Are water vole resistant to anticoagulant rodenticides following field treatments?

The anti-vitamin Ks (AVKs) are widely used to control rodent populations. They inhibit Vitamin K regeneration by the Vitamin K Epoxide Reductase (VKOR) and cause a fatal hemorrhagic syndrome. Because of repeated use, some populations of commensal rodents have expressed resistance to these compounds. In Franche-Comté (France), the water vole exhibits cyclic population outbreaks. A second generation AVK, bromadiolone, has been used for the last 20 years to control vole populations. The aim of this study is to determine whether these repeated treatments could have led to the development of resistance to AVKs in water vole populations. We conducted enzymatic and genetic studies on water voles trapped in treated and non treated plot. The results indicate that voles from the most heavily treated area exhibit enzymatic changes in VKOR activity hence arguing for resistance to AVKs and that an intronic haplotype on the vkorc1 gene seems to be associated with these enzymatic changes.

Distribution of VKORC1 single nucleotide polymorphism in wild Rattus norvegicus in France.

BACKGROUND: Anticoagulant rodenticides are commonly used to control rodent pests all over the world. These pesticides inhibit one enzyme of the vitamin K cycle, Vkorc1, and thus prevent blood clotting and cause death by haemorrhage. Resistance to anticoagulants was first observed in Scotland in 1958, and more potent anticoagulants have been developed to overcome this obstacle. Unfortunately, these chemicals are very toxic and cannot be used everywhere. Some authors have shown that resistance to anticoagulants seems closely linked with single nucleotide polymorphism (SNP) in the Vkorc1 gene. RESULTS: This study draws a map of SNP and haplotypes found in Vkorc1 in rats from different areas of France. Some of them had never been described before. Moreover, the Y139F mutation, described previously in France and Belgium, is the most frequent in France. This mutation is known to be associated with a strong resistance to anticoagulants, and it was found in 28% of the samples. CONCLUSION: This biomolecular approach to studying and detecting resistance is easier to carry out than the phenotypic approach measuring blood coagulation time because it can be conducted on biological samples from dead animals, and it is less dangerous for the operator.

Consequences of the Y139F Vkorc1 mutation on resistance to AVKs: in-vivo investigation in a 7th generation of congenic Y139F strain of rats.

OBJECTIVES: In humans, warfarin is used as an anticoagulant to reduce the risk of thromboembolic clinical events. Warfarin derivatives are also used as rodenticides in pest control. The gene encoding the protein targeted by anticoagulants is the Vitamin K-2,3-epoxide reductase subunit 1 (VKORC1). Since its discovery in 2004, various amino acid and transcription-regulatory altering VKORC1 mutations have been identified in patients who required extreme antivitamin K dosages, or wild populations of rodents that were difficult to control with anticoagulant rodenticides. One unresolved question concerns the dependency of the VKORC1 on the genetic background in humans and rodents that respond weakly or not at all to anticoagulants. Moreover, an important question requiring further analyses concerns the role of the Vkorc1 gene in mediating resistance to more recently developed warfarin derivatives (superwarfarins). METHODS: In this study, we bred a quasicongenic rat strain by using a wild-caught anticoagulant resistant rat as a donor to introduce the Y>F amino acid change at position 139 in the Vkorc1 into the genetic background of an anticoagulant susceptible Spraque-Dawley recipient strain. RESULTS AND CONCLUSION: In this manuscript we report the prothrombin times measured in the F7 generation after exposure to chlorophacinone, bromadiolone, difenacoum and difethialone. We observed that the mutation Y139F mediates resistance in an otherwise susceptible genetic background when exposed to chlorophacinone and bromadiolone. However, the physiological response to the super-warfarins, difenacoum and difethialone, may be strongly dependent on other genes located outside the congenic interval (28.3 cM) bracketing the Vkorc1 in our F7 generation congenic strain.

Heterogeneity of the coumarin anticoagulant targeted vitamin K epoxide reduction system. Study of kinetic parameters in susceptible and resistant mice (Mus musculus domesticus).

Vitamin K epoxide reductase (VKOR) activity in liver microsomes from a susceptible and a genetically warfarin-resistant strain of mice (Mus Musculus domesticus) was analyzed to determine the mechanism of resistance to this 4-hydroxycoumarin derivative. Kinetic parameters for VKOR were calculated for each strain by incubating liver microsomes with vitamin K epoxide +/- warfarin. In susceptible mice, an Eadie-Hofstee plot of the data was not linear and suggested the involvement of at least two different components. Apparent kinetic parameters were obtained by nonlinear regression using a Michaelis--Menten model, which takes into account two enzymatic components. Component A presents a high Km and a high Vm, and as a consequence only an enzymatic efficiency Vm/Km was obtained (0.0024 mL/min/mg). Estimated warfarin Ki was 0.17 microM. Component B presented an apparent Km of 12.73 microM, an apparent Vm of 0.32 nmol/min/mg, and an apparent Ki for warfarin of 6.0 microM. In resistant mice, the enzymatic efficiency corresponding to component A was highly decreased (0.0003-0.00066 mL/min/mg) while the Ki for warfarin was not modified. The apparent Vm of component B was poorly modified between susceptible and resistant mice. The apparent Km of component B observed in resistant mice was similar to the Km observed in susceptible mice. These modifications of the catalytic properties are associated with a single nucleotide polymorphism (T175G) in the VKOR-C1 gene, which corresponds to a Trp59Gly mutation in the protein.