Assessing the Risks to Bats from Plant Protection Products: A Review of the Recent European Food Safety Authority Statement Regarding Toxicity and Exposure Routes.

Wild birds and mammals that feed in agricultural habitats are potentially exposed to pesticides through various routes. Until recently, it has been implicitly assumed that the existing European Union risk assessment scheme for birds and mammals also covered bats (Chiroptera). However, recent publications raised concerns and, in 2019, a scientific statement was published by the European Food Safety Authority (EFSA) that concluded that bats were not adequately covered by the current risk assessment scheme. We review the evidence presented and assumptions made in the EFSA bat statement relating to toxicity, bioaccumulation, and exposure pathways (oral, dermal, and inhalation), in terms of their relevance for bats potentially foraging in agricultural areas in the European Union; we highlight where uncertainties remain and how these could be addressed. Based on our review, it is clear that there is still much uncertainty with regard to the appropriateness of the assumptions made in the EFSA bat statement. Significantly more information needs to be gathered to answer fundamental questions regarding bat behavior in agricultural landscapes, together with the relative sensitivity of bats to pesticide exposure. Given the current critical information gaps, it is recommended that quantitative risk assessments for bats not be performed for pesticides until more robust, reliable, and relevant data are available. The risk to bats can then be compared with that for birds and ground-dwelling mammals, to determine the protectiveness of the existing scheme and thus whether a bat scenario is indeed required and under what circumstances. Environ Toxicol Chem 2021;40:2978-2989. © 2021 Cambridge Environmental Assessments, part of RSK ADAS Ltd. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Prevalence of bacterial contamination in 50% dextrose vials in varying storage conditions after multiple punctures.

OBJECTIVE: To determine the risk of bacterial growth in single use 50% dextrose vials punctured multiple times and stored in various hospital environments. MATERIALS AND METHODS: Three groups of three 50% dextrose vials were stored in our hospital intensive care unit at either ambient light or in a darkened drawer at room temperature or refrigerated at 4°C. One vial in each group was punctured either once, once weekly or once daily for 28 days and samples taken for bacterial culture every 7 days until completion of the project. A fourth group of three vials were inoculated with several species of bacteria and stored in our microbiology laboratory under the environmental conditions described above with cultures performed every 7 days for 28 days. In addition, the water activity of 50% dextrose was determined using commercial laboratory equipment. RESULTS: Scant growth of Escherichia coli and Enterobacter agglomerans was detected in cultures performed on day 7, but not subsequent time points, from the inoculated refrigerated vials. The vial punctured once daily for 28 days and stored under refrigerated conditions showed growth of Bacillus subtilis on day 28. All remaining bottles had no bacterial growth at any time point or environmental condition. The water activity of 50% dextrose was 093 at 24°C and 092 at 4°C. CLINICAL SIGNIFICANCE: Bacterial growth in 50% dextrose vials was uncommon even when inoculated with pathogens. Bacterial growth only occurred in refrigerated storage conditions. The water activity of 50% dextrose is not low enough to inhibit all bacterial and fungal growth.

Endotoxin-induced HIF-1alpha stabilisation in equine endothelial cells: synergistic action with hypoxia.

OBJECTIVE AND DESIGN: Hypoxia may enhance the deleterious effects of lipopolysaccharide (LPS) in the endotoxaemic horse. This study has examined some of the actions of LPS and hypoxia, alone and in combination, on cultured equine digital vein endothelial cells (EDVEC) and the signalling molecules involved. METHODS: EDVEC were exposed to LPS, 5% O(2) and LPS then 5% O(2) for up to 24 h. HIF-1alpha stabilisation, neutrophil adhesion and EDVEC permeability were assessed by immunoblotting, measurement of myeloperoxidase and movement of FITC-dextran, respectively. Pharmacological inhibitors were used to assess the roles of p38 MAPK and HIF-1alpha. RESULTS: LPS and hypoxia significantly increased HIF-1alpha stabilisation, neutrophil adhesion and EDVEC permeability and the effects of the two stimuli in combination on HIF-1alpha stabilisation and neutrophil adhesion were more than additive. The effect of LPS, but not 5% O(2), on neutrophil adherence required activation of p38 MAPK, whereas EDVEC permeability in response to both stimuli was dependent on p38 MAPK and HIF-1alpha. CONCLUSIONS: Exposure of EDVEC to LPS prior to induction of hypoxia up-regulates responses that may enhance LPS-induced tissue damage in the endotoxaemic horse. Inhibitors of p38 MAPK or HIF-1alpha could reduce such unwanted effects.

Regulation of platelet activating factor-induced equine platelet activation by intracellular kinases.

Lipopolysaccharide (LPS) can activate equine platelets directly or indirectly, via leukocyte-derived platelet activating factor (PAF). Thromboxane (Tx) production by LPS-stimulated equine platelets requires p38 MAPK and this kinase has been suggested as a therapeutic target in endotoxaemia. The present study has utilised selective inhibitors to investigate the role of p38 MAPK and two other kinases, phosphatidylinositol-3 kinase (PI3K) and protein kinase C (PKC), in regulating PAF-induced Tx production, aggregation and 5-HT release in equine platelets, and the modification of these responses by LPS. LPS enhanced PAF-induced 5-HT release, an effect that was reduced by the p38 MAPK inhibitor, SB203580 (60 +/- 8% reduction; n = 6). SB203580 did not affect responses to PAF alone; whereas inhibition of PKC reduced PAF-induced 5-HT release, Tx production and aggregation (maximal inhibition by the PKCdelta inhibitor, rottlerin: 69 +/- 13%, 63 +/- 14% and 97 +/- 1%, respectively; n = 6). Wortmannin and LY249002, which inhibit PI3K, also caused significant inhibition of PAF-induced aggregation (maximal inhibition 78 +/- 3% and 88 +/- 2%, respectively; n = 6). These data suggest that inhibition of platelet p38 MAPK may be of benefit in equine endotoxaemia by counteracting some of the effects of LPS. However, detrimental effects of platelet activation mediated by PAF and not enhanced by LPS are unlikely to be markedly affected.

Plasma concentrations of endotoxin and platelet activation in the developmental stage of oligofructose-induced laminitis.

The link between the fermentation of carbohydrate in the equine large intestine and the development of acute laminitis is poorly understood. Absorption of endotoxin (lipopolysaccharide; LPS) into the plasma has been observed in one experimental model of laminitis, but does not cause laminitis when administered alone. Thus, the potential role of endotoxin is unclear. Platelet activation has previously been demonstrated in the developmental stage of laminitis. Equine platelets are more sensitive than leukocytes to activation by endotoxin, and can be activated directly by LPS in the low pg/ml range, activating p38 MAP kinase and releasing serotonin (5-HT) and thromboxane. The objectives of this study were firstly to determine whether endotoxin and platelet activation could be measured in the plasma of horses in the developmental phase of laminitis induced with oligofructose. Secondly, the time course of events involving platelet activation and platelet-derived vasoactive mediator production was investigated. Laminitis was induced in six Standardbred horses by the administration of 10 g/kg bwt of oligofructose. Plasma samples were obtained every 4h, and platelet pellets were obtained by centrifugation. LPS was measured using a kinetic limulus amebocyte lysate assay, and platelet activation was assessed by Western blotting for the phosphorylated form of p38 MAP kinase. Plasma 5-HT was assayed by HPLC with electrochemical detection and thromboxane B(2) was measured by radioimmunoassay. Clinical signs of laminitis and histopathologic changes were observed in lamellar sections from five of the six horses. Onset of lameness was between 20 and 30 h after the administration of oligofructose. LPS increased above the limit of detection (0.6 pg/ml) to reach a peak of 2.4+/-1.0 pg/ml at 8 h. TNFalpha was also detectable in the plasma from 12 to 24 h. There was a time-dependent increase in platelet p38 MAPK phosphorylation, which peaked at approximately 12 h (3.8+/-1.3 fold increase); plasma 5-HT and thromboxane increased steadily after this time (2.9+/-0.6 and 11.3+/-5.0 fold increases, respectively). These data indicate that small quantities of endotoxin may move into the circulation from the large intestine after the sharp decrease in pH that occurs as a result of carbohydrate fermentation. Correlating these findings with in vitro studies suggests that LPS may primarily activate platelets, leading indirectly to the activation of leukocytes. Therefore, endotoxin may contribute in the initiation of the early inflammatory changes observed in experimental models of acute laminitis.

Endotoxin-induced activation of equine platelets: evidence for direct activation of p38 MAPK pathways and vasoactive mediator production.

OBJECTIVE AND DESIGN: The aim of this study was to determine the effects of endotoxin on p38 MAPK activation in equine platelets and leukocytes in vivo and in vitro and its role in thromboxane (Tx) production with reference to equine endotoxaemia. METHODS: Six adult Thoroughbred horses were used for in vivo infusion studies and separate in vitro studies. For in vivo studies, following collection of a pre-infusion sample, horses were infused with E. Coli O55:B5 LPS (30 ng/kg; 30 min) during and after which platelets were harvested. For in vitro studies isolated platelets and leukocytes were exposed to LPS (10 pg/ml-1 microg/ml). p38 MAPK activity was assessed by SDS-PAGE followed by immunoblotting. TxA2 release was measured by radioimmunoassay. RESULTS: LPS infusion caused increased phospho-p38 MAPK in equine platelets and leukocytes (1492 +/- 486 % and 83 +/- 45 above basal, respectively) from 10 min after the start of the infusion, which returned to basal by 60 min. In vitro, platelets were 1,000 times more sensitive to LPS than leukocytes in terms of both TxA2 production (EC50 66 pg/ml versus 110 ng/ml, respectively) and p38 MAPK phosphorylation (EC50 11.1 +/- 2 pg/ml versus 14.8 +/- 4 ng/ml, respectively). p38 MAPK inhibitors SB203580 and PD169316 attenuated LPS-induced TxA2 release in platelets, but not leukocytes. CONCLUSIONS: In vivo, LPS stimulates TxA2 production and p38 MAPK phosphorylation in equine platelets and leukocytes at a concentration within a similar range to those reported in clinical endotoxaemia. These data suggest that LPS-induced eicosanoid production in the early phase of clinical endotoxaemia may involve direct effects of LPS upon platelets, mediated via activation of p38 MAPK.

Reactive oxygen species generation and histamine release by activated mast cells: modulation by nitric oxide synthase inhibition.

1. We have examined the generation of intracellular reactive oxygen species (ROS) and release of histamine by rat peritoneal mast cells (RPMC) in response to stimulation with antigen (ovalbumin), compound 48/80, nerve growth factor (NGF) and substance P (SP). 2. We have also examined the effects of the non-specific nitric oxide synthase inhibitor, L-NAME (100 microM) upon the release of histamine and generation of intracellular ROS in response to the named secretagogues. 3. Ovalbumin (100 - 1000 microg ml-1), compound 48/80 (0.1 - 100 microg ml-1), NGF (0.1 - 100 microg ml-1), and SP (5 - 50 microM), caused a concentration-dependent release of histamine from RPMC. 4. Ovalbumin (1 ng ml-1 - 0.1 microg ml-1), compound 48/80 (1 - 100 microg ml-1), NGF (1 pg ml-1 - 1 microg ml-1), and SP (0.005 - 50 microM) caused a concentration-dependent generation of intracellular ROS by RPMC. 5. Pre-incubation of RPMC with L-NAME (100 microM) caused a significant enhancement of both histamine release and intracellular ROS from RPMC in response to ovalbumin, compound 48/80, NGF and SP. 6. Our data demonstrate that NGF, SP and ovalbumin are capable of causing intracellular ROS generation by RPMC at lower concentrations than those causing significant histamine release and we speculate that this may contribute to the activation of cytokine production. 7. The data also show that NO modulates histamine release, and ROS generation in response to the secretagogues used. This may have significance in pathologies where NO synthesis is decreased, leading to an increased activation of mast cells.

Determination of regional myocardial blood flow using fluorescent microspheres.

Determination of blood flow in tissues at risk for infarction is necessary for in vivo studies of the pathologic effects of vascular occlusion. Such blood flow measurements are traditionally carried out using radioactive microspheres. These are expensive and may pose a significant hazard to laboratory personnel. In addition, disposal of radioactive wastes, especially radioactive carcasses, is an increasingly expensive and inconvenient process. To avoid these problems, we have developed a modified procedure using fluorescent microspheres. To measure blood flow, spheres are injected intravenously and a blood sample is taken. Following cardiectomy, appropriate segments of left ventricular myocardium are digested with proteases to release the spheres, which are then purified through sucrose gradients. The spheres may be counted manually using an epifluorescence microscope, or by flow cytometry. Comparison of this method with the traditional radioactive microsphere procedure reveals similar results for the two methods. Fluorescent microspheres should provide a useful and accurate alternative method for measuring blood flow in studies of vascular disease.