R Eddie Clutton

A role for solvents in the toxicity of agricultural organophosphorus pesticides

Organophosphorus (OP) insecticide self-poisoning is responsible for about one-quarter of global suicides. Treatment focuses on the fact that OP compounds inhibit acetylcholinesterase (AChE); however, AChE-reactivating drugs do not benefit poisoned humans. We therefore studied the role of solvent coformulants in OP toxicity in a novel minipig model of agricultural OP poisoning. Gottingen minipigs were orally poisoned with clinically relevant doses of agricultural emulsifiable concentrate (EC) dimethoate, dimethoate active ingredient (AI) alone, or solvents. Cardiorespiratory physiology and neuromuscular (NMJ) function, blood AChE activity, and arterial lactate concentration were monitored for 12 h to assess poisoning severity. Poisoning with agricultural dimethoate EC40, but not saline, caused respiratory arrest within 30 min, severe distributive shock and NMJ dysfunction, that was similar to human poisoning. Mean arterial lactate rose to 15.6 [SD 2.8] mM in poisoned pigs compared to 1.4 [0.4] in controls. Moderate toxicity resulted from poisoning with dimethoate AI alone, or the major solvent cyclohexanone. Combining dimethoate with cyclohexanone reproduced severe poisoning characteristic of agricultural dimethoate EC poisoning. A formulation without cyclohexanone showed less mammalian toxicity. These results indicate that solvents play a crucial role in dimethoate toxicity. Regulatory assessment of pesticide toxicity should include solvents as well as the AIs which currently dominate the assessment. Reformulation of OP insecticides to ensure that the agricultural product has lower mammalian toxicity could result in fewer deaths after suicidal ingestion and rapidly reduce global suicide rates.

Opioid Analgesia in Horses

Opioid analgesics have been the foundation of human pain management for centuries, and their value in animals has increased since it was proposed that it is the veterinarians duty to alleviate pain whenever it may occur. Compared with other domesticated species, the horse has benefitted less from the increased understanding of opioid pharmacology in animals, because early literature was overlooked and later work, which examined adverse side effects rather than analgesia, concluded that analgesic and excitatory doses were irreconcilably close. More recent studies have indicated a widening role for opioid analgesics in equine pain management, and radioligand studies have revealed a basis for the equine response pattern to opioid analgesics.

Simultaneous quantification of the organophosphorus pesticides dimethoate and omethoate in porcine plasma and urine by LC-ESI-MS/MS and flow-injection-ESI-MS/MS.

Dimethoate is an organophosphorus toxicant used in agri- and horticulture as a systemic broad-spectrum insecticide. It also exhibits toxic activity towards mammalian organism provoked by catalytic desulfuration in the liver producing its oxon-derivative omethoate thus inhibiting acetylcholinesterase, initiating cholinergic crisis and ultimately leading to death by respiratory paralysis and cardiovascular collapse. Pharmaco- and toxicokinetic studies in animal models help to broaden basic understanding of medical intervention by antidotes and supportive care. Therefore, we developed and validated a LC-ESI-MS/MS method suitable for the simultaneous, selective, precise (RSD(intra-day) 1-8%; RSD(inter-day) 5-14%), accurate (intra-day: 95-107%; inter-day: 90-115%), and robust quantification of both pesticides from porcine urine and plasma after deproteinization by precipitation and extensive dilution (1:11,250 for plasma and 1:40,000 for urine). Accordingly, lower limits of quantification (0.24-0.49 microg/ml plasma and 0.78-1.56 microg/ml urine) and lower limits of detection (0.12-0.24 microg/ml plasma and 0.39-0.78 microg/ml urine) were equivalent to quite low absolute on-column amounts (1.1-2.1 pg for plasma and 2.0-3.9 pg for urine). The calibration range (0.24-250 microg/ml plasma and 0.78-200 microg/ml urine) was subdivided into two linear ranges (r(2)>or=0.998) each covering nearly two orders of magnitude. The lack of any interfering peak in 6 individual blank specimens from plasma and urine demonstrated the high selectivity of the method. Furthermore, extensive sample dilution causing lowest concentration of potentially interfering matrix ingredients prompted us to develop and validate an additional flow-injection method (FI-ESI-MS/MS). Validation characteristics were as good as for the chromatographic method but sample throughput was enhanced by a factor of 6. Effects on ionization provoked by plasma and urine matrix from 6 individuals as well as in the presence of therapeutics (antidotes) administered in an animal study were investigated systematically underlying in the reliability of the presented methods. Both methods were applied to porcine samples derived from an in vivo animal study.

The influence of body mass and thoracic dimensions on arterial oxygenation in anaesthetized horses and ponies

OBJECTIVE To examine the relationship between body mass and thoracic dimensions on arterial oxygen tensions (PaO(2)) in anaesthetized horses and ponies positioned in dorsal recumbency. STUDY DESIGN Prospective clinical study. ANIMALS Thirty six client-owned horses and ponies, mean [+/-SD (range)] age 8.1 +/- 4.8 (1.5-20) years and mean body mass 467 +/- 115 (203-656) kg. METHODS Before general anaesthesia, food and water were withheld for 12 and 1 hours respectively. Body mass (kg), height at the withers (H), thoracic circumference (C), thoracic depth (length between dorsal spinous process and sternum; D), thoracic width (between point of shoulders; W), and thoracic diagonal length (point of shoulder to last rib; L) were measured. Pre-anaesthetic medication was with intravenous (IV) romifidine (0.1 mg kg(-1)). Anaesthesia was induced with an IV ketamine (2.2 mg kg(-1)) and diazepam (0.05 mg kg(-1)) combination and maintained with halothane in 1:1 oxygen:nitrous oxide (N(2)O) mixture. Animals were positioned in dorsal recumbency and allowed to breathe spontaneously. Nitrous oxide was discontinued after 10 minutes, and arterial blood samples obtained and analysed for gas tensions at 15, 30 and 60 minutes after connection to the anaesthetic breathing circuit. Data were analysed using anova and Pearsons correlation co-efficient. RESULTS The height per unit body mass (H kg(-1)) and thoracic circumference per unit body mass (C kg(-1)) correlated strongly (r = 0.85, p < 0.001 and r = 0.82, p < 0.001 respectively) with arterial oxygen tensions (PaO(2)) at 15 minutes. CONCLUSIONS There is a strong positive correlation between H kg(-1) and C kg(-1) and PaO(2) after 15 minutes of anaesthesia in halothane-anaesthetized horses positioned in dorsal recumbency. CLINICAL RELEVANCE Readily obtained linear measurements (height and thoracic circumference) and body mass may be used to predict the ability of horses to oxygenate during anaesthesia.

Rapid and Complete Bioavailability of Antidotes for Organophosphorus Nerve Agent and Cyanide Poisoning in Minipigs After Intraosseous Administration

STUDY OBJECTIVE Management of chemical weapon casualties includes the timely administration of antidotes without contamination of rescuers. Personal protective equipment makes intravenous access difficult but does not prevent intraosseous drug administration. We therefore measured the systemic bioavailability of antidotes for organophosphorus nerve agent and cyanide poisoning when administered by the intraosseous, intravenous, and intramuscular routes in a small study of Göttingen minipigs. METHODS Animals were randomly allocated to sequentially receive atropine (0.12 mg/kg by rapid injection), pralidoxime (25 mg/kg by injection during 2 minutes), and hydroxocobalamin (75 mg/kg during 10 minutes) by the intravenous or intraosseous route, or atropine and pralidoxime by the intramuscular route. Plasma concentrations were measured for 6 hours to characterize the antidote concentration-time profiles for each route. RESULTS Maximum plasma concentrations of atropine and pralidoxime occurred within 2 minutes when administered by the intraosseous route compared with 8 minutes by the intramuscular route. Maximum plasma hydroxocobalamin concentration occurred at the end of the infusion when administered by the intraosseous route. The mean area under the concentration-time curve by the intraosseous route was similar to the intravenous route for all 3 drugs and similar to the intramuscular route for atropine and pralidoxime. CONCLUSION This study showed rapid and substantial antidote bioavailability after intraosseous administration that appeared similar to that of the intravenous route. The intraosseous route of antidote administration should be considered when intravenous access is difficult.

Animal welfare in biomedical publishing.

A recurrent responsibility of the President of the Association of Veterinary Anaesthetists is to respond to AVA members complaining of ‘‘transgressions’’ against animals appearing in the biomedical literature. These transgressions take various forms, for example: the misuse of anaesthetics and, or analgesics in laboratory animal species; the unethical use of client-owned animals in clinical veterinary research; the inappropriate use of control groups and placebos in pain trials, etc. That veterinary anaesthetists appear to be particularly sensitive to these transgressions is hardly surprising given their background, training and motivation. That they are likely to identify them is similarly unsurprising given their inclination to review both veterinary and medical journals on subjects ranging from the physiological and pharmacological sciences to clinical medicine and surgery. What is surprising is that despite clear recommendations from the National Institute of Health (NIH), the Institute for Laboratory Animal Research (ILAR) and other authorities, such transgressions continue to occur and continue to be published. There are at least two reasons for this. First, unless they have received appropriate animal training, which is mandatory for anyone wishing to obtain a personal license and conduct animal experiments in the UK, the editors of many biomedical journals, being non-veterinarians, do not have an inkling as to what constitutes appropriate standards of animal care. This unpalatable truth is readily confirmed by reviewing the ‘‘Guidelines to Contributors’’ of sufficient biomedical journals to encounter the inference in several that the welfare requirement of laboratory animals will be overlooked if the scientific importance of the study justifies it. A second reason for the problem is the increasingly international identity of prestigious medical journals coupled with an increasing submission rate from countries whose cultures allow a different perspective on animal welfare than those of ‘‘developed’’ nations. In the past, it was easy for a journal with a clear national association to cite that country’s legislation in rejecting material describing animal abuse. This becomes less straightforward when the publishing house has offices in the capital city of a developing nation which has no animal protection legislation on its statute books. At the 2008 (London) meeting of the International Association of Veterinary Editors (IAVE) a presentation that I gave (REC) prompted a demand for a consensually formed, compulsory animal welfare element to be added to the ‘‘Guidelines to Contributors’’ of journals subscribing to the IAVE. It was envisioned that this element would state unequivocally that any article describing animal abuse would not be published. It was also anticipated that all member journals of the IAVE would adopt these recommendations as a means of identifying and limiting the problem. In addition to continuing to emphasize the need to optimize laboratory animal welfare, it was hoped the guidelines would extend to the welfare of production animals, client-owned animals and those in public ownership, i.e. wild animals. (A more detailed description of the development of this project can be seen at http://www.veteditors.org/ethicsconsen susguidelines.html). The first draft of the consensual document was based on an amalgamation of the welfare sections of the ‘‘Guidelines for Contributors’’ from journals represented at the 2008 IAVE Congress. In addition, the Guidelines for Contributors from journals with a clear commitment to animal welfare were also studied and some of their requirements incorporated. The first draft report was circulated for approval by IAVE members in November 2008 and whilst it received widespread support, several editors felt it to be too prescriptive and strident in its demands for high standards of animal care in published material. An amended version was reviewed at the 2009 Seattle IAVE meeting and was amended once again. At the 2010

PREPARE: guidelines for planning animal research and testing:

There is widespread concern about the quality, reproducibility and translatability of studies involving research animals. Although there are a number of reporting guidelines available, there is very little overarching guidance on how to plan animal experiments, despite the fact that this is the logical place to start ensuring quality. In this paper we present the PREPARE guidelines: Planning Research and Experimental Procedures on Animals: Recommendations for Excellence. PREPARE covers the three broad areas which determine the quality of the preparation for animal studies: formulation, dialogue between scientists and the animal facility, and quality control of the various components in the study. Some topics overlap and the PREPARE checklist should be adapted to suit specific needs, for example in field research. Advice on use of the checklist is available on the Norecopa website, with links to guidelines for animal research and testing, at https://norecopa.no/PREPARE.

The bispectral index during recovery from halothane and sevoflurane anaesthesia in horses

OBJECTIVE To record the bispectral index (BIS) when horses moved during either halothane or sevoflurane anaesthesia and when they made volitional movements during recovery from these anaesthetics. STUDY DESIGN Randomized prospective clinical study. ANIMALS Twenty-five client-owned horses undergoing surgery aged 8.8 (± 5.3; 1-19) years (mean ± SD; range). METHODS Baseline BIS values were recorded before pre-anaesthetic medication (BISB) and during anaesthesia (BISA) maintained with halothane (group H; n = 12) or sevoflurane (group S; n =13) at approximately 0.8-0.9 × minimum alveolar concentrations (MAC). Bispectral indices were recorded during the surgery when unexpected movement occurred (BISMA), during recovery when the first movement convincingly associated with consciousness was observed (BISM1) and once sternal recumbency was achieved (BISST). RESULTS No significant difference in BISM1 was found between halothane- (85 ± 7; 75-93) and sevoflurane- (87 ± 10; 70-98) anaesthetized horses although BISA was significantly (p = 0.0002) lower in group S (62 ± 7; 53-72) than group H (74 ± 7; 60-84). Differences between BISM1 and BISA were significant in sevoflurane (p = 0.00001) and halothane recipients (p = 0.002) but were greater in group S (25 ± 9; 4-38) compared with group H (12 ± 10; -9-25). In six of eight horses, BISMA values ranged between those recorded during anaesthesia and at first movement. CONCLUSIONS AND CLINICAL RELEVANCE Bispectral indices appear to approximate levels of unconsciousness, suggesting that monitoring the BIS may assist equine anaesthesia. However, it does not predict intra-operative movement.

A Review of Pain Assessment in Pigs

There is a moral obligation to minimize pain in pigs used for human benefit. In livestock production, pigs experience pain caused by management procedures, e.g., castration and tail docking, injuries from fighting or poor housing conditions, “management diseases” like mastitis or streptococcal meningitis, and at parturition. Pigs used in biomedical research undergo procedures that are regarded as painful in humans, but do not receive similar levels of analgesia, and pet pigs also experience potentially painful conditions. In all contexts, accurate pain assessment is a prerequisite in (a) the estimation of the welfare consequences of noxious interventions and (b) the development of more effective pain mitigation strategies. This narrative review identifies the sources of pain in pigs, discusses the various assessment measures currently available, and proposes directions for future investigation.

Cardiovascular and autonomic nervous effects of edrophonium and atropine combinations during neuromuscular blockade antagonism in sheep.

OBJECTIVE To study heart rate (HR), arterial blood pressure (BP) and autonomic nervous (AN) effects of edrophonium-atropine combinations during neuromuscular blockade (NMB) antagonism in sheep. EXPERIMENTAL DESIGN Randomized, prospective and experimental study. ANIMALS Seventy-eight Scottish blackface ewes; mean age: 4.5 years; mean body mass: 54 kg. METHODS After induction with IV etomidate (0.5 mg kg(-1)) and midazolam (0.5 mg kg(-1)), anaesthesia was maintained with halothane and NMB produced with atracurium or mivacurium. In the first study (n = 53), the electrocardiographic (ECG), HR, BP and AN effects of low (40 microg kg(-1)) and high (80 microg kg(-1)) atropine doses combined with either of two edrophonium doses (0.5 or 1.0 mg kg(-1)) were investigated. These variables were also measured in a second study when edrophonium (1.0 mg kg(-1)) was administered 5 minutes before atropine (80 microg kg(-1)) and vice versa. Data were analysed using one-way within-subjects and repeated measures anova. RESULTS In the first study, all combinations reversed NMB but significantly (p < 0.001) increased HR and BP within 2 minutes without arrhythmias. In the second study, edrophonium (1.0 mg kg(-1)) significantly increased HR and BP, saliva flow (n = 1) and lung sounds (n = 3) and caused ECG changes (n = 1). Cardiovascular changes were partially reversed by atropine (80 microg kg(-1)) administered 5 minutes later. Administered first, atropine (80 microg kg(-1)) significantly decreased HR and BP effects which were fully (HR) and partially (BP) reversed by edrophonium (1 mg kg(-1)) administered 5 minutes later. CONCLUSION AND CLINICAL RELEVANCE The cardiovascular effects of edrophonium and atropine were opposite to those reported in humans and dogs. Edrophonium (0.5 mg kg(-1)) and atropine (80 microg kg(-1)) caused the mildest HR changes without ECG and noncardiac AN disturbances, and is recommended for the antagonism of NMB in sheep.