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Pesticide Resistance: Strategies and Tactics for Management. 1986. National Academy Press, Washington, D.C. Resistance to 4-Hydroxycoumann Anticoagulants in Rodents ALAN D. MACNICOLL There are few reported cases of development of resistance to pesticides in vertebrates. The most widespread and well-documented example is resistance to warfarin in rodents. It has been demon- strated in Rattus norvegicus and Mus musculus that inheritance of warfarin resistance is monogenic and the gene is closely linked to that for coat color. The biochemistry and mechanism of resistance in the latter species has not been investigated thoroughly, but war- farin resistance may be associated with an altered metabolism of the anticoagulant. Wa~arin resistance in R. norvegicus is probably as- sociated with alterations in a vitamin K metabolizing enzyme or enzymes. Second-generation anticoagulants, which are more toxic than warfarin, were introduced in the 1970s and were considered elective in controlling warfarin-resistant rodent infestations. Some warfarin-resistant populations may also be cross-resistant to other 4-hydroxycoumarin anticoagulant rodenticides, and control of these infestations with more toxic compounds is less effective than using warfarin to control anticoagulant-susceptible rodents. INTRODUCTION The incidence of inheritable resistance to pesticides in vertebrates is re- markably low. The mosquito fish Gambusia Alibis (Vinson et al., 1963; Boyd and Ferguson, 1964) and other fish species (Ferguson et al., 1964; Ferguson and gingham, 1966) have developed resistance to chlorinated hy- drocarbon pesticides. Also, two frog species may have developed resistance to DDT (Boyd et al., 19631. Incidences of inheritable pesticide resistance in 87
88 MECHANISMS OF RESISTANCE TO PESTICIDES mammals are confined almost exclusively to rodents. Differential suscepti- bility to fluoroacetate, however, has been reported in some areas of Australia in populations of the grey kangaroo and tammar wallaby, as well as the bush rat Rattus fuscipes (Oliver et al., 19791. Inheritable tolerance in these species is thought to be a result of the abundance in some parts of Australia of leguminous plants that naturally produce fluoroacetate. Genetically determined resistance in humans to coumarin anticoagulant drugs, some of which are also used as rodenticides, was first reported in 1964 (O' Reilly et al., 1964~. Resistance to coumarin anticoagulants in rodents is the most widespread and thoroughly investigated example of inheritable pesticide resistance in vertebrates and will be discussed in detail. A laboratory mouse strain has been developed that showed a 1.7-fold tolerance to DDT when compared to the original susceptible strain (Ozburn and Morrison, 19621. This was achieved by treating nine successive gen- erations with DDT and breeding the survivors. Probably more significant was the discovery that pine voles, Microtus pinetorium, trapped in orchards with a history of endrin treatment, had a 12-fold resistance to this compound, compared with voles trapped in untreated orchards (Webb and Horsfall, 19671. These resistant animals also showed a two-fold cross-resistance to dieldrin, a stereoisomer of endrin. This example of inheritable resistance may be associated with alterations in the metabolism of endrin, as indicated by studies on the hepatic, microsomal, mixed-function oxidase system of endrin-resistant and endrin-susceptible strains (Webb et al., 1972; Hartgrove and Webb, 19731. INCIDENCE AND GENETICS OF WARFARIN RESISTANCE IN RODENTS Warfarin resistance in R. norvegicus was first noted in Scotland in 1958 (Boyle, 1960) and subsequently on the Wales-England border (Drummond and Bentley, 1967) and in Denmark (Lund, 1964), Holland (Ophof and Langveld, 1969), Germany (Telle, 1967), and the United States (Jackson and Kaukeinen, 19721. These initial observations were not isolated, and in 1979, it was reported in 36 out of 77 American cities surveyed that more than 10 percent of each R. norvegicus population was warfarin-resistant (Jackson and Ashton, 19801. With evolutionary pressure from the continued use of warfarin, some resistant populations can spread to cover areas of several thousand square kilometers (Greaves, 19701. Inheritance of warfarin resistance in R. norvegicus is due to the inheritance of an autosomal gene, closely linked to the gene controlling coat color, which has been mapped in linkage group I (Greaves and Ayres, 19694. Further genetic studies (Greaves and Ayres, 1977, 1982) on warfarin resistance in wild rats from Wales, Scotland, and Denmark showed that there are at least
RESISTANCE TO 4-HYDROXYCOUMARIN ANTICOAGULANTS 89 three multiple alleles of the warfarin resistance gene Rw. Strains of R. norv- egicus derived from wild Welsh or Danish rats have an increased requirement for vitamin K (Pool et al., 1968; Hermodson et al., 1969; Greaves and Ayres, 1973, 1977; Martin, 1973), but only the Welsh resistance gene is described as dominant (Greaves and Ayres, 1969, 19821. Inheritable warfarin resistance in Rattus rattus has been observed in the United Kingdom (Greaves et al., 1973a, 1976), Australia (Saunders, 1978), and the United States (Jackson and Ashton, 1980~. Warfarin resistance in this species was a significant problem in 4 of 12 American cities where populations had been sampled. Warfarin resistance in the house mouse Mus musculus has followed a similar pattern to that of R. norvegicus. Problems in controlling house mice (Dodsworth, 1961) were initially thought to be due to inheritance of more than one gene (Rowe and Redfern, 1965; Roll, 19661. Subsequent investi- gations (Wallace and MacSwiney, 1976) demonstrated a major warfarin resistance gene, War, that was closely linked to coat color and located on chromosome 7 in the mouse, which is analogous to linkage group I in the rat. Monitoring of warfarin resistance in the house mouse is not routine, but resistance seems to be widespread (Jackson and Ashton, 19801. WARFARIN ACTION AND RESISTANCE MECHANISM The naturally occurring anticoagulant dicoumarol (structure I in Figure 2) was isolated from moldy sweet clover hay in 1939 (Link, 19441. Following observations that cattle that were fed on spoiled sweet clover hay developed a fatal haemorrhagic malady, dicoumarol was subsequently clinically used as a prophylactic agent against thrombosis. Oral vitamin K3 (menadione: structure V in Figure 2) or vitamin Kit were antidotal in excessive hypo- prothrombinaemia (Cromer and Barker, 1944; Lehmann, 19431. This natu- rally occurring coumarin was also considered for rodent control, but it was replaced by a more toxic synthetic analogue, warfarin (structure II in Figure 21. Warfarin was also more suitable than dicoumarol for routine clinical use and for 30 years has been widely used both as a drug and as a rodenticide (Shapiro, 1953; Clatanoff et al., 19541. Despite this widespread dual use of warfarin and the known role of vitamin K as an antidote, little progress was made in elucidating the mode of action of warfarin until the mid-1970s. Vitamin K and warfarin are antagonistic in their effects on the synthesis of blood-clotting factors II, VII, IX, and X. In 1974 y-carboxyglutamic acid residues (GLA) were discovered (Stenflo et al., 1974) in prothrombin (factor II), which were not present in the altered proteins in the blood of cows or humans treated with coumarin anticoagulants. Post-translational ~y-carboxylation of glutamyl residues appears to require the hydroquinone (or reduced form) of vitamin K as a cofactor (Sadowski et al.,
go MECHANISMS OF RESISTANCE TO PESTICIDES Glutamic acid \ y-Carboxy ghutanic acid OH \ O2 + CO2 / o FIR Carboxylase OH ~NAD(P)+ ~\ O ~ ~ _ NAD(P)H ~3 /.. 0~ 11 Ha 11 o FIGURE 1 Schematic representation of the vitamin K cycle. 1980), and vitamin K 2,3-epoxide is a product of this reaction (Larson et al., 198 1 ). An enzyme cycle (Figure 1 ~ exists in liver microsomes to generate vitamin K hydroquinone from the epoxide, with the quinone form of the vitamin as an intermediate product (Fasco and Principe, 1980; Fasco et al., 19821. Administration of warfarin and vitamin Kit to rats increased the ratio of vitamin Kit 2,3-epoxide to vitamin Kit quinone in plasma and liver, when compared with animals that received vitamin Kit alone (Bell and Caldwell, 1973~. This effect was more pronounced in warfarin-susceptible than in warfarin-resistant animals. Further studies confirmed the hypothesis that 4- hydroxycoumarin anticoagulants act by inhibiting the enzyme vitamin K epoxide reductase (Ren et al., 1974, 1977; Shearer et al., 19741. In addition, Sf-)-warfarin was more effective in inhibiting prothrombin synthesis and vitamin K epoxide reductase activity than the Rt + )-enantiomer (Bell and Ren, 1981~. An efficient method for determining the warfarin resistance genotype in R. norvegicus was based partly on the effect of coadministration of vitamin Kit 2,3-epoxide and warfarin on prothrombin synthesis (Martin et al., 1 9791. Analysis of blood-clotting time 24 hours after treatment showed that rats that were either homozygous or heterozygous for the Welsh warfarin resistance gene had normal prothrombin levels, but homozygous-susceptible animals had elongated clotting times. The implication was that warfarin- resistant animals were able to utilize vitamin K 2,3-epoxide in the presence
RESISTANCE TO 4-HYDROXYCOUMARIN ANTICOAGULANTS 91 of warfarin. Other studies showed that warfarin metabolism and excretion were not significantly altered in warfarin-resistant strains of R. norvegicus when compared with a related susceptible strain (Hermodson et al., 1969; Townsend et al., 19751. This evidence, and some from other studies not described above, led to the common belief that 4-hydroxycoumarin anticoagulants inhibit the enzyme vitamin K epoxide reductase, which is altered in warfarin-resistant rats (R. norvegicus), and therefore indirectly inhibits the synthesis of vitamin K- dependent clotting factors. These hypotheses can be questioned on a number of points. All of the supporting evidence has been obtained from investi- gations of the metabolism of vitamin Kit (phylloquinone) and its epoxide, but this form of vitamin K is present only in plant material (McKee et al., 1939~. Vertebrates (Dialameh et al., 1971) as well as invertebrates (Burt et al., 1977) and bacteria (Tishler and Sampson, 1948), synthesize compounds of the vitamin K2 (menaquinone) series. Compounds of the vitamin K2 series have a variable-length polyisoprene (unsaturated) substituent at the 3-position of the 2-methyl 1,4-naphthoquinone nucleus, whereas the side chain of phyl- loquinone is 20 carbon atoms long and has only one double bond. Synthesis of vitamin K2~20', the equivalent of phylloquinone, by chick liver microsomes is inhibited by warfarin in vitro (Dialameh, 1978), and the effects of the Sk - ~ and Rid + )-enantiomers are proportional to the effects on prothrombin synthesis. In addition, menadione (vitamin K3) is as effective as phylloqui- none (when administered intravenously) in relieving vitamin K deficiency in chicks (Dam and Sondergaard, 1953), but it is not as effective an antidote to warfarin (Green, 1966; Griminger, 1966~. Studies on vitamin K metabolism in warfarin-resistant R. norvegicus until recently have only been carried out using animals derived from wild Welsh rats (Pool et al., 1968; Greaves and Ayres, 19691. These rat strains un- doubtedly have an altered hepatic microsomal vitamin K epoxide reductase with reduced sensitivity to warfarin. The activity of this enzyme is, however, as sensitive to warfarin in a strain derived from wild Scottish warfarin- resistant rats as the enzyme from a closely related susceptible strain (MacNicoll, 19854. Studies of warfarin inhibition in vitro of NADH and dithiothreitol- dependent vitamin K reductase (Fasco and Principe, 1980; MacNicoll et al., 1984) have shown that this enzyme is as sensitive to warfarin as vitamin K epoxide reductase, but it probably is not the same enzyme. Similar inves- tigations of the vitamin K-dependent~y-glutamyl carboxylase, however, have shown that this third enzyme of the vitamin K cycle is relatively insensitive to warfarin (Hildebrandt and Suttie, 1982) and is probably not inhibited directly in vivo by 4-hydroxycoumarin anticoagulants. The hypothesis that inhibition of vitamin K epoxide reductase is the only effect of warfarin on vitamin K-dependent protein synthesis and that reduced warfarin sensitivity of this enzyme is the result of expression of all of the different allelic forms
92 MECHANISMS OF RESISTANCE TO PESTICIDES of the warfarin resistance gene in R. norvegicus is, therefore, questionable (Bechtold et al., 1983; MacNicoll, 1985; Preusch and Sutte, 19841. Other hypotheses on the mechanism of warfarin resistance in R. norvegicus have been largely discounted. For example, Ernster et al. (1972) observed that the activity of the enzyme DT-diaphorase was considerably lower in a soluble fraction prepared from the livers of warfarin-resistant rats when com- pared with preparations from susceptible animals. This enzyme is present (in different forms) in several liver fractions, utilizes NADH or NADPH as cofactors, and reduces quinone groups in a number of substrates including menadione (vitamin K3) (Ernster et al., 1960~. DT-diaphorase is also highly sensitive to dicoumarol, and it was concluded (Ernster et al., 1972) that altered activity of this enzyme was a result of expression of the warfarin resistance gene. A later study (Greaves et al., 1973b), however, clearly demonstrated that the different enzyme activities were more correctly as- signed to differences between the Wistar stock, from which the warfarin- resistant animals were derived, and the Sprague-Dawley strain, which was used for the susceptible comparison in the earlier study. This enzyme has been implicated in the production of vitamin K hydroquinone in viva. Highly purified rat-liver cytosolic DT-diaphorase reduced vitamin Kit (Fasco and Principe, 1982~; this reduction was dicoumarol- but not warfarin-sensitive. The results are inconsistent with the warfarin-sensitive NADH or DDT- dependent vitamin K~ hydroquinone formation observed with crude rat-liver microsomal fractions. Recent studies (Lied et al., 1982; Talcott et al., 1983) on the action of DT-diaphorase in detoxification or activation of a wide range of quinones, including some antimalarial drugs, suggests that the capacity of this enzyme for vitamin K reduction is not associated with the ribosomal synthesis of vitamin K-dependent clotting factors. A more recent hypothesis on the mechanism of warfarin resistance in R. norvegicus was based on the formation of 2- or 3-hydroxyvitamin K~ from the epoxide by liver microsomal fractions (Fasco et al., 19831. These putative metabolites were detected in greater quantities in incubations with prepara- tions from warfarin-resistant rats when compared with preparations from susceptible animals. This observation was associated with the reduced activity of vitamin K epoxide reductase in that resistant strain. A second report, however, showed that under certain conditions these hydroxylated com- pounds were formed by a chemical reaction in control incubations (Hilde- brandt et al., 19841. The apparent increase in metabolism to these compounds by liver microsomes from resistant animals probably reflected the reduced rate of metabolism to the quinone form of the vitamin. The detection of hydroxyvitamin K~ in the blood of warfarin-resistant rats that had received an intravenous injection of vitamin K 2,3-epoxide (Preusch and Suttie, 1984), therefore, is probably not associated directly with expression of the warfarin resistance gene.
RESISTANCE TO 4-HYDROXYCOUMARIN ANTICOAGULANTS 93 Little if any work has been carried out on the mechanism of warfarin resistance in R. rattus, but there have been many studies conducted on M. musculus. Observations of the effect of warfarin on mortality and blood clotting in wild warfarin-resistant and -susceptible house mice indicated that resistant animals developed a tolerance to daily doses of warfarin (admin- istered intravenously) up to 100 mg/kg, and susceptible animals developed a tolerance to doses of 1 mg/kg administered at 21-day intervals (Rowe and Redfern, 19681. Female mice were particularly tolerant to warfarin. Animals trapped in areas with control problems had normal clotting times when fed a diet containing 0.025 percent warfarin for 21 days. As mentioned above, warfarin resistance in M. musculus is due to inher- itance of the gene War located on chromosome 7 (Wallace and MacSwiney, 1976~. This resistance may be related to a gene on the same chromosome (Wood and Conney, 1974), which is expressed as an increased rate of hy- droxylation of coumarin. Subsequent investigation of 16 different strains demonstrated that warfarin resistance and rapid coumarin hydroxylation were not coinherited (Lush and Arnold, 19751. Warfarin resistance in this species may be inversely related to hexobarbitone sleeping time, but it is not stim- ulated by phenobarbitone (Lush, 19761. The report suggested that warfarin resistance in the house mouse may be due to an increased rate of warfarin hydroxylation. There are no reports of vitamin K deficiency in warfarin- resistant mouse strains, and it is possible that resistance in this species is related to alterations in warfarin rather than vitamin K metabolism. SECOND-GENERATION ANTICOAGULANT RODENTICIDES The three compounds (Figure 2) based on 4-hydroxycoumarin, commonly known as the second-generation anticoagulant rodenticides, are difenacoum (structure III: when radical = hydrogen), brodifacoum (structure III: when radical = bromine), and bromadiolone (structure IV). The mechanism of action of these compounds is assumed to be the same as for warfarin. The increased toxicity is assigned to the highly lipophilic nature of the substituents at the 3-position of the 4-hydroxycoumarin nucleus (Hadler and Shadbolt, 1975; Dubock and Kaukeinen, 1978~. Initial laboratory studies and field trials indicated that these compounds could effectively control warfarin-re- sistant rat and mouse populations (Hadler, 1975; Hadler et al., 1975; Hadler and Shadbolt, 1975; Redfern et al., 1976; Rennison and Dubock, 1978; Redfern and Gill, 1980; Lund, 1981; Richards, 1981; Rowe et al., 19811. Studies in vitro on the mode of action of difenacoum (Whitlon et al., 1978; Hildebrandt and Suttie, 1982) and in vivo on difenacoum and brodifacoum (Breckenbridge et al., 1978; Leck and Park, 1981) indicated that these com- pounds inhibited the enzyme vitamin K epoxide reductase and were effective in both warfarin-susceptible and -resistant R. norvegicus.
94 MECHANISMS OF RESISTANCE TO PESTICIDES OH AH r - ~'CH I OH ¢4 III OR o m:CH3 o I K, Ra CH' of CH2` ACHE` iCH3 Kit R=CH' off ~H2` ACHE ACHE CH3 CH 3 K3 R-H OH ~ 11 [^H ~C'H EACH II OH ~ OH AH C H`3`Br V FIGURE 2 Chemical structures: I. Dicoumarol. II. Warfare. III. When the radical (R) is hydrogen, the compound is difenacoum. When R is bromine, the compound is bro- difacoum. IV. Bromadiolone. V. Vitamin K. Some early reports on field trials of difenacoum and bromadiolone ex- pressed concern about apparent incidences of cross-resistance observed in some warfarin-resistant populations of R. norvegicus and M. musculus. A laboratory test for difenacoum resistance in R. norvegicus was developed a few years after this compound was introduced as a rodenticide (Redfern and Gill, 19781. A significant widespread incidence of difenacoum resistance was detected in rat populations across an area of English farmland (Greaves et al., 1982a) where a monogenic form of resistance to warfarin had been present for several years. Resistance to difenacoum suggested that this was an example of another allele of the warfarin resistance gene, since no dif- ficulty had been experienced previously in controlling warfarin-resistant pop- ulations of R. norvegicus (Rennison and Dubock, 1978~. Further field trials
RESISTANCE TO 4-HYDROXYCOUMARIN ANTICOAGULANTS 95 of bromadiolone, brodifacoum, and difenacoum in this area showed that these compounds were not as effective in controlling the R. norvegicus populations as warfarin was for controlling warfarin-susceptible infestations (Greaves et al., 1982b). Continued use of 4-hydroxycoumarin anticoagulants in this area may apply evolutionary pressure favoring animals that may be resistant to this whole class of compounds. Since there are several forms of the warfarin resistance gene in R. norvegicus, and inherited resistance in R. rattus and M. musculus, it may be difficult to control rodent infestations in other areas using 4-hydroxycoumarin anticoagulants. CONCLUSION The development of resistance to 4-hydroxycoumarin anticoagulants in rodents may have implications for resistance to other pesticides. Studies on the biochemistry and pharmacology of warfarin resistance may have provided misleading information. Almost all such studies used rat strains derived from wild Welsh rats, and comparative studies have not always used a suitable susceptible control. At least one hypothesis of the mechanism of resistance was erroneously based on a strain difference. The current theory on altered vitamin K epoxide reductase activity may apply only to animals whose re- sistance is associated with an increased susceptibility to vitamin K deficiency. When the highly toxic second-generation anticoagulants were developed, most of the evidence for the control of warfarin-resistant R. norvegicus was based on studies using rats of the Welsh resistant strains. Control of rat infestations in Wales and several other areas was achieved with these com- pounds, but in other areas resistance to the new compounds developed or was already present. It is important, therefore, that appropriate comparative studies are carried out and that when similar compounds are introduced to control pesticide-resistant populations, the potential for cross-resistance is fully investigated. There is not a logical explanation for the apparent confinement of cross- resistance to 4-hydroxycoumarin anticoagulants to the United Kingdom. The long history of widespread use of anticoagulants for rodent control may be significant, but so could the established system for detecting and monitoring rodenticide resistance, which may not be so well developed in other countries. It is likely, therefore, that the continued use of 4-hydroxycoumarin antico- agulants in areas with known warfarin-resistant populations could result in rodent infestations that are difficult to control with any of this class of compounds. REFERENCES Bechtold, H., D. Trenk, T. Meinertz, M. Rowland, and E. Jahnchen. 1983. Cyclic interconversions of vitamin Kit and vitamin Kit 2,3-epoxide in man. Br. J. Clin. Pharmacol. 16:683-689.
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