalian test systems with entirely negative results (Bootsma et al., 1971; Witherup et al., 1971; Buselmaier et al., 1972; Dean, 1972; Dean and Thorpe, 1972a, b; Dean et al., 1972; Epstein et al., 1972; Voogd et al., 1972; Blair et al., 1976; Dean and Blair, 1976; NCI report, 1977; Molina et al., 1978). The results of recent microbial and biochemical investigations have provided an explanation as to why dichlorvos induces mutations in micro-organisms but fails to produce genotoxic effects in mammals. It is, therefore, apposite to review the biochemistry of this compound, particularly those aspects of possible relevance to genotoxicity.
Determinants of Toxicity Five factors are recognized as governing the nature and magnitude of toxic effects resulting from the exposure of an organism to a foreign compound: 1. The chemical structure and reactivity of the compound in the microenvironment of the target. 2. The topography, specificities and activities of enzymes catalysing the metabolism of the compound. 3. The physico-chemical properties of the target. 4. The target dose, defined as the concentration of the compound or its toxic metabolites at the target locus and the duration of such exposure (determined by the route of exposure, dose level, exposure time and also by the efficiencies and capacities of protective barriers such as cell membranes and detoxifying enzyme systems). 5. The efticiencies, capacities and fidelity of operation of repair systems and modulating factors. Leaving aside rate and route of exposure, the four latter factors are largely determined by genetic structure, cell phenotype and cellular organisation and they are responsible for differences in response among tissues, individuals and species. In those instances where the target locus has been identified, it is usually possible to obtain pertinent information concerning all of these determinants of toxicity. In other instances, where the critical target loci have not been established, e.g., chemical carcinogenesis, the information is, of necessity, less precise.
Target Locus for Chemical Mutagens The genetic material, DNA, is established as the ultimate target of chemical mutagens. In many instances, DNA is also a primary target. For example, the primary mutagenic lesions induced by the powerful alkylating agents are ascribed to direct
The Chemicaland BiochemicalReactivityof Dichlorvos interactions between the electrophilic centre(s) in such compounds and nucleophilic centres in DNA. These initial structural changes in DNA are realised as mutations either by direct miscoding during DNA replication or indirectly as a consequence of misrepair or failure to repair. During the last l0 years it has been recognised that many chemicals are converted into electrophilic reactants by biotransformation of the parent compounds during metabolism within exposed animals. Such metabolites may be capable of reacting with DNA. Consequently, in any mechanistic consideration of chemical mutagenesis, attention must be focussed not only upon the parent compound but also upon the intermediates and products generated during the metabolism of these compounds. It is clear that electrophilic reactivity per se does not automatically confer mutagenic or other overt toxic properties upon a substance (for a review see Miller and Miller, 1974). Thus, the chemistry of the individual electrophiles and their concentrations at target sites are critical determinants of the nature and magnitude of any primary toxic effects. Furthermore, studies of the relationships between methylation reactions and mutagenesis suggest that not all of the primary structural modifications introduced into DNA by reaction with electrophiles are pro-mutagenic. For example, largely because of its quantitative significance it was generally presumed that methylation at N 7 of guanine moieties of DNA was causally related to mutations induced by methylating agents. However, while genetic damage resulting from scissions of 7-methylguanine moieties and subsequent misrepair or failure to repair cannot be discounted, in vitro experiments with synthetic polymers containing varying proportions of 7-methylguanine have demonstrated that the presence of this base does not give rise to errors at the level of transcription (Ludlum, 1970) or translation (Wilhelm and Ludlum, 1966). In addition, methylation of N 7 of guanine moieties in DNA correlates poorly with carcinogenic effects (Kleihues and Magee, 1973). On the other hand, methylations at O6 of guanine and certain other centres, although quantitatively less important than methylation at N 7 of guanine, distort base pairing, affect the secondary structure of DNA and are generally regarded as pro-mutagenic (Lawley, 1974). Current evidence indicates that a good correlation exists between the persistence of these latter molecular lesions and the susceptibility of tissues to tumour formation (Margison and Kleihues, 1975). The Chemistry of Diehlorvos As a mixed triester of phosphoric acid, dichlorvos possesses twe. methyl groups and an electron-withdrawing dichlorovinyl group. The electron-withdrawing capacity of the attached oxygen atoms and of the dichlorovinyl group result in a marked residual positive charge at phosphorus.
The Chemistry of the Phosphoryl Centre The reactivity of nucleophiles for electrophiles varies markedly according to the structures of the reactants. Thus, the electrophilic phosphorus atom is particularly susceptible to attack by those nucleophiles classified as "hard" (Pearson and Song-
4
A.S. Wright et al.
stad, 1967). The general mechanism of nucleophilic substitution reactions at the phosphoryl centre is illustrated in Figure 2. In addition to contributing to the withdrawal of electrons from the phosphorus atom, thus activating this centre towards nucleophilic attack, the diehlorovinyloxy group is a better leaving group than methoxy. Consequently, nucleophilic substitution reactions at phosphorus lead to scission of the P - O (dichlorovinyl) bond and to the dimethylphosphorylation of the attacking nucleophile. This reaction mechanism (Fig. 2) is fundamental to both the insecticidal action of dichlorvos and the detoxification of dichlorvos by mammals. Thus, dichlorvos exerts its acute toxic action by inhibiting the enzyme acetylcholinesterase (Braid and Nix, 1969). This is a general reaction of the insecticidal dialkyl phosphoric acid triesters and is thought to be achieved by attack on phosphorus by the serine hydroxyl (or corresponding oxyanion) located at the active centre of the enzyme. This affords, in the ease of dichlorvos, dimethylphosphorylation of the active site and the loss of enzyme activity (Fig. 3) (for reviews see Aldridge and Riener, 1972; Matsumura, 1975). This type of reaction mechanism also applies when the attacking nucleophile is water (Fig. 4). Although the most abundant nucleophile in living organisms, water is also one of the slowest reacting of all nucleophiles. Thus, in order to be effective in affording protection to other nucleophiles, e.g., the dimethylphosphorylation of serinyl moieties in important proteins, the reaction of dichlorvos with water must be accelerated. In mammals, this acceleration is achieved by the catalytic action of esterases. These esterases (arylesterases) are of a different type to those inhibited by dichlorvos and do not contain serine at the active centre (Augustinsson, 1964). Such enzymes are present in all mammalian tissues, including blood. Consequently, the dimethylphosphorylation of water proceeds extremely efficiently and constitutes the predominant CH3-O (~- 0 CH3-O
CH3-O ~ ~0
" 10 mg/kg body weight) (Wennerberg, 1973).
Conclusions
In vitro bacterial tests that have been employed to assess the mutagenicity of dichlorvos take account of all spontaneous reactions that are dependent upon the presence of the parent compound, e.g., methylation reactions, dimethylphosphorylation reactions with attendant generation of dichloroacetaldehyde together with any theoretical reaction such as transfer of the dichloroalkene moiety. Comparisons of quantitative data relating mutagenesis and methylation reactions indicate that the mutagenicity of dichlorvos in simple bacterial systems is due solely to methylation of bacterial DNA. 9 Methylation of DNA could not be demonstrated in mammals under realistic exposure conditions or after exposure to high doses of this compound. The failure of dichlorvos to induce genotoxic effects in mammals is attributed to the very efficient metabolism of dichlorvos in mammals which results in the destruction of the methylating potential of this compound.
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The Chemical and Biochemical Reactivity of Dichlorvos
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