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International audience; Cyclic water vole population explosions can be controlled in some European countries with anticoagulant rodenticides leading sometimes to wildlife poisonings due to the toxin's tissue persistence. Here, we analyzed the pharmacokinetics of rodenticide residues in voles and we explored potential ways of improving the mass application of these agents based on the concept of stereoisomers.We demonstrated the dramatic persistence of bromadiolone in vole tissues with a hepatic half-life of about 10-30 days, while the tissue persistence of chlorophacinone is rather short with a hepatic half-life of about one day. The dramatic persistence of bromadiolone is due to the trans-isomer group (the major compound in bromadiolone), while the cis-isomer group has a short half-life. Because of resistance to chlorophacinone, the cisbromadiolone isomers may constitute an excellent compromise between efficacy and ecotoxicological risk to control voles. A mathematical model is proposed to favor the development of baits mixed with cis-isomer groups.

International audience; Vitamin K antagonists (VKA) are not recommended during pregnancy because warfarin (a first-generation VKA) is associated with a malformation syndrome "the fetal warfarin syndrome" (FWS). VKA are also used for rodent management worldwide. Recently, the Committee for Risk Assessment responsible for the European chemical legislation for advances on the safe use of chemicals had classed 8 anticoagulant used as rodenticides in the reprotoxic category 1A or 1B. This classification emerges from a read-across prediction of toxicity considering the warfarin malformation syndrome. Herein, our study explores the teratogenicity of warfarin at the human therapeutic dose and that of bromadiolone, a second-generation anticoagulant rodenticide. Using a rat model, our study demonstrates that warfarin used at the therapeutic dose is able to induce teratogenicity, while in the same conditions bromadiolone does not induce any teratogenic effect, challenging the classification of all VKA as reprotoxic molecules.

International audience; The need for the control of rodent populations with anticoagulant rodenticides remains actual, and enantioselective analytical methods are mandatory to understand ecotoxicity issues of those chiral pesticides. This study presents two enantioselective methods to achieve the residue levels and differentiated persistence of the four stereoisomers of difethialone (called in this work E1-trans, E2-cis, E3-cis and E4-trans), which is one of the most toxic second generation anticoagulant rodenticide. Their enantiomeric fraction evaluation in biological matrices of rats was determined by two LC-MS/MS methods. The first one (chiral-LC-MS/MS) combined a chiral column employed in reversed-phase mode (with acetonitrile-water mobile phase) to be compatible with mass spectrometry detection. The second one was also a LC-MS/MS method but with a reversed phase column after a derivatization step with (1S)-(-)-camphanic chloride. Extraction process combined Solid-Liquid extraction and sorbent cartridges. The methods were fully validated. The chiral column was chosen as a reference method for our laboratory because it was quicker and cheaper, and enantioresolution and sensitivity were better. This chiral-LC-MS/MS method was used to measure the enantiomeric fraction of the four stereoisomers of difethialone in rodent biological matrices (liver, plasma, blood and feces) of female rats treated with 3.5 mg/kg of difethialone. The results showed that metabolism is not the same for all the stereoisomers: cis-E3-difethialone was the most persistent, and E4-trans-difethialone was the most quickly eliminated.This chiral-LC-MS/MS method will be used to study the pharmacokinetics of the four stereoisomers of difethialone, and for ecotoxicological surveillance to evaluate the specific persistence of each stereoisomer of difethialone in case of secondary exposure of wildlife non-target species.

The use of pesticides can affect non-target species by causing population declines through indirect intoxication. Small mustelids (SMs; weasels, Mustela nivalis L.; stoats, Mustela erminea L.) consume water voles (WVs, Arvicola scherman S.) and can be exposed to bromadiolone, an anticoagulant rodenticide used in some countries to reduce WV damage to grasslands. Here, we investigated whether bromadiolone affected SM abundance.We monitored SM abundance using footprint tracking tunnels in spring and autumn at ten sites. Among these sites, four were treated with bromadiolone, while six were not treated. We found reduced SM abundance at these four sites from spring to autumn (treated sites, mean ± SE SM abundance change = -1.68 ± 0.42; untreated sites, 0.29 ± 0.25). Using a linear model, we observed that SM abundance decreased as a function of the quantity of bromadiolone applied during the 3 months before the autumn estimate. We found that WV abundance increased at treated sites (linear model, treated sites, mean ± SE WV abundance change = 1.4 ± 0.4; untreated sites, 0.33 ± 0.25). Thus, at treated sites, SM abundance declined despite increased food availability. By analyzing residues in vole livers and SM scats we showed that SMs may be exposed to bromadiolone at the sites where this compound was used.This study is the first to document the relationship between SM abundance and bromadiolone usage for small mammal control. Declines in SM abundance were observed at treated sites, where bromadiolone residue was found in SM scats. This correlative approach suggests that bromadiolone treatment may lead to seasonal SM declines and associated WV increases. © 2018 Society of Chemical Industry.

International audience; In this work, a serie of cyclocoumarol derivatives was designed, synthesized, characterized and studied for their potentialities as selective inhibitors of COX-2. All target compounds have been screened for their anti-inflammatory activity by the assay of PGE2 production. Among them, compound 5d exhibited the most potent inhibitory activity with a PGE2 inhibition compared to NS-398 (79% and 88% respectively) and showed non-inhibitory activity towards the COX-1 enzyme. Docking studies revealed the capacity of this compound to occupy the selective COX-2 cavity establishing additional hydrogen bonds between the oxygen of the methoxy group and the His90 and Arg513 of the binding site of the enzyme.

Since the discovery of Warfarin in the 1940s, the design of new warfarin-derived anticoagulants for rodent management has been challenging, with mainly structural modifications performed on the C3 position of the coumarin skeleton. In order to better understand the pharmacomodulation of such derivatives, we have synthesized a family of C3 (linear and branched) alkyl-4-hydroxycoumarins, which led to the identification of compounds 5e and 5f as potential short-term active anticoagulants.

International audience; 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.

International audience; BACKGROUNDIn 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. RESULTSFrom 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. CONCLUSIONThe 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.

International audience; Worldwide, agricultural uses of anticoagulant rodenticides (ARs) cause poisonings of non-target wildlife as observed in France where bromadiolone is used to control water vole outbreaks. Following bromadiolone field application, a part of the vole population may die above-ground of the treated plots and thus, can represent an important risk of secondary poisoning for scavengers. In this study, water voles were trapped in a non-treated area and their carcasses were placed aboveground in plots located in an area where a vole outbreak occurred. Then, the environmental persistence, the diurnal and nocturnal scavenging rates of water vole carcasses were assessed in autumn 2011 and in spring 2012. The diurnal scavenger species were also identified. The environmental persistence of the carcasses to reach at least a scavenging rate of 87.5 % was 0.5–1.5 day. The average rates of diurnal and nocturnal scavenging ranged from 67 to 100 % and 5 to 100 %, respectively. They depended on the composition of the scavenger community present near the monitored plots; diurnal scavenging rates being higher with corvids than with raptors. In autumn, the red kite and the common buzzard were the main scavengers in one of the plots, what suggests a high risk of poisoning for these raptors during post-nuptial migration. So, the collection of vole carcasses after treatments and the limitations of bromadiolone applications when high densities of predators/scavengers are observed could be implemented to mitigate the risks of secondary poisoning.

International audience; In Europe, bromadiolone, an anticoagulant rodenticide authorized for plant protection, may be applied intensively in fields to control rodents. The high level of poisoning of wildlife that follows such treatments over large areas has been frequently reported. In France, bromadiolone has been used to control water voles (Arvicola terrestris) since the 1980s. Both regulation and practices of rodent control have evolved during the last 15 years to restrict the quantity of poisoned bait used by farmers. This has led to a drastic reduction of the number of cases of poisoned wildlife reported by the French surveillance network SAGIR. During the autumn and winter 2011, favorable weather conditions and high vole densities led to the staging of several hundreds of Red Kites (Milvus milvus) in the Puy-de-Dôme department (central France). At the same time, intensive treatments with bromadiolone were performed in this area. Although no misuse has been mentioned by the authorities following controls, 28 Red Kites and 16 Common Buzzards (Buteo buteo) were found dead during surveys in November and December 2011. For all these birds, poisoning by bromadiolone as the main cause of death was either confirmed or highly suspected. Other observations suggest a possible impact of bromadiolone on the breeding population of Red Kites in this area during the spring 2011. French regulation of vole control for plant protection is currently under revision, and we believe this event calls for more sustainable management of rodent outbreaks. Based on large-scale experiments undertaken in eastern France, we propose that direct control of voles at low density (with trapping or limited chemical treatments) and mechanical destruction of vole tunnels, mole control, landscape management, and predator fostering be included in future regulation because such practices could help resolve conservation and agricultural issues.

International audience

International audience; 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- Comte´ (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.

International audience; In many countries, the fox (Vulpes vulpes), predator of small mammals, is particularly affected by anticoagulant rodenticides such as bromadiolone due to secondary poisoning. Nevertheless, to date, no method of exposure monitoring is applicable in the field over large areas, and no toxicological data are available concerning sensitivity of foxes to bromadiolone. The aim of this work was to compare excretion kinetics of bromadiolone in fox faeces with clinical and haemostatic effects after repeated exposure to intoxicated voles. A sensitive method for the quantification of bromadiolone excretion in fox faeces and plasma was developed, using liquid chromatography combined with electrospray ionisation mass spectrometry (LC-ESI-MS). The LoD was 0.9 μg/kg and 0.15 μg/L, and the LoQ was 3.0 μg/kg and 0.5 μg/L, in faeces and in plasma, respectively. Four captive foxes were fed for 2 or 5 days with water voles (Arvicola terrestris Sherman) spiked with bromadiolone at concentrations close to those measured in the field. Faeces and blood were collected for bromadiolone titration, and blood-clotting tests were performed to monitor fox health daily during ten days and then every 3-4 days until the end of the experiment (D28). Then, after euthanasia, a complete necropsy was performed, and levels of bromadiolone residues in the liver were determined. Bromadiolone residues were detected in faeces 15 hours after the first exposure. They increased dramatically during the exposure period and then gradually decreased, but they remained detectable at the end of the experiment, i.e., 26 days after the last exposure. Bromadiolone residues in plasma showed a similar pattern but were no longer detectable 7 to 24 days after the last exposure. Two foxes presented very severe external haemorrhages, requiring the administration of the antidote vitamin-K1. Bromadiolone residues in faeces and their relationships with exposure and other directmarkers that were measured is discussed. Liver residues and the toxicity data of our study will help to interpret data from fox carcasses collected by wildlife disease surveillance networks. These findings provide a basis for programs aiming to monitor the exposure of wild fox populations to bromadiolone using non-invasive methods based on standard sampling and analysis of residues in faeces.

International audience; 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.

International audience; We document the kinetics of bromadiolone in two rodent populations after a field control of water voles, and their implications for predator exposure. Water voles and common voles were trapped aboveground and underground from 1 to 135 days after bromadiolone treatment in the field. Livers, digestive tracts, and rests of the body were analyzed separately. Our results indicate that 99.6% of the water voles trapped underground and 41% of the common voles trapped aboveground contain bromadiolone residues. Concentrations were maximal between 3.3 and 6.5 days after treatment, according to the tissues examined and the model applied for water voles, and after 1.3 to 3.7 days for common voles. Water voles appeared available almost exclusively for foraging predators. Common voles, found less likely to be poisoned and exhibiting weaker concentrations, were mainly sampled aboveground. The liver, primarily eaten by some predators and scavengers, contains a larger bromadiolone quantity (59% of the total amount found in water voles). The rejection of the digestive tract by those species may lead to a subsequent consumption of voles with higher bromadiolone concentrations (from +3.8 to +5.8% of concentration) and provide a moderate risk increase. After 135 days, eight of the ten water voles and one of the two common voles exhibited detectable residues. Additionally, one specimen presented higher concentrations than the others, and similar to those measured in Voles trapped between the first 15-20 days. This may have consequences on predator intoxications several months after treatment. These results integrate individual differences for the two main rodent species present in treated areas. Implications for predator exposure were investigated at the end of the study and suggest that, if the risk of secondary poisoning is maximal during the first 15-20 days when the rodent densities remain high, exposure conditions are maintained for at least 135 days.

Anticoagulant rodenticides are commonly used to control rodent pests all over the world. These pesticides inhibit the vitamin K cycle in the liver, prevent blood clotting and cause death by hemorrhage. Despite the fact that they were intended for the control of commensal rodents, they may affect non target species, either directly by consumption of contaminated cereal-based baits, or indirectly by consumption of contaminated preys (secondary poisoning). Secondary poisoning of non target species by anticoagulant rodenticides has been confirmed in many mammals and birds species, such as mustelids, foxes and birds of prey (Shore et al., 1996; Berny et al., 1997; Birks 1998; Stone et al., 1999; Stone et al., 2003; Harding 2004; Fournier-Chambrillon et al., 2004). Nevertheless, poisoning of non target species by direct consumption of anticoagulant rodenticides in the form of contaminated baits is poorly documented. Loire Atlantique is a French department well known for its wetlands and marshes in which anticoagulant rodenticides are commonly used especially against coypus (Myocastor coypus) because of the damage to cereal fields, rivers and water ponds banks. The aim of this study was to estimate the exposure of raptors and waterbirds to anticoagulant rodenticides in Loire Atlantique.

International audience; This paper documents the exposure pattern of a population of small mammals to bromadiolone over time in a field-scale follow up. This is the first assessment of the field-scale effect of such control operation on the availability of bromadiolone-exposed A. terrestris prey to nontarget predator species. It indicates that an important risk of poisoning of nontarget species does exist during large-scale field control operations with bromadiolone, which is contradictory to results obtained from laboratory experiments in the early 1980s and consistent with the secondary poisoning hazards due to repeated exposure regularly reported during the past 20 years.

We report here the synthesis of aromatic coumarins and aromatic alpha-quinolones which were evaluated in vitro for their protective potentialities against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage on human liver cell death, i.e., human hepatoma HepG2 cell line and human hepatocytes in primary culture. We found that the presence of a benzylidene at the 3-position or a heterocycle with N and S heteroatoms on the benzopyrone or quinolone system was essential for the protective effect of these compounds against t-BHP-induced decrease in viability of cells. We found also that a methoxy group on the aromatic ring systems decreased this potential. t-BHP-induced cytotoxicity in primary cultures of human hepatocytes could be therefore prevented by these compounds suggesting that they could display hepatoprotective effects in humans.