RESUMO
Poisoning caused by coumarin-type anticoagulant rodenticides (ARs) stands as the predominant method for controlling rodents globally. ARs, through secondary poisoning, pose a significant threat to predators due to their lethal and sublethal effects. We examined the concentration of accumulated ARs in liver samples of mostly road-killed steppe polecats (Mustela eversmanii) and European polecats (M. putorius) collected throughout Hungary between 2005 and 2021. The steppe polecat samples were found mainly from Eastern Hungary, while European polecats from Western Hungary. We measured the concentration of six residues by HPLC-FLD. Our analysis revealed the presence of one first-generation and four second-generation ARs in 53% of the steppe polecat (36) and 39% of the European polecat (26) samples. In 17 samples we detected the presence of at least two AR compounds. Although we did not find significant variance in AR accumulation between the two species, steppe polecats displayed greater prevalence and maximum concentration of ARs, whereas European polecat samples exhibited a more diverse accumulation of these compounds. Brodifacoum and bromadiolone were the most prevalent ARs; the highest concentrations were 0.57 mg/kg and 0.33 mg/kg, respectively. The accumulation of ARs was positively correlated with human population density and negatively correlated with the extent of the more natural habitats in both species. To the best of our knowledge, this is the first study to demonstrate anticoagulant rodenticide exposure in steppe polecats globally, and for European polecats in Central European region. Although the extent of AR accumulation in European polecat in Hungary appears comparatively lower than in many other European countries, the issue of secondary poisoning remains a serious problem as these ARs intrude into food webs. Reduced and more prudent usage of pesticides would provide several benefits for wildlife, included humans. However, we advocate a prioritization of ecosystem services through the complete prohibition of the toxicants.
Assuntos
Anticoagulantes , Monitoramento Ambiental , Rodenticidas , Animais , Anticoagulantes/análise , Hungria , Mustelidae , Poluentes Ambientais/análiseRESUMO
Envenoming resulting from snakebites is recognized as a priority neglected tropical disease by The World Health Organization. The Bothrops genus, consisting of different pitviper species, is considered the most medically significant taxa in Central and South America. Further research into Bothrops venom composition is important to aid in the development of safer and more effective snakebite treatments. In addition, the discovery of Bothrops toxins that could potentially be used for medical or diagnostic purposes is of interest to the pharmaceutical industry. This study aimed to employ high-throughput (HT) venomics to qualitatively analyze venom composition while utilizing coagulation bioassays for identifying coagulopathic toxins and characterizing coagulopathic activity in various Bothrops venoms. Using the recently demonstrated HT venomics workflow in combination with post-column coagulopathic bioassaying, focus was placed at anticoagulant toxins. Well-known procoagulant toxins were also investigated, taking into account that using the HT venomics workflow, procoagulant toxins are especially prone to denaturation during the reversed-phase chromatographic separations performed in the workflow. The findings revealed that the venoms of B. atrox and B. jararaca harbored procoagulant toxins, whereas those of B. alternatus and B. neuwiedi contained both procoagulant and anticoagulant toxins. In general, anticoagulation was associated with phospholipases A2s, while procoagulation was associated with snake venom metalloproteinases and snake venom serine proteases. These results showed the identification of coagulopathic venom toxins in the Bothrops venoms analyzed using multiple analytical methods that complement each other. Additionally, each venom underwent qualitative characterization of its composition.
Assuntos
Coagulação Sanguínea , Bothrops , Venenos de Crotalídeos , Ensaios de Triagem em Larga Escala , Animais , Venenos de Crotalídeos/química , Coagulação Sanguínea/efeitos dos fármacos , Bioensaio , Anticoagulantes/farmacologia , Anticoagulantes/química , Anticoagulantes/análise , HumanosRESUMO
Pesticide contamination poses a significant threat to non-target wildlife, including amphibians, many of which are already highly threatened. This study assessed the extent of pesticide exposure in dead frogs collected during a mass mortality event across eastern New South Wales, Australia between July 2021 and March 2022. Liver tissue from 77 individual frogs of six species were analysed for >600 legacy and contemporary pesticides, including rodenticides. More than a third (36 %) of the liver samples contained at least one of the following pesticides: brodifacoum, dieldrin, DDE, heptachlor/heptachlor epoxide, fipronil sulfone, and 2-methyl-4-chlorophenoxyacetic acid (MCPA). Brodifacoum, a second-generation anticoagulant rodenticide, was found in four of the six frog species analysed: the eastern banjo frog (Limnodynastes dumerilii), cane toad (Rhinella marina), green tree frog (Litoria caerulea) and Peron's tree frog (Litoria peronii). This is the first report of anticoagulant rodenticide detected in wild amphibians, raising concerns about potential impacts on frogs and extending the list of taxa shown to accumulate rodenticides. Dieldrin, a banned legacy pesticide, was also detected in two species: striped marsh frog (Limnodynastes peronii) and green tree frog (Litoria caerulea). The toxicological effects of these pesticides on frogs are difficult to infer due to limited comparable studies; however, due to the low frequency of detection the presence of these pesticides was not considered a major contributing factor to the mass mortality event. Additional research is needed to investigate the effects of pesticide exposure on amphibians, particularly regarding the impacts of second-generation anticoagulant rodenticides. There is also need for continued monitoring and improved conservation management strategies for the mitigation of the potential threat of pesticide exposure and accumulation in amphibian populations.
Assuntos
Anticoagulantes , Anuros , Monitoramento Ambiental , Praguicidas , Rodenticidas , Animais , Rodenticidas/análise , Anticoagulantes/análise , New South Wales , AustráliaRESUMO
Second-generation anticoagulant rodenticides (SGARs) are widely used to control rodent populations, resulting in the serious secondary exposure of predators to these contaminants. In the United Kingdom (UK), professional use and purchase of SGARs were revised in the 2010s. Certain highly toxic SGARs have been authorized since then to be used outdoors around buildings as resistance-breaking chemicals under risk mitigation procedures. However, it is still uncertain whether and how these regulatory changes have influenced the secondary exposure of birds of prey to SGARs. Based on biomonitoring of the UK Common Buzzard (Buteo buteo) collected from 2001 to 2019, we assessed the temporal trend of exposure to SGARs and statistically determined potential turning points. The magnitude of difenacoum decreased over time with a seasonal fluctuation, while the magnitude and prevalence of more toxic brodifacoum, authorized to be used outdoors around buildings after the regulatory changes, increased. The summer of 2016 was statistically identified as a turning point for exposure to brodifacoum and summed SGARs that increased after this point. This time point coincided with the aforementioned regulatory changes. Our findings suggest a possible shift in SGAR use to brodifacoum from difenacoum over the decades, which may pose higher risks of impacts on wildlife.
Assuntos
Anticoagulantes , Rodenticidas , Animais , Anticoagulantes/análise , Rodenticidas/análise , Animais Selvagens , Aves , Reino Unido , Monitoramento AmbientalRESUMO
The widespread use of anticoagulant rodenticides (ARs) poses a worldwide threat to farmland wildlife. These compounds accumulate in tissues of both target and non-target species, potentially endangering both direct consumers and their predators. However, investigations on ARs in blood of free-ranging predatory birds are rare. Here, the long-eared owl (Asio otus) has been used as a model predator to assess AR exposure in different agricultural landscapes from a Mediterranean semiarid region. A total of 69 owlets from 38 nests were blood-sampled over 2021 and 2022, aiming to detect AR residues and explore factors that determine their exposure, such as land uses. In addition, prothrombin time (PT) test was conducted to assess potential effects of AR contamination. Overall, nearly all the samples (98.6 %) tested positive for at least one compound and multiple ARs were found in most of the individuals (82.6 %). Among the ARs detected, flocoumafen was the most common compound (88.4 % of the samples). AR total concentration (ΣARs) in blood ranged from 0.06 to 34.18 ng mL-1, detecting the highest levels in the most intensively cultivated area. The analysis of owl pellets from 19 breeding territories showed relevant among-site differences in the contribution of rodents and birds into the diet of long-eared owls, supporting its high dietary plasticity and indicating AR presence at multiple trophic levels. Moreover, a positive and significant correlation was found between ΣARs and PT (Rho = 0.547, p < 0.001), which demonstrates the direct effect of ARs on free-living nestlings. Our results provide a preliminary overview of AR exposure in a little-studied owl species inhabiting agricultural and rural landscapes. Despite the low detected levels, these findings indicate widespread exposure -often to multiple compounds- from early life stages, which raises concern and draws attention to an ongoing and unresolved contamination issue.
Assuntos
Rodenticidas , Estrigiformes , Animais , Anticoagulantes/análise , Rodenticidas/análise , Tempo de Protrombina , Animais SelvagensRESUMO
The study deals with the environmental residues of anticoagulant rodenticides (ARs) in Slovenia to evaluate the toxicological risk of secondary poisoning of red foxes (Vulpes vulpes) as representatives of non-target wildlife, and in relation to the investigated use patterns of ARs and specific local parameters in Slovenia. From 2019 to 2022, 148 liver tissue samples of adult red foxes were collected from almost all state geographical regions. The samples were extracted with methanol/water (2:1, v/v), cleaned-up using a solid supported liquid-liquid extraction, and measured by liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) with reporting limits of 0.5 to 5.0 ng/g. Residues of at least one rodenticide were detected in 77.7 % of the samples. The second generation ARs of bromadiolone, brodifacoum and difenacoum were the most frequently found, appearing in 75.0, 51.4, and 18.9 % of the samples, respectively. Concentrations of pooled ARs ranged from 1.5 to 2866.5 ng/g with mean and median values of 601.4 and 350.2 ng/g, respectively. We determined bromadiolone and brodifacoum at concentrations of ≥800 ng/g in 10.8 and 10.1 % of the samples, and 1.4 and 0.7 % of the samples contained residues >2000 ng/g, respectively. These concentrations are much higher than those found in comparable studies in Europe and elsewhere in the world. Residues of ARs were detected in all monitored statistical regions of Slovenia, with higher concentrations in the eastern parts of the country. First generation ARs were found in only 9.5 % of samples, and residues were below 10 ng/g with one exception (coumatetralyl with 55 ng/g). The results of the study indicate a serious toxicological risk for red foxes in Slovenia as part of the Western Balkans, and will contribute to the growing body of knowledge about the protection of European ecosystems, as wildlife is not limited by national borders.
Assuntos
Anticoagulantes , Rodenticidas , Animais , Anticoagulantes/análise , Rodenticidas/análise , Raposas , Espectrometria de Massas em Tandem/métodos , Eslovênia , Ecossistema , Fígado/química , Animais Selvagens , Península BalcânicaRESUMO
Apixaban and dabigatran are the two major direct oral anticoagulant drugs to treat thromboembolic disordered patients. Increasing the clinical application for the thromboembolic disorder and monitoring the concentrations of apixaban, dabigatran, and their metabolites are essential in most clinical circumstances. In this work, we developed a rapid analytical methodology comprising of vortex-assisted salt-enhanced liquid-liquid microextraction technique coupled with UHPLC-MS/MS for the extraction and simultaneous determination of two major direct oral anticoagulant drugs (apixaban, dabigatran), and their two major metabolites from plasma, serum, and urine samples of patients. The developed method was optimized with various procedural steps and validated to study the analytical merits. The developed method yielded a good detection limit of 0.01 â¼ 0.37 ng/mL, 0.01 â¼ 0.32 ng/ml, and 0.01 â¼ 0.27 ng/mL for four target analytes in the plasma, serum, and urine matrices. Moreover, extraction recoveries ranged from 85.11 - 113.57% (for plasma), 89.63 - 110.47% (for serum), and 87.44 -106.79% (for urine samples) with 8.78% RSD. In addition, the method exhibited good R2 values of 0.999 for all four target analytes, and the specificity and carryover study revealed no carryover effect from the UHPLC-MS/MS system for determining the apixaban, dabigatran, and their metabolites. Due to the above advantages, the developed analytical technique was applied to examine 11 real-time clinical patients' samples, and the observed results were satisfactory for all three different sample matrices. Therefore, this analytical method can be applied for biomonitoring apixaban, dabigatran, and their two major metabolites with high sensitivity in a short time for various clinical applications.
Assuntos
Dabigatrana , Rivaroxabana , Humanos , Dabigatrana/análise , Espectrometria de Massas em Tandem/métodos , Monitoramento Biológico , Anticoagulantes/uso terapêutico , Anticoagulantes/análise , Cromatografia Líquida de Alta Pressão/métodosRESUMO
INTRODUCTION: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest. OBJECTIVES: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), describing its kinetic mechanism and anticoagulant effect. METHODS: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time. RESULTS: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10-6 M) and Ki (1.05 x 10-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 µM, prolonging clotting time by 2.6 times. CONCLUSION: Our results suggest that BpuTI can be a biological tool to be used in blood clotting studies.
Assuntos
Bauhinia , Inibidores da Tripsina , Animais , Bovinos , Inibidores da Tripsina/farmacologia , Inibidores da Tripsina/química , Bauhinia/metabolismo , Tripsina/análise , Tripsina/química , Tripsina/metabolismo , Espectrometria de Massas em Tandem , Sementes/química , Anticoagulantes/farmacologia , Anticoagulantes/análise , Anticoagulantes/químicaRESUMO
Anticoagulant rodenticides (ARs) influence predator populations and threaten the stability of ecosystems. Understanding the prevalence and impact of rodenticides in predators is crucial to inform conservation planning and policy. We collected dead birds of four nocturnal predatory species across differing landscapes: forests, agricultural, urban. Liver samples were analysed for eight ARs: three First Generation ARs (FGARs) and five SGARs (Second Generation ARs). We investigated interspecific differences in liver concentrations and whether landscape composition influenced this. FGARs were rarely detected, except pindone at low concentrations in powerful owls Ninox strenua. SGARs, however, were detected in every species and 92 % of birds analysed. Concentrations of SGARs were at levels where potential toxicological or lethal impacts would have occurred in 33 % of powerful owls, 68 % of tawny frogmouths Podargus strigoides, 42 % of southern boobooks N. bookbook and 80 % of barn owls Tyto javanica. When multiple SGARs were detected, the likelihood of potentially lethal concentrations of rodenticides increased. There was no association between landscape composition and SGAR exposure, or the presence of multiple SGARs, suggesting rodenticide poisoning is ubiquitous across all landscapes sampled. This widespread human-driven contamination in wildlife is a major threat to wildlife health. Given the high prevalence and concentrations of SGARs in these birds across all landscape types, we support the formal consideration of SGARs as a threatening process. Furthermore, given species that do not primarily eat rodents (tawny frogmouths, powerful owls) have comparable liver rodenticide concentrations to rodent predators (southern boobook, eastern barn owl), it appears there is broader contamination of the food-web than anticipated. We provide evidence that SGARs have the potential to pose a threat to the survival of avian predator populations. Given the functional importance of predators in ecosystems, combined with the animal welfare impacts of these chemicals, we propose governments should regulate the use of SGARs.
Assuntos
Rodenticidas , Estrigiformes , Animais , Humanos , Anticoagulantes/toxicidade , Anticoagulantes/análise , Rodenticidas/toxicidade , Rodenticidas/análise , Monitoramento Ambiental , EcossistemaRESUMO
Anticoagulant rodenticides (ARs) are used to control rodent populations. Poisoning of non-target species can occur by accidental consumption of commercial formulations used for rodent control. A robust method for determining ARs in animal tissues is important for animal postmortem diagnostic and forensic purposes. We evaluated an ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) method to quantify 8 ARs (brodifacoum, bromadiolone, chlorophacinone, coumachlor, dicoumarol, difethialone, diphacinone, warfarin) in a wide range of animal (bovine, canine, chicken, equine, porcine) liver samples, including incurred samples. We further evaluated UPLC-MS in 2 interlaboratory comparison (ILC) studies; one an ILC exercise (ICE), the other a proficiency test (PT). The limits of detection of UPLC-MS were 0.3-3.1 ng/g, and the limits of quantification were 0.8-9.4 ng/g. The recoveries obtained using UPLC-MS were 90-115%, and relative SDs were 1.2-13% for each of the 8 ARs for the 50, 500, and 2,000 ng/g spiked liver samples. The overall accuracy from the laboratories participating in the 2 ILC studies (4 and 11 laboratories for ICE and PT studies, respectively) were 86-118%, with relative repeatability SDs of 3.7-11%, relative reproducibility SDs of 7.8-31.2%, and Horwitz ratio values of 0.5-1.5. Via the ILC studies, we verified the accuracy of UPLC-MS for AR analysis in liver matrices and demonstrated that ILC can be utilized to evaluate performance characteristics of analytical methods.
Assuntos
Anticoagulantes , Técnicas de Química Analítica , Cumarínicos , Indanos , Rodenticidas , Animais , Técnicas de Química Analítica/métodos , Técnicas de Química Analítica/normas , Técnicas de Química Analítica/veterinária , Rodenticidas/análise , Anticoagulantes/análise , Fígado/química , Espectrometria de Massa com Cromatografia Líquida , Indanos/análise , Cumarínicos/análise , Limite de Detecção , Reprodutibilidade dos TestesRESUMO
Blood platelets play a crucial role in hemostasis, the process responsible for keeping blood flowing in the circulatory system. However, unnecessary platelet activation can lead to aggregation at the site of atherosclerotic plaque rapture and the formation of a thrombus, which promotes atherothrombotic diseases. Various dietary components, such as phenolic compounds, are known to demonstrate antiplatelet and anticoagulant properties, and it is possible that these could form an important element in the prophylaxis and therapy of cardiovascular diseases. Our present study examined the biological activity of isorhamnetin (1) and two isorhamnetin derivatives, (2): 3-O-beta-glucoside-7-O-alpha-rhamnoside and (3): 3-O-beta-glucoside-7-O-alpha-(3â³'-isovaleryl)-rhamnoside, isolated from the phenolic fraction of sea buckthorn fruit, against human washed blood platelets and human whole blood in vitro. The anti-platelet and anticoagulant potential was determined using (A) flow cytometry, (B) the thrombus-formation analysis system (T-TAS) and (C) colorimetry. The results of the T-TAS test indicate that the AUC10 (Area Under the Curve) of the tested phenolic compounds (compounds 1, 2 and 3; 50 µg/mL) was markedly reduced compared to the control values. Moreover, flavonol demonstrated anti-platelet potential, including anti-adhesive activity, with these effects being more intense in compound 2 than isorhamnetin. Different actions of flavonol on platelet activation may depend on their binding ability to various receptors on blood platelets. However, the mechanism of their anti-platelet potential requires further additional studies, including in vitro and in vivo experiments.
Assuntos
Hippophae , Anticoagulantes/análise , Anticoagulantes/farmacologia , Flavonóis/análise , Frutas/química , Glucosídeos/análise , Hippophae/química , Humanos , Fenóis/química , Quercetina/análogos & derivadosRESUMO
BACKGROUND: Ex vivo assays of platelet function critically inform mechanistic and clinical hematology studies, where effects of divergent blood processing methods on platelet composition are apparent, but unspecified. OBJECTIVE: Here, we evaluate how different blood anticoagulation options and processing times affect platelet function and protein content ex vivo. METHODS: Parallel blood samples were collected from healthy human donors into sodium citrate, acid citrate dextrose, EDTA or heparin, and processed over an extended time course for functional and biochemical experiments, including platelet proteome quantification with multiplexed tandem mass tag (TMT) labeling and triple quadrupole mass spectrometry (MS). RESULTS: Each anticoagulant had time-dependent effects on platelet function in whole blood. For instance, heparin enhanced platelet agonist reactivity, platelet-monocyte aggregate formation and platelet extracellular vesicle release, while EDTA increased platelet α-granule secretion. Following platelet isolation, TMT-MS quantified 3357 proteins amongst all prepared platelet samples. Altogether, >400 proteins were differentially abundant in platelets isolated from blood processed at 24 h versus 1 h post-phlebotomy, including proteins pertinent to membrane trafficking and exocytosis. Anticoagulant-specific effects on platelet proteomes included increased complement system and decreased α-granule proteins in platelets from EDTA-anticoagulated blood. Platelets prepared from heparinized blood had higher levels of histone and neutrophil-associated proteins in a manner related to neutrophil extracellular trap (NET) formation and platelet:NET interactions in whole blood ex vivo. CONCLUSION: Our results demonstrate that different anticoagulants routinely used for blood collection have varying effects on platelets ex vivo, where methodology-associated alterations in platelet proteome may influence mechanistic, translational and biomarker studies.
Assuntos
Plaquetas , Proteoma , Anticoagulantes/análise , Anticoagulantes/farmacologia , Ácido Edético/análise , Ácido Edético/farmacologia , Heparina/farmacologia , Humanos , Proteoma/análise , Proteoma/farmacologiaRESUMO
Anticoagulant rodenticides (ARs) are used globally to control rodent pest infestations in both urban and agricultural settings. It is well documented that non-target wildlife, including predatory birds, are at risk for secondary anticoagulant exposure and toxicosis through the prey they consume. However, there have been no large-scale studies of AR exposure in raptors in Ontario, Canada since new Health Canada legislation was implemented in 2013 in an attempt to limit exposure in non-target wildlife. Our objective was to measure levels of ARs in wild raptors in southern Ontario to assess their exposure. We collected liver samples from 133 raptors representing 17 species submitted to the Canadian Wildlife Health Cooperative (CWHC) in Ontario, Canada, between 2017 and 2019. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantitatively assess the level of exposure to 14 first- and second-generation ARs. Detectable levels of one or more ARs were found in 82 of 133 (62%) tested raptors, representing 12 species. The most commonly detected ARs were bromadiolone (54/133), difethialone (40/133), and brodifacoum (33/133). Of AR-positive birds, 34/82 (42%) contained residues of multiple (> 1) anticoagulant compounds. Our results indicate that AR exposure is common in raptors living in southern Ontario, Canada. Our finding that brodifacoum, difethialone, and bromadiolone were observed alone or in combination with one another in the majority of our sampled raptors indicates that legislative changes in Canada may not be protecting non-target wildlife as intended.
Assuntos
4-Hidroxicumarinas , Aves Predatórias , Rodenticidas , 4-Hidroxicumarinas/análise , Animais , Anticoagulantes/análise , Aves , Cromatografia Líquida , Ontário , Rodenticidas/análise , Espectrometria de Massas em TandemRESUMO
The powerful owl (Ninox strenua) is a threatened apex predator that consumes mainly arboreal marsupial prey. Low density populations reside in urban landscapes where their viability is tenuous. The catalyst for this research was the reported death of eight powerful owls around Melbourne, Australia, in less than one year (2020/2021). Eighteen deceased owls were toxicologically screened. We assessed toxic metals (Mercury Hg, Lead Pb, Cadmium Cd and Arsenic As) and anticoagulant rodenticides (ARs) in liver (n = 18 owls) and an extensive range of agricultural chemicals in muscle (n = 14). Almost all agricultural chemicals were below detection limits except for p,p-DDE, which was detected in 71% of birds at relatively low levels. Toxic metals detected in some individuals were generally at low levels. However, ARs were detected in 83.3% of powerful owls. The most common second-generation anticoagulant rodenticide (SGAR) detected was brodifacoum, which was present in every bird in which a rodenticide was detected. Brodifacoum was often present at toxic levels and in some instances at potentially lethal levels. Presence of brodifacoum was detected across the complete urban-forest/agriculture gradient, suggesting widespread exposure. Powerful owls do not scavenge but prey upon arboreal marsupials, and generally not rodents, suggesting that brodifacoum is entering the powerful owl food web via accidental or deliberate poisoning of non-target species (possums). We highlight a critical need to investigate SGARs in food webs globally, and not just in species directly targeted for poisoning or their predators.
Assuntos
Rodenticidas , Estrigiformes , Agricultura , Animais , Anticoagulantes/análise , Monitoramento Ambiental , Florestas , Rodenticidas/análiseRESUMO
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.
Assuntos
Rodenticidas , Anticoagulantes/análise , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Toxicologia Forense , Rodenticidas/análise , Rodenticidas/química , Espectrometria de Massas em Tandem/métodosRESUMO
Wild raptors are widely used to assess exposure to different environmental contaminants, including anticoagulant rodenticides (ARs). ARs are used on a global scale for rodent control, and act by disruption of the vitamin K cycle that results in haemorrhage usually accompanied by death within days. Some ARs are highly persistent and bioaccumulative, which can cause significant exposure of non-target species. We characterized AR exposure in a heterogeneous sample of dead raptors collected over 12 years (2008-2019) in south-eastern France. Residue analysis of 156 liver samples through LC-MS/MS revealed that 50% (78/156) were positive for ARs, with 13.5% (21/156) having summed second-generation AR (SGAR) concentrations >100 ng/g ww. While SGARs were commonly detected (97.4% of positive samples), first-generation ARs were rarely found (7.7% of positive samples). ARs were more frequently detected and at greater concentration in predators (prevalence: 82.5%) than in scavengers (38.8%). Exposure to multiple ARs was common (64.1% of positive samples). While chlorophacinone exposure decreased over time, an increasing exposure trend was observed for the SGAR brodifacoum, suggesting that public policies may not be efficient at mitigating risk of exposure for non-target species. Haemorrhage was observed in 88 birds, but AR toxicosis was suspected in only 2 of these individuals, and no difference in frequency of haemorrhage was apparent in birds displaying summed SGAR levels above or below 100 ng/g ww. As for other contaminants, 17.2% of liver samples (11/64) exhibited Pb levels compatible with sub-clinical poisoning (>6 µg/g dw), with 6.3% (4/64) above the threshold for severe/lethal poisoning (>30 µg/g dw). Nine individuals with Pb levels >6 µg/g dw also had AR residues, demonstrating exposure to multiple contaminants. Broad toxicological screening for other contaminants was positive for 18 of 126 individuals, with carbofuran and mevinphos exposure being the suspected cause of death of 17 birds. Our findings demonstrate lower but still substantial AR exposure of scavenging birds compared to predatory birds, and also illustrate the complexity of diagnosing AR toxicosis through forensic investigations.
Assuntos
Rodenticidas , Animais , Anticoagulantes/análise , Aves , Cromatografia Líquida , Monitoramento Ambiental , Rodenticidas/análise , Espectrometria de Massas em TandemRESUMO
Antiplatelet and anticoagulant drugs are classified antithrombotic agents with the purpose to reduce blood clot formation. For a successful treatment of many known complex cardiovascular diseases driven by platelet and/or coagulation activity, the need of more than one antithrombotic agent is inevitable. However, combining drugs with different mechanisms of action enhances risk of bleeding. Dual anticoagulant and antiplatelet (APAC), a novel semisynthetic antithrombotic molecule, provides both anticoagulant and antiplatelet properties in preclinical studies. APAC is entering clinical studies with this new exciting approach to manage cardiovascular diseases. For a better understanding of the biological function of APAC, comprehensive knowledge of its structure is essential. In this study, atomic force microscopy (AFM) was used to characterize APAC according to its structure and to investigate the molecular interaction of APAC with von Willebrand factor (VWF), since specific binding of APAC to VWF could reduce platelet accumulation at vascular injury sites. By the optimization of drop-casting experiments, we were able to determine the volume of an individual APAC molecule at around 600 nm3, and confirm that APAC forms multimers, especially dimers and trimers under the experimental conditions. By studying the drop-casting behavior of APAC and VWF individually, we depictured their interaction by using an indirect approach. Moreover, in vitro and in vivo conducted experiments in pigs supported the AFM results further. Finally, the successful adsorption of APAC to a flat gold surface was confirmed by using photothermal-induced resonance, whereby attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) served as a reference method.
Assuntos
Anticoagulantes/análise , Heparina/análogos & derivados , Microscopia de Força Atômica/métodos , Inibidores da Agregação Plaquetária/análise , Proteoglicanas/análise , Heparina/análise , Humanos , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
Carthami flos, commonly known as Honghua in China, is the dried floret of safflower and widely acknowledged as a blood stasis promoting herb. The study aimed at investigating the relationship between thrombin and carthami flos through a high-performance thrombin affinity chromatography combined with a high-performance liquid chromatography-tandem mass spectrometry system. First, thrombin was immobilized on the glutaraldehyde-modified amino silica gel to prepare the thrombin affinity stationary phase, which was packed into a small column (1.0 × 2.0 mm, id) for recognizing the anticoagulant active components of carthami flos. The target component was enriched and analyzed by the high-performance liquid chromatography-tandem mass spectrometry system. Finally, hydroxysafflor yellow A was screened out and identified as the active component. The anticoagulant effects of hydroxysafflor yellow A were analyzed by anticoagulant experiments in vitro, and the interaction of hydroxysafflor yellow A with thrombin was investigated by the molecular docking method. The results proved that hydroxysafflor yellow A (30 µg/mL, 0.05 mM) and carthami flos extract (30 µg/mL) could prolong activated partial thrombin time and thrombin time by 50 and 11%, respectively. Moreover, hydroxysafflor yellow A exhibits a good hydrogen bond field and stereo field matching with thrombin. Overall, it was concluded that hydroxysafflor yellow A might exert an anticoagulation effect by interacting with thrombin and thus could be potential anticoagulant drugs for the prevention and treatment of venous thrombosis.
Assuntos
Anticoagulantes/análise , Carthamus tinctorius/metabolismo , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/metabolismo , Espectrometria de Massas em Tandem/métodos , Trombina/química , Animais , Chalcona/análogos & derivados , Chalcona/química , Ligação de Hidrogênio , Técnicas In Vitro , Masculino , Simulação de Acoplamento Molecular , Pós , Quinonas/química , Coelhos , Reprodutibilidade dos Testes , Trombina/análise , Tempo de Trombina , Trombose Venosa/tratamento farmacológicoRESUMO
Anticoagulant rodenticides (ARs) used to control mammalian pest populations cause secondary exposure of predatory species throughout much of the world. It is important to understand the drivers of non-target AR exposure patterns as context for assessing long-term effects and developing effective mitigation for these toxicants. In Australia, however, little is known about exposure and effects of ARs on predators. We detected AR residues in 74% of 50 opportunistically collected carcasses of the Tasmanian wedge-tailed eagle (Aquila audax fleayi), an endangered apex predator. In 22% of birds tested, or 31% of those exposed, liver concentrations of second generation ARs (SGARs) were >0.1 mg/kg ww. Eagles were exposed to flocoumafen, a toxicant only available from agricultural suppliers, at an exceptionally high rate (40% of birds tested). Liver SGAR concentrations were positively associated with the proportion of agricultural habitat and human population density in the area around where each eagle died. The high exposure rate in a species not known to regularly prey upon synanthropic rodents supports the hypothesis that apex predators are vulnerable to SGARs. Our results indicate that AR exposure constitutes a previously unrecognized threat to Tasmanian wedge-tailed eagles and highlight the importance of efforts to address non-target AR exposure in Australia.
Assuntos
Rodenticidas , Animais , Anticoagulantes/análise , Austrália , Ecossistema , Monitoramento Ambiental , Rodenticidas/análise , Rodenticidas/toxicidadeRESUMO
The common vampire bat (Desmodus rotundus) is a hematophagous species responsible for paralytic rabies and bite damage that affects livestock, humans and wildlife from Mexico to Argentina. Current measures to control vampires, based upon coumarin-derived poisons, are not used extensively due in part to the high cost of application, risks for bats that share roosts with vampires and residual environmental contamination. Observations that vampire bat bites may induce resistance in livestock against vampire bat salivary anticoagulants encourage research into novel vaccine-based alternatives particularly focused upon increasing livestock resistance to vampire salivary components. We evaluated the action of vampire bat saliva-Freund's incomplete adjuvant administered to sheep with anticoagulant responses induced by repeated vampire bites in a control group and examined characteristics of vampire bat salivary secretion. We observed that injections induced a response against vampire bat salivary anticoagulants stronger than by repeated vampire bat bites. Based upon these preliminary findings, we hypothesize the utility of developing a control technique based on induction of an immunologically mediated resistance against vampire bat anticoagulants and rabies virus via dual delivery of appropriate host and pathogen antigens. Fundamental characteristics of host biology favor alternative strategies than simple culling by poisons for practical, economical, and ecologically relevant management of vampire populations within a One Health context.