Virginie Lattard

Biochemical characterization of spontaneous mutants of rat VKORC1 involved in the resistance to antivitamin K anticoagulants

Antivitamin K anticoagulants have been commonly used to control rodent pest all over the world for more than 50 years. These compounds target blood coagulation by inhibiting the vitamin K epoxide reductase (VKORC1), which catalyzes the reduction of vitamin K 2,3-epoxide to vitamin K. Resistance to anticoagulants has been reported in wild rat populations from different countries. From these populations, several mutations of the rVkorc1 gene have been reported. In this study, rat VKORC1 and its most frequent mutants L120Q-, L128Q-, Y139C-, Y139S- and Y139F-VKORC1 were expressed as membrane-bound proteins in Pichia pastoris and characterized by the determination of kinetic and inhibition parameters. The recombinant rVKORC1 showed similar properties than those of the native proteins expressed in the rat liver microsomes, validating the expression system as a good model to study the consequences of VKORC1 mutations. The determination of the inhibition parameters towards various antivitamin K anticoagulants demonstrated that mutations at Leu-120, Leu-128 and Tyr-139 confer the resistance to the first generation AVKs observed in wild rat populations.

VKORC1L1, an Enzyme Rescuing the Vitamin K 2,3-Epoxide Reductase Activity in Some Extrahepatic Tissues during Anticoagulation Therapy

Background: Effective involvement of VKORC1L1 in vitamin K epoxide reductase activity, target of vitamin K antagonists (VKAs), is still unclear. Results: VKORC1L1 is not inhibited by VKAs and catalyzes VKOR activity in extrahepatic tissues. Conclusion: During long term anticoagulation the limited unwanted side effects of VKAs are due to VKORC1L1. Significance: Potential pharmaco-toxicologic effects of specific VKORC1L1 inhibitors should be assessed. Vitamin K is involved in the γ-carboxylation of the vitamin K-dependent proteins, and vitamin K epoxide is a by-product of this reaction. Due to the limited intake of vitamin K, its regeneration is necessary and involves vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is known to be supported by VKORC1 protein, but recently a second gene, VKORC1L1, appears to be able to support this activity when the encoded protein is expressed in HEK293T cells. Nevertheless, this protein was described as being responsible for driving the vitamin K-mediated antioxidation pathways. In this paper we precisely analyzed the catalytic properties of VKORC1L1 when expressed in Pichia pastoris and more particularly its susceptibility to vitamin K antagonists. Vitamin K antagonists are also inhibitors of VKORC1L1, but this enzyme appears to be 50-fold more resistant to vitamin K antagonists than VKORC1. The expression of Vkorc1l1 mRNA was observed in all tissues assayed, i.e. in C57BL/6 wild type and VKORC1-deficient mouse liver, lung, and testis and rat liver, lung, brain, kidney, testis, and osteoblastic cells. The characterization of VKOR activity in extrahepatic tissues demonstrated that a part of the VKOR activity, more or less important according to the tissue, may be supported by VKORC1L1 enzyme especially in testis, lung, and osteoblasts. Therefore, the involvement of VKORC1L1 in VKOR activity partly explains the low susceptibility of some extrahepatic tissues to vitamin K antagonists and the lack of effects of vitamin K antagonists on the functionality of the vitamin K-dependent protein produced by extrahepatic tissues such as matrix Gla protein or osteocalcin.

VKORC1 mutations detected in patients resistant to vitamin K antagonists are not all associated with a resistant VKOR activity

Summary.  Background: The VKORC1 gene codes for the VKORC1 enzyme, which is responsible for the reduction of vitamin K epoxide into vitamin K. VKORC1 enzyme is the target of vitamin K antagonists (VKA). Twenty‐eight rare single mutations in the VKORC1 coding sequence have been reported from resistant patients receiving unusually high doses of VKA to achieve therapeutic anticoagulation.

Development of an Ecofriendly Anticoagulant Rodenticide Based on the Stereochemistry of Difenacoum.

Difenacoum, an antivitamin K anticoagulant, has been widely used as rodenticide to manage populations of rodents. Difenacoum belongs to the second generation of anticoagulant, and, as all the molecules belonging to the second generation of anticoagulant, difenacoum is often involved in primary poisonings of domestic animals and secondary poisonings of wildlife by feeding contaminated rodents. To develop a new and ecofriendly difenacoum, we explored in this study the differences in properties between diastereomers of difenacoum. Indeed, the currently commercial difenacoum is a mixture of 57% of cis-isomers and 43% of trans-isomers. Cis- and trans-isomers were thus purified on a C18 column, and their respective pharmacokinetic properties and their efficiency to inhibit the coagulation of rodents were explored. Tissue persistence of trans-isomers was shown to be shorter than that of cis-isomers with a half-life fivefold shorter. Efficiency to inhibit the vitamin K epoxide reductase activity involved in the coagulation process was shown to be similar between cis- and trans-isomers. The use of trans-isomers of difenacoum allowed to drastically reduce difenacoum residues in liver and other tissues of rodents when the rodent is moribund. Therefore, secondary poisonings of wildlife should be decreased by the use of difenacoum largely enriched in trans-isomers.

New insights into the catalytic mechanism of vitamin K epoxide reductase (VKORC1) - The catalytic properties of the major mutations of rVKORC1 explain the biological cost associated to mutations

The systematic use of antivitamin K anticoagulants (AVK) as rodenticides caused the selection of rats resistant to AVKs. The resistance is mainly associated to genetic polymorphisms in the Vkorc1 gene encoding the VKORC1 enzyme responsible for the reduction of vitamin K 2,3‐epoxide to vitamin K. Five major mutations, which are responsible for AVK resistance, have been described. Possible explanations for the biological cost of these mutations have been suggested. This biological cost might be linked to an increase in the vitamin K requirements. To analyze the possible involvement of VKORC1 in this biological cost, rVKORC1 and its major mutants were expressed in Pichia pastoris as membrane‐bound proteins and their catalytic properties were determined for vitamin K and 3‐OH‐vitamin K production. In this report, we showed that mutations at Leu‐120 and Tyr‐139 dramatically affect the vitamin K epoxide reductase activity. Moreover, this study allowed the detection of an additional production of 3‐hydroxyvitamin K for all the mutants in position 139. This result suggests the involvement of Tyr‐139 residue in the second half‐step of the catalytic mechanism corresponding to the dehydration of vitamin K epoxide. As a consequence, the biological cost observed in Y139C and Y139S resistant rat strains is at least partially explained by the catalytic properties of the mutated VKORC1 involving a loss of vitamin K from the vitamin K cycle through the formation of 3‐hydroxyvitamin K and a very low catalytic efficiency of the VKOR activity.

Core-shell LC–MS/MS method for quantification of second generation anticoagulant rodenticides diastereoisomers in rat liver in relationship with exposure of wild rats

Second generation anticoagulant rodenticides (SGARs), pesticides used worldwide to control rodent populations, exist in two diastereoisomer chemical species because they own two stereogenic centers. A core-shell LC-MS/MS multi-residue method for comprehensive quantitative analysis of the diastereoisomers of five SGARs as well as three first generation anticoagulant rodenticide molecules has been fully validated in liver of rats according to a bioanalytical guideline. A core-shell column (superficially porous particles) has been chosen for its ability to separate the diastereomers of bromadiolone, difenacoum, brodifacoum, flocoumafen and difethialone and for its robustness to rat liver extracts. The highly selective chromatographic separation of the diastereoisomers contributes to good signal to noise ratios and then enhances the sensitivity of the method compared to the ones of fully porous columns. An elution gradient has been optimized with 10mM ammonium acetate and acetonitrile as aqueous/organic mobile phase respectively. Triple quadrupole mass detector has been used to achieve specifity and LLOQ from 0.92 to 2.2ng/g for each diastereoisomer, or first generation anticoagulant rodenticides. Then we evidenced diastereoisomeric ratios in liver of rats issued from not controlled exposure of wild rats (Rattus norvegicus) trapped in a French Parisian park through a campaign of rodent eradication. We compared them to diastereoisomeric ratios in SGARs commercial baits that contain both isomers, and showed that one of the two diastereoiomers had nearly disappeared in liver of rats. The proportions of cis-bromadiolone and trans-difenacoum were really lowered compared to the baits: 5/7 and 9/12 rats had only trans-bromadiolone and cis-difenacoum hepatic residues respectively. Liver persistence of the two diastereoisomers of bromadiolone and difenacoum was different due to differences in their pharmacokinetics in wild rats. The new core-shell LC-MS/MS method is particularly well adapted for further exploration of diastereoisomers ratios in rodent and predatory wildlife biological samples in order to evaluate ecological consequences of actual baits, to explore new formulated baits with a good balance between efficacity (ability to kill rodents) and diastereoisomers persistence, and hopefully to mitigate exposure of non-target species.

Management of rodent populations by anticoagulant rodenticides: Toward third-generation anticoagulant rodenticides

Second-generation anticoagulant rodenticides (SGARs) have been used since the 1980s for pest management. They are highly efficient even in warfarin-resistant rodents. Nevertheless, because of their tissue persistence, nontarget poisoning by SGARs is commonly described in wildlife. Due to this major problem, a new generation of anticoagulants must be developed to limit this risk. This study proposes a method of developing a new generation of anticoagulant rodenticides by revisiting the old SGARs based on the concept of stereochemistry. Each current SGAR is a mixture of diastereomers. Diastereomers of each compound were purified, and their biologic properties were compared by determining their ability to inhibit vitamin K epoxide reductase (VKOR) activity involved in the activation of vitamin K-dependent clotting factors and their toxicokinetic properties. Systematically, for each SGAR, both diastereomers are as effective in inhibiting VKOR activity. However, their toxicokinetic properties are very different, with one of the two diastereomers always more rapidly cleared than the other one. For all SGARs except flocoumafen, the less persistent diastereomer is always the less predominant isomer present in the current mixture. Therefore, the development of baits containing only the less persistent diastereomer would avoid the ecotoxicological risk associated with their use without decreasing their efficacy.

Evidence of a target resistance to antivitamin K rodenticides in the roof rat Rattus rattus: identification and characterisation of a novel Y25F mutation in the Vkorc1 gene

BACKGROUND In spite of intensive use of bromadiolone, rodent control was inefficient on a farm infested by rats in Zaragoza, Spain. While metabolic resistance was previously described in this rodent species, the observation of a target resistance to antivitamin K rodenticides had been poorly documented in Rattus rattus. RESULTS From rats trapped on the farm, cytochrome b and Vkorc1 genes were amplified by PCR and sequenced in order to identify species and detect potential Vkorc1 mutations. VKORC1-deduced amino acid sequences were thus expressed in Pichia pastoris, and inhibition constants towards various rodenticides were determined. The ten rats trapped on the farm were all identified as R. rattus. They were found to be homozygous for the g.74A>T nucleotide replacement in exon 1 of the Vkorc1 gene, leading to p.Y25F mutation. This mutation led to increased apparent inhibition constants towards various rodenticides, probably caused by a partial loss of helical structure of TM4. CONCLUSION The p.Y25F mutation detected in the Vkorc1 gene in R. rattus trapped on the Spanish farm is associated with the resistance phenotype to bromadiolone that has been observed. It is the first evidence of target resistance to antivitamin K anticoagulants in R. rattus.

Identification and characterization of the FMO2 gene in Rattus norvegicus: a good model to study metabolic and toxicological consequences of the FMO2 polymorphism.

BACKGROUND In lung of many animal species flavin-containing monooxygenase 2 (FMO2) is a 535-amino acid residues drug-metabolizing enzyme. In humans FMO2 exhibits a genetic polymorphism. The major allele encodes a truncated FMO2, the minor allele a full-length FMO2. In laboratory rats we previously reported a FMO2 gene encoding a truncated FMO2 (432-AA residues). In these strains, a double deletion leads to the appearance of a premature stop codon. All laboratory rat strains were derived from the same wild ancestor, Rattus norvegicus. METHODS A PCR-based method able to specifically recognize either the wild-type or the mutant allele was developed to investigate a putative FMO2 polymorphism in a population of wild rats. The FMO2 gene was analyzed in 42 wild rats. RESULTS A genetic FMO2 polymorphism similar to that described in humans was found in R. norvegicus. We observed three different genotypes: homozygotes for the wild-type FMO2 (33.3%), homozygotes for the mutant FMO2 (38.1%) and heterozygotes (28.6%). Comparative FMO2 mRNA and protein expressions in lungs were studied by reverse transcription-PCR and western blotting. FMO2 mRNA expression was identical between the three groups. In contrast, major differences in the expression of FMO2 protein were detected. FMO2 was strongly expressed in lungs of homozygotes for the wild-type FMO2, faintly expressed in lungs of heterozygotes and non-expressed in lungs of homozygotes for the mutant FMO2. Comparative catalytic properties of lung microsomes were studied by the determination of the oxygenation of methimazole. FMO2 genetic polymorphism was associated with major differences in the S-oxidative metabolism.

Cis-bromadiolone diastereoisomer is not involved in bromadiolone Red Kite ( Milvus milvus) poisoning

Anticoagulant rodenticides (ARs) are widely used pesticides to control rodent populations. Bromadiolone, a second generation anticoagulant rodenticide (SGARs), is authorized in France to control the population of water voles (Arvicola scherman). The persistence of SGARs in rodents is responsible for secondary exposure or poisoning of predators and scavengers, and is of ecological concern for the conservation of endangered species. Commercial formulations are a mixture of two diastereoisomers of bromadiolone: 70-90% is trans-bromadiolone and 10-30% is cis-bromadiolone. Both diastereoisomers have been shown to inhibit coagulation function with the same potency. On the other hand, cis-bromadiolone has been shown to be less tissue-persistent than trans-bromadiolone in rats. This difference led to residue levels in rats with substantially weakened proportion in cis-bromadiolone compared to the composition of baits. In this study, a multi-residue LC-MS/MS method for the quantification of the diastereoisomers of SGARs was used to investigate their proportions in field samples of predators. In 2011, 28 red kites (Milvus milvus) were found dead within a few months of bromadiolone application in grassland to control water vole outbreaks. In this study, we report the concentrations of the two diastereoisomers of bromadiolone measured in the livers of thirteen red kites. Exposure to bromadiolone was apparent in all the kites with hepatic concentrations of trans-bromadiolone ranging from 390 to 870ng/g (89 to 99% of summed SGARs). However, cis-bromadiolone was not detected in 5 of 13 red kites and was present at very low concentrations (below 2.2ng/g) in 8 of 13 kites, demonstrating that cis-bromadiolone is not involved in this red kite poisoning event. The results suggest that a change of the proportions of bromadiolone diastereoisomers in baits could reduce the risk of secondary poisoning of predators, but retain primary toxicity for control rodent outbreaks.