Comparative pharmacokinetics of difethialone stereoisomers in male and female rats and mice: development of an intra- and inter-species model to predict the suitable formulation mix.

The ecotoxicity of anticoagulants used for rodent pests' management is a major concern, particularly with second generation anticoagulants, which are more persistent in the body of rodents and therefore more likely to cause secondary exposure in their predators. One of the solutions envisaged to mitigate this risk is to use stereoisomers of these anticoagulants, each of which has particular pharmacokinetics. However, the few studies published to date have considered only one species and one sex. Here, we study the pharmacokinetics of the 4 stereoisomers of 3.4 mg/kg of difethialone in rats (Rattus norvegicus) and 3 mg/kg in mice (Mus musculus) in both sexes and propose a model to choose the optimal stereoisomer efficacy/ecotoxicity mixture for the management of all these animals. Our results show that while the most persistent stereoisomer (E3-cis) is common to both species and sexes, the pharmacokinetics of the other stereoisomers show marked differences between sexes and species. Thus, the area under curve (AUC) of E4-trans in male rats is four times lower than in females or mice, making it a priori unusable in male rats. Conversely, our modeling seems to show that the E1-trans stereoisomer seems to offer the best compromise AUC persistence. In conclusion, we highlight that studies on anticoagulants must necessarily integrate research on the effect of gender and species both on efficacy and with regard to the ecotoxicity of these molecules.

Mass spectrometry characterization of anticoagulant rodenticides and hydroxyl metabolites.

RATIONALE: Anticoagulant rodenticides (ARs) are used worldwide for rodent population control to protect human health and biodiversity, and to prevent agricultural and economic losses. Rodents may develop a metabolic resistance to ARs. In order to help understand such metabolic resistance, mass spectrometry was used to position the hydroxylated group of hydroxyl metabolites of second-generation ARs (SGARs). METHODS: Most AR pesticides are derived from the 4-hydroxycoumarin/thiocoumarin family. We used low-resolution and high-resolution mass spectrometry to understand the fragmentation pathways of the ARs and their respective metabolites, and to better define the structure of their tandem mass spectrometry product ions. RESULTS: Seven specific product ions were evidenced for five ARs, with their respective chemical structures. Those ions were obtained as well from the mass spectra of the hydroxyl metabolites of four SGARs, difenacoum (DFM), brodifacoum (BFM), difethialone (DFTL) and flocoumafen (FLO), with different positions of the hydroxyl group. CONCLUSIONS: The differences in chemical structure between DFM on the one hand and BFM, FLO and DFTL on the other could explain the differences in bioavailability between these two groups of molecules. The defined product ions will be used to investigate the part played by the metabolic issue in the field resistance of SGARs.

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.

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.

Plant Secondary Metabolites as Rodent Repellents: a Systematic Review.

The vast number of plant secondary metabolites (PSMs) produced by higher plants has generated many efforts to exploit their potential for pest control. We performed a systematic literature search to retrieve relevant publications, and we evaluated these according to PSM groups to derive information about the potential for developing plant-derived rodent repellents. We screened a total of 54 publications where different compounds or plants were tested regarding rodent behavior/metabolism. In the search for widely applicable products, we recommend multi-species systematic screening of PSMs, especially from the essential oil and terpenoid group, as laboratory experiments have uniformly shown the strongest effects across species. Other groups of compounds might be more suitable for the management of species-specific or sex-specific issues, as the effects of some compounds on particular rodent target species or sex might not be present in non-target species or in both sexes. Although plant metabolites have potential as a tool for ecologically-based rodent management, this review demonstrates inconsistent success across laboratory, enclosure, and field studies, which ultimately has lead to a small number of currently registered PSM-based rodent repellents.

Synthesis and methemoglobinemia-inducing properties of benzocaine isosteres designed as humane rodenticides.

A number of isosteres (oxadiazoles, thiadiazoles, tetrazoles and diazines) of benzocaine were prepared and evaluated for their capacity to induce methemoglobinemia-with a view to their possible application as humane pest control agents. It was found that an optimal lipophilicity for the formation of methemoglobin (metHb) in vitro existed within each series, with 1,2,4-oxadiazole 3 (metHb%=61.0±3.6) and 1,3,4-oxadiazole 10 (metHb%=52.4±0.9) demonstrating the greatest activity. Of the 5 candidates (compounds 3, 10, 11, 13 and 23) evaluated in vivo, failure to induce a lethal end-point at doses of 120mg/kg was observed in all cases. Inadequate metabolic stability, particularly towards hepatic enzymes such as the CYPs, was postulated as one reason for their failure.

Synthesis and methemoglobinemia-inducing properties of analogues of para-aminopropiophenone designed as humane rodenticides.

A number of structural analogues of the known toxicant para-aminopropiophenone (PAPP) have been prepared and evaluated for their capacity to induce methemoglobinemia--with a view to their possible application as humane pest control agents. It was found that an optimal lipophilicity for the formation of methemoglobin (metHb) in vitro existed for alkyl analogues of PAPP (aminophenones 1-20; compound 6 metHb% = 74.1 ± 2). Besides lipophilicity, this structural sub-class suggested there were certain structural requirements for activity, with both branched (10-16) and cyclic (17-20) alkyl analogues exhibiting inferior in vitro metHb induction. Of the four candidates (compounds 4, 6, 13 and 23) evaluated in vivo, 4 exhibited the greatest toxicity. In parallel, aminophenone bioisosteres, including oximes 30-32, sulfoxide 33, sulfone 34 and sulfonamides 35-36, were found to be inferior metHb inducers to lead ketone 4. Closer examination of Hammett substituent constants suggests that a particular combination of the field and resonance parameters may be significant with respect to the redox mechanisms behind PAPPs metHb toxicity.

Simultaneous determination of nine anticoagulant rodenticides in soil and water by LC-ESI-MS.

A new and sensitive analytical method is presented to determine nine anticoagulant rodenticide (chlorophacinone, bromadiolone, pindone, diphacinone, warfarin, coumatetralyl, brodifacoum, floucomafen, and difenacoum) residues in water and soil samples by LC-ESI-MS. Rodenticides were extracted from soil using a methanol and ammonium formate 30 mM mixture, while ethyl acetate was employed in the water samples. A Gemini 5 µm C18 column was employed, and a mobile phase comprising a mixture of ammonium formate 30 mM and di-n-butylamine 30 mM in water (pH 3.5), ammonium formate 30 mM and di-n-butylamine 20 mM in water (pH 4.4), ammonium formate 30 mM in water (pH 6.5), and methanol in a gradient elution mode was selected. The method was fully validated and it was found to be selective and precise in terms of linearity and accuracy. Extraction recoveries ranged from 90 to 104% for the compounds studied, while the detection and quantification limits were between 0.09 and 2.2 µg/kg in soil or 0.08 and 1.7 µg/L in water. The method was applied to simultaneously measure these compounds in water and soil samples.

Occupational phosphine gas poisoning at veterinary hospitals from dogs that ingested zinc phosphide--Michigan, Iowa, and Washington, 2006-2011.

Zinc phosphide (Zn3P2) is a readily available rodenticide that, on contact with stomach acid and water, produces phosphine (PH3), a highly toxic gas. Household pets that ingest Zn3P2 often will regurgitate, releasing PH3 into the air. Veterinary hospital staff members treating such animals can be poisoned from PH3 exposure. During 2006-2011, CDC's National Institute for Occupational Safety and Health (NIOSH) received reports of PH3 poisonings at four different veterinary hospitals: two in Michigan, one in Iowa, and one in Washington. Each of the four veterinary hospitals had treated a dog that ingested Zn3P2. Among hospital workers, eight poisoning victims were identified, all of whom experienced transient symptoms related to PH3 inhalation. All four dogs recovered fully. Exposure of veterinary staff members to PH3 can be minimized by following phosphine product precautions developed by the American Veterinary Medical Association (AVMA). Exposure of pets, pet owners, and veterinary staff members to PH3 can be minimized by proper storage, handling, and use of Zn3P2 and by using alternative methods for gopher and mole control, such as snap traps.

Antemortem and postmortem rodenticide analysis in forensic toxicology as a part of an LC-MS/MS-based multi-target screening strategy.

Since rodenticides represent a substance group relevant in toxicological analyses, the aim of this work was the development of a complex multi-target screening strategy for the identification with liquid chromatography-tandem mass spectrometry. A simple protein precipitation was used as the sample preparation strategy. Further, a Luna 5 µm C18 (2) 100 Å, 150 × 2 mm analytical column was applied for the separation of relevant analytes with a Shimadzu HPLC. Signal detection was performed with a SCIEX API 5500 QTrap MS/MS system. The rodenticides investigated (α-chloralose, brodifacoum, bromadiolone, coumatetralyl, difenacoum, and warfarin) could be incorporated effectively into a multi-target screening strategy covering about 250 substances representing different groups with a limit of detection appropriate for substance identification. The strategy can easily be modified to perform semi-quantitative measurements for this substance group and could be supplemented by quantification based on standard addition.

Enantiomer fraction evaluation of the four stereoisomers of second-generation anticoagulant rodenticides in biological matrices with polysaccharide-based chiral selectors and liquid chromatography tandem mass spectrometry.

Numerous cases of wildlife exposure to five second-generation anticoagulant rodenticides have been reported worldwide, and residues of these chiral pesticides in biological matrices are still quantified by achiral liquid chromatography methods. However, they are a mixture of cis- and trans-diastereomers, thus a mixture of four stereoisomers. Their persistence must be evaluated in a differentiated way in the food chain of concerned predator species in order to reduce the environmental impact. This article presents an evaluation of the chiral selectivity of five polysaccharide-based chiral selectors for the four stereoisomers of bromadiolone, difenacoum, brodifacoum, flocoumafen and difethialone. Different chromatographic parameters, influencing the chiral separation, such as organic modifier (acetonitrile, methanol), percentage of formic acid and water content in the mobile phase are systematically tested for all columns. It was shown that little amount of water added to the acetonitrile mobile phase may influence the retention behaviors between reversed phase and HILIC-like modes, and consequently the enantiomer elution order of the four stereoisomers. On the contrary, reversed phase is always the observed mode for the methanol water mobile phase. A suitable combination of all these parameters is presented for each second-generation anticoagulant rodenticide with a description of the enantioresolution, the enantiomer elution order and the retention times of the respective stereoisomers. A method is validated for all stereoisomers of each second-generation anticoagulant rodenticide with chicken liver and according to an official bioanalytical guideline. As an example, the enantiomer fraction is evaluated in the liver of a raptor species (rodent predator) exposed to bromadiolone and difenacoum. The results showed that only one enantiomer of trans-bromadiolone and one enantiomer of cis-difenacoum is present in hepatic residues, although all four stereoisomers are present in bromadiolone and difenacoum rodenticide baits.

Chiral liquid chromatography-tandem mass spectrometry analysis of superwarfarin rodenticide stereoisomers - Bromadiolone, difenacoum and brodifacoum - In human plasma.

Superwarfarins are second-generation long-acting anticoagulant rodenticides that can cause unintended human and wildlife toxicity due, in part, to their prolonged half-lives. Commercially available superwarfarin rodenticides are synthesized as racemates with two asymmetric carbons, producing four stereoisomers. To support studies of human plasma half-lives of individual superwarfarin stereoisomers, a method was developed based on LC-MS/MS to separate and quantify stereoisomers of the commercially important superwarfarins bromadiolone, difenacoum and brodifacoum. Human plasma samples were prepared using protein precipitation and centrifugation. Chiral-phase HPLC separation was carried out on-line with tandem mass spectrometric quantitative analysis of the eluting stereoisomers using selected-reaction monitoring with positive ion electrospray on a triple quadrupole mass spectrometer. All four stereoisomers of each superwarfarin were resolved within 12.5 min with calibration curves spanning 2-3 orders of magnitude and lower limits of quantitation between 0.87 and 2.55 ng/mL. This method was used to determine the half-lives of superwarfarin stereoisomers in plasma from patients who had inhaled synthetic cannabinoid products contaminated with superwarfarins. These data may be used to guide the development of safer next generation anticoagulant rodenticides stereoisomers.

Water vole management - Could anticoagulant rodenticides stereochemistry mitigate the ecotoxicity issues associated to their use?

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 cis-bromadiolone 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.

Accidental chlorophacinone exposure of lactating ewes: Clinical follow-up and human health dietary implications.

Anticoagulant rodenticides are widely used for rodent control in agricultural and urban settings. Their intense use can sometimes result in accidental exposure and even poisoning of livestock. Can milk, eggs or meat derived from such accidently exposed animals be consumed by humans? Data on the pharmacokinetics of chlorophacinone in milk of accidently exposed ewes were used to estimate the risk associated with its consumption. Three days after accidental ingestion, chlorophacinone was detected in plasma of 18 ewes, with concentrations exceeding 100 ng/mL in 11 animals. Chlorophacinone was detected in milk on day 2 post-exposure and remained quantifiable for at least 7 days in milk of these 11 ewes. Concentrations in milk were much lower than in plasma and decreased quickly (mean half-life of 2 days). This study demonstrated dose-dependent mammary transfer of ingested chlorophacinone. Variation in prothrombin time (PT) on Day 3 suggested that some of the ewes that ingested chlorophacinone may have been adversely affected, but PT did not facilitate estimation of the quantity of chlorophacinone consumed. Using safety factors described in the literature, consumption of dairy products derived from these ewes after a one-week withdrawal period would pose low risk to consumers.

[Spectroscopic study on interaction of rodenticide brodifacoum with bovine serum albumin].

The mutual interaction of bovine serum albumin (BSA) with brodifacoum (3-[3-(4'-bromophenyl-4) 1,2,3,4-tetralin-10]-4-hydroxyl-coumarin), an anticoagulant rodenticide, was investigated by ultra-violet spectroscopy, flurorescence spectroscopy and synchronous fluorescence spectroscopy under physiological conditions. It was proved that the intrinsic fluorescence quenching of BSA by brodifacoum was the result of the formation of brodifacoum-BSA complex. And this quenching is mainly due to static fluorescence quenching. The quenching rate constant (K(sv)), binding site number (n) and binding constant (KA) at different temperatures were calculated from the double reciprocal Lineweaver-Burk plots and the quenching function of lg[(F0 - F)/F] - lg[Q] plots. The thermodynamic parameters indicated that the process of binding was a spontaneous molecular interaction and the hydrophobic force played a major role in stabilizing the brodifacoum BSA complex. The binding distance r between brodifacoum and BSA was 2.84 and 2.87 nm at 20 and 30 degrees C, respectively, which was obtained based on Forster theory of non-radiation energy transfer. The synchronous spectroscopy of BSA and brodifacoum-BSA revealed that the BSA conformation had changed in the presence of brodifacoum. The binding mode and interaction mechanism were suggested as follows: brodifacoum molecules are closed with amino acid residues with electric charge on the hydrophobic cavities of BSA by electrostatic interaction, and binded to the Trp212 residues inside of BSA hydrophobic cavities by hydrophobic interaction force, thereby changed the microenvironment around the Trp residues. The interaction prevented the energy transfer between Tyr and Trp residues, moreover, caused to a non-radiation energy transfer from Trp residues in BSA to brodifacoum, and finally leaded of the quenching the intrinsic fluorescence of BSA.

[Study of the interaction mechanism between brodifacoum and DNA by spectroscopy].

The interaction between brodifacoum (3-[3-(4'-bromophenyl-4) 1,2,3,4-tetralin-10]-4-hydroxyl-coumarin) (BDF), an anticoagulant rodenticide, and calf thymus DNA (ct-DNA) was studied by UV spectrum and fluorescence spectrum. The results were summarized as follows: There was a hypochromic effect of low concentration ct-DNA on the UV spectra. The fluorescence quenching studies showed a regular decrease in the fluorescence intensity after addition of ct-DNA by the static quenching mode with a quenching constant (Ksv) of 1.21 x 10(4) L x mol(-1) at 27 degrees C. The BDF possibly bonded to ct-DNA mainly via Van der Waals forces by the corresponding thermodynamics parameter. KI quenching experiment found that there was not obvious protection of ct-DNA to BDF. The fluorescence intensity of BDF/ct-DNA system changed with the variation in ionic strength Quenching of ct-DNA on the fluorescence of BDF/beta-CD inclusion complex was reduced in contrast with the free BDF, which showed that beta-CD could provide BDF with protection. So the comprehensive interaction mode of BDF with ct-DNA may be the groove binding by the above results. It was indicated that there had been static-electro interaction between BDF and ct-DNA at the same time. The conjunct action of Van der Waals forces and electrostatic attraction favorably provide BDF bonding interaction in the groove of ct-DNA.

PREVALENCE OF ANTICOAGULANT RODENTICIDES IN FECES OF WILD RED FOXES (VULPES VULPES) IN NORWAY.

High occurrence of anticoagulant rodenticides (ARs) in wildlife is a rising concern, with numerous reports of secondary exposure through predation. Because of widespread distribution of the red fox (Vulpes vulpes), they may act as sentinels for small mammal-hunting predators in rural, suburban, and urban areas. No AR surveillance in wild mammals with analyses of residues in feces has been conducted throughout a single country. We collected 163 fecal samples from presumed healthy red foxes from 18 out of 19 counties in Norway. The foxes were shot during regular hunting between January and December 2016 and samples collected directly after death. Fecal samples were analyzed for six ARs: brodifacoum, bromadiolone, coumatetralyl, difenacoum, difethialone, and flocoumafen. We detected ARs in 54% (75/139) of the animals. Brodifacoum was most frequently detected (46%; 64/139), followed by coumatetralyl (17%; 23/139), bromadiolone (16%; 22/139), difenacoum (5%; 7/139), difethialone (1%; 2/139), and flocoumafen (1%; 2/139). More than one substance was detected in 40% (30/75) of the positive foxes, and 7% (5/75) of these animals were exposed to four different ARs. There were no statistically significant seasonal, age, or sex differences in foxes after exposure to one AR compound. We found a significant difference in occurrence of brodifacoum and coumatetralyl in foxes from different geographical areas. These findings demonstrate fecal analyses as a valuable method of detecting AR exposure in red foxes. We suggest using direct fecal sampling with analyses as a method to evaluate the occurrence of ARs in live endangered wildlife in connection with radio tagging or collaring operations.

The stereoisomerism of second generation anticoagulant rodenticides: a way to improve this class of molecules to meet the requirements of society?

Second generation anticoagulant rodenticides (SGAR) are generally highly efficient for rodent management even towards warfarin-resistant rodents. Nevertheless, because of their long tissue-persistence, they are very associated with non-target exposure of wildlife and have been identified as 'Candidates for Substitution' by the European Union's competent authority. A promising way to reduce ecotoxicity issues associated to SGAR could be the improvement of SGAR based on their stereoisomery, and due to this improvement, positioning about SGAR might be reconsidered. © 2018 Society of Chemical Industry.

The research of synthesis and bioactivity of the novel indanedione anticoagulant rodenticides.

Three new anticoagulant rodenticides R1, R2, and R3 were designed. The containing fluorine acute toxicity groups was added to 1,3-indandione derivative. Analysis method of synthesized anticoagulant rodenticides were IR, 1H NMR. Acute bioactivity of the new rodenticides was evaluated, including the coefficient of absorb food and median lethal dose (LD50). Average coefficient of absorbed food for R1 was 0.54 and LD50 of R1 was 2.15 mg/kg. Average coefficient of absorbed food for R2 was 0.59 and the acute oral LD50 of R2 was 2.65 mg/kg. Average feeding coefficient of R3 was 0.68 and the acute oral LD50 of R3 was 3.12 mg/kg. Experiments showed that rat's death peak was at about 72 h and rodenticides had good characteristics of acute medicine. The LD50 of three new fluoride anticoagulant rodenticides showed that they had good palatability for big white rats, and they had a strong poison effect on rodent. The result of all experiments proved that the synthesis of 1,3-indan diketone used as parent compound of new anticoagulant rodenticides could replace the current 4-hydroxyl coumarin as parent compound of the second-generation anticoagulant rodenticides. 1,3-Indan diketone would be widely used as parent compound of anticoagulant rodenticides.

Evidence of bromethalin toxicosis in feral San Francisco "Telegraph Hill" conures.

During 2018, four free-ranging conures, from a naturalized flock in San Francisco, presented with a characteristic set of neurologic signs that had been reported in other individuals from this flock. The cause of morbidity or mortality in historic cases has not been identified. From these four subjects, fresh feces were collected during their initial days of hospitalization and submitted to the University of Georgia Infectious Diseases Laboratory and Center for Applied Isotope Studies for bromethalin and desmethyl-bromethalin quantitation. Using High Performance Liquid Chromatography, the laboratory detected bromethalin, a non-anticoagulant, single-dose rodenticide, in fecal samples from three subjects; half of these samples were also positive for desmethyl-bromethalin, bromethalin's active metabolite. In three subjects that died, the UGA laboratory screened brain and liver samples and found bromethalin in all samples; desmethyl-bromethalin was detected in all but one brain sample, which was below the detection limit. Our findings suggest the conures are more resistant to bromethalin than are other species in which bromethalin has been studied, and/or that the conures may be ingesting the toxin at a sublethal dose. More data is needed to better assess the long-term effects of bromethalin on animals exposed at the subacute/chronic levels, and also to better understand the compartmentalization of bromethalin and desmethyl-bromethalin in a wider variety of species.