Use of real-time near-infrared fluorescence to assess gastric viability in dogs with gastric dilatation volvulus: A case-control study.

OBJECTIVE: To describe near-infrared fluorescence (NIRF) for assessment of gastric viability and describe NIRF's influence on the surgeon's operative strategy in dogs with gastric dilatation and volvulus (GDV). STUDY DESIGN: Prospective clinical trial. ANIMALS: Twenty dogs with GDV and 20 systemically healthy dogs. METHODS: Following gastric derotation, the surgeon's subjective assessment of gastric viability was recorded prior to near-infrared imaging. Changes in the surgeon's initial assessment of viability based on the visual pattern of gastric fluorescence was recorded. If nonviable (lack of defined vessels), a partial gastrectomy was performed and submitted for histopathology. The stapled gastrectomy line was imaged. Viable (defined vessels) and nonviable fluorescence intensities were compared with healthy dogs undergoing surgery for nongastrointestinal disease. RESULTS: Subjective assessment diagnosed 17 viable and three nonviable GDVs (2 fundi; 1 cardia). Near-infrared imaging demonstrated nonviable gastric fluorescence in 4 dogs (3 fundi/cardia; 1 fundus). The surgeon's margins for resection were altered in 3/20 dogs. Fluorescence intensity (cardia, fundus, body, pylorus) was lower in GDV viable (30.59%, p = .04; 38.17%, p < .01; 51.18%, p < .01; 44.12%, p= .01) and nonviable (11.00%, p < .01; 4.33%, p < .01; 57.67%, p = .22; 54.33%, p = .72) dogs compared to healthy controls (44.7%, 70.05%, 84.00%, 63.95%). Fundic fluorescence was less in nonviable gastric tissue in comparison with viable gastric tissue (p = .03). Fluorescence of the gastrectomy staple line approximated that of viable tissue. CONCLUSION: Near-infrared fluorescence can identify histologically confirmed nonviable gastric tissue. CLINICAL SIGNIFICANCE: These results provide enough evidence to support the implementation of NIRF as an adjunct to gross examination of the gastric wall in dogs with GDV.

Investigating mitochondria-immune responses in zebrafish, Danio rerio (Hamilton, 1822): A case study with the herbicide dinoseb.

The dinitrophenol herbicide dinoseb is an uncoupler of mitochondrial oxidative phosphorylation (OXPHOS). Studies in fish demonstrate impaired OXPHOS is associated with altered immune system responses and locomotor activity in fish. The objective of this study was to determine the effect of dinoseb on zebrafish (Danio rerio) during early stages of development. We measured oxygen consumption rates of embryos, transcripts related to OXPHOS, growth, and the immune system (cytokines and immune-signaling transcripts), and locomotor activity. We hypothesized that OXPHOS of fish would be impaired in vivo, leading to altered basal immune system expression and locomotor activity. Oxidative respiration assessments in embryos revealed that dinoseb decreased both mean basal respiration and oligomycin-induced ATP-linked respiration. Expression levels of cytochrome c oxidase complex IV, 3-hydroxyacyl-COA dehydrogenase and superoxide dismutase 1 were decreased in larvae following exposure to dinoseb while succinate dehydrogenase complex flavoprotein subunit A, insulin growth factor 1 (igf1) and igf2a mRNA were increased in abundance. Immune-related transcripts chemokine (C-X-C motif) ligand 1 and matrix metallopeptidase 9 (MMP-9) were decreased in expression levels while toll-like receptor 5a and 5b were increased in expression. In addition, a visual motor response test was conducted on both 6 and 7 dpf larvae to determine if dinoseb impaired locomotor activity. Dinoseb decreased locomotor activity in 7 dpf larvae but not 6 dpf. This study improves knowledge of toxicity mechanisms for dinoseb in early stages of fish development and demonstrates that mitochondrial toxicants may disrupt immune signaling in zebrafish.

Assessing the toxicity of the benzamide fungicide zoxamide in zebrafish (Danio rerio): Towards an adverse outcome pathway for beta-tubulin inhibitors.

Commercial benzamide fungicides are applied to crops to control damage caused by oomycete fungi and are used as veterinary pharmaceuticals in aquaculture. The mechanism of action of these fungicides is to induce mitotic arrest via binding to beta-tubulin, thus inhibiting tubulin polymerization. However, there are little toxicity data available for benzimidazole fungicides in fish. To address this knowledge gap, we conducted zebrafish embryo toxicity tests to assess deformities, survival, and sub-lethal responses following exposure to zoxamide (0, 0.5, 1.0, 2.5, 5.0 and 10 µM zoxamide). We hypothesized that skeletal deformities would be prevalent in zebrafish due to its mechanism of inhibiting beta-tubulin polymerization. Zoxamide was relatively toxic to zebrafish embryos and larvae, and survival was reduced ∼50 % at 2 days post fertilization (dpf) with 10 µM exposure and over time at 6 dpf, 2.5 µM exposure reduced survival by ∼20 %. Frequency of hatch was also reduced/delayed in zebrafish at 3 dpf with >2.5 µM zoxamide. Pericardial edema, body length shortening, and spine curvature were observed in larvae exposed to >5 µM. Mitochondrial bioenergetics were assessed in ∼30 hpf embryos (24-hour exposure) using an XFe24 Flux Analyzer and regression analysis revealed a negative relationship between basal respiration and zoxamide concentration. Superoxide dismutase 1 and caspase 3 mRNA levels were both decreased in 6 dpf larvae exposed to 2.5 µM zoxamide, but were not changed in expression at 0.5 nor 1 µM zoxamide. Continuous 6-day exposure to zoxamide reduced larval activity at 2.5 µM; conversely a 24-hour exposure (at 5-6 dpf) induced hyperactivity at 5 µM suggesting dose and time dependent effects on fish behavior. Based on sub-lethal endpoints, we conceptualize an adverse outcome pathway for chemicals that inhibit tubulin polymerization.

Investigation into the sub-lethal effects of the triazole fungicide triticonazole in zebrafish (Danio rerio) embryos/larvae.

Global use of azole fungicides is expected to increase over the next several years. Triticonazole is a triazole fungicide that is used for turf protection, residential, and other commercial applications. As such, it can enter local rural and urban water systems via run-off and rain events. Early life stages of aquatic organisms can be susceptible to pesticides that enter the water, but in the case of triticonazole, data on the potential for subacute toxicity are lacking. Here, we determined the effects of triticonazole on development, oxygen consumption rates, and locomotor activity in zebrafish to address this knowledge gap. Wild-type zebrafish (ABTu strain) embryos and larvae were exposed to triticonazole (1-100 µM) in early development for different lengths of time depending on the assay conducted. Triticonazole did not affect survival nor induce significant deformity (pericardial edema, skeletal defects) in zebrafish at doses up to 100 µM. Oxygen consumption rate was measured in embryos after 24 and 48 hour exposure to triticonazole beginning at ∼6 hpf using the XFe flux analyzer. Triticonazole did not affect basal respiration, oligomycin-induced ATP linked respiration, FCCP-induced maximum respiration, proton leak, spare capacity, nor non-mitochondrial respiration at doses up to 100 µM for 24 hours, even for exposure up to 250 µM for 48 hours. To determine whether the fungicide affected larval swimming activity, the visual motor response test was conducted following triticonazole exposure for 6 days. Larval zebrafish exposed to triticonazole showed hypoactivity in the dark following a 100 µM treatment, suggesting that the fungicide can affect the locomotor activity of zebrafish, albeit at relatively high levels. Given the fact that sublethal biological responses were absent at lower environmentally relevant concentrations, we conclude that triticonazole, relative to other triazole fungicides and types of pesticides, exhibits a relatively low risk of toxicity to the early life stages of fish.

The organochlorine pesticide toxaphene reduces non-mitochondrial respiration and induces heat shock protein 70 expression in early-staged zebrafish (Danio rerio).

Toxaphene is a restricted-use pesticide produced by reacting chlorine gas with camphene. It was heavily used as a pesticide for agricultural purposes in the 1960-1970s, but despite being banned >30 years ago, it can remain elevated in the soil due to its resistance to metabolic degradation; this has led to longstanding concerns about elevated levels of toxaphene and other organochlorine pesticides (OCPs) in the environment. The objective of this study were to determine the effects of waterborne exposure to toxaphene on early life stages of zebrafish. Based on the LC50, zebrafish embryos were exposed to control (embryo rearing media or DMSO) or to one dose of toxaphene ranging between 0.011 and 111.1 µg/mL from 6 h post fertilization (hpf) up to 120 hpf. Significant mortality and hatch time delays were observed in embryos exposed to toxaphene (at or above 0.11 and 1.11 µg/mL, depending on the assay). Higher prevalence of deformities was noted at higher doses (≥0.011 µg/mL), and these included pericardial edema and skeletal deformities. As energy production is important for normal development, mitochondrial bioenergetics were assessed in embryos following toxaphene exposure. Embryos exposed to 11.1 or 111 µg/mL toxaphene for 24 h showed lower non-mitochondrial respiration (~30%) compared to both solvent and no treatment controls. Expression of transcripts related to oxidative damage responses and apoptosis were measured and heat shock protein 70 was significantly increased with 111 µg/mL toxaphene (14.5 fold), while the expression levels of caspase 3, caspase 9, and superoxide dismutase 1 were not changed. These data demonstrate that developmental deformities induced by toxaphene include pericardial edema and skeletal deformity, and that toxaphene can affect oxidative phosphorylation in early staged zebrafish.

Sub-lethal effects of the triazole fungicide propiconazole on zebrafish (Danio rerio) development, oxidative respiration, and larval locomotor activity.

Propiconazole is a triazole fungicide used in agriculture. Via run-off, it can enter the aquatic environment, and can adversely affect organisms. However, data are scarce on how propiconazole may affect early developmental life stages of fish. The objectives of this study were to evaluate the potential sub-lethal effects of propiconazole during zebrafish development. Wildtype zebrafish (ABTu strain) embryos and larvae were exposed to propiconazole (0.1-100 µM) for up to 150 hours post fertilization (hpf) depending upon the endpoint measured. Propiconazole decreased survival and induced hypopigmentation in fish at 100 µM compared to the water and solvent controls. Pericardial edema was also noted in embryos and larvae (beginning at 2-3 dpf) exposed to 100 µM propiconazole. To visualize the effects of propiconazole on the circulatory system in more detail, we exposed transgenic zebrafish (globin-LCR:eGFP) to the fungicide. Hematopoietic changes were observed within 48 h of exposure to 100 µM, and localization of blood cells in the cardic region became diffuse, indicating pooling of blood in the pericardial region. We measured oxidative respiration in embryos as sufficient ATP is needed for development. Exposure to 100 µM propiconazole (~6-30 hpf) reduced basal respiration (~50%), oligomycin-induced ATP linked respiration (~70%), proton leak (~30%), and non-mitochondrial respiration (~50%), indicating compromised mitochondrial bioenergetics. A Visual Motor Response (VMR) test was used to measure dark photokinesis behavior in larval fish exposed to propiconazole for a 6-day period. Larval fish exposed to the highest concentration in the assay (10 µM) showed evidence of hypoactivity. This study demonstrates that propiconazole can induce hypopigmentation in zebrafish, disrupt mitochondrial bioenergetics, and can alter locomotor activity. However, these sub-lethal responses were observed at concentrations above what is typically detected in the environment.

Tebuconazole reduces basal oxidative respiration and promotes anxiolytic responses and hypoactivity in early-staged zebrafish (Danio rerio).

Triazole fungicides are increasingly used in North America to combat mold and fungi, in order to protect vegetables, citrus, ornamental plants and field crops. To determine the biological impacts of tebuconazole in non-target aquatic organisms, early life stage zebrafish were exposed to 0.1-100 µM tebuconazole for 120 h (5 dpf). There was a significant increase in mortality over time and at 100 µM, only 50% of the animals survived 96 h compared to >95% for all other experimental groups. There was evidence for increased hatching time with 10 µM tebuconazole compared to the control group (~7 h longer at 50% total hatch) or a lack of hatch observed with 100 µM. Oxidative respiration and behavior were evaluated to assess whether the fungicide impaired energy-associated processes. Oxygen consumption rates in embryos (exposed from ~6 hpf) were determined with exposure to 2.5, 25, 50, 100 µM tebuconazole for 24 h using the XFe24 Extracellular Flux Analyzer. Embryos treated with 100 µM showed a ~60% reduction in basal respiration, indicating impaired oxygen consumption and/or changes in resource allocation (e.g. anti-oxidant production, metabolite synthesis). Environmentally-relevant concentrations of tebuconazole did not affect oxidative phosphorylation. As behavior is a sensitive endpoint for toxicity, we measured the dark photokinesis response and conducted a light-dark preference test in 6 dpf larvae following a sub-chronic exposure to 0.1, 1 and 10 µM tebuconazole beginning with 6 hpf embryos. It was observed in two independent experiments for dark photokinesis that 10 µM tebuconazole reduced total distance moved (i.e. hypoactivity) in the dark period by ~25-35%. In the light-dark preference test, there was an increase for mean time in dark zone (~100% increase in the average time/visits per second) and frequency in dark zone (increase of ~35% in average number of visits) with tebuconazole, suggestive of anxiolytic behavior at environmentally-relevant doses. This study demonstrates that exposure to tebuconazole can affect survival, hatch time, oxidative phosphorylation, and behavioral activity of early-staged zebrafish. While survival, hatch time, and mitochondrial bioenergetics were not different than control fish at environmentally-relevant levels of tebuconazole, behavioral responses were detected at concentrations reported in some aquatic environments.

Elucidating Conserved Transcriptional Networks Underlying Pesticide Exposure and Parkinson's Disease: A Focus on Chemicals of Epidemiological Relevance.

While a number of genetic mutations are associated with Parkinson's disease (PD), it is also widely acknowledged that the environment plays a significant role in the etiology of neurodegenerative diseases. Epidemiological evidence suggests that occupational exposure to pesticides (e.g., dieldrin, paraquat, rotenone, maneb, and ziram) is associated with a higher risk of developing PD in susceptible populations. Within dopaminergic neurons, environmental chemicals can have an array of adverse effects resulting in cell death, such as aberrant redox cycling and oxidative damage, mitochondrial dysfunction, unfolded protein response, ubiquitin-proteome system dysfunction, neuroinflammation, and metabolic disruption. More recently, our understanding of how pesticides affect cells of the central nervous system has been strengthened by computational biology. New insight has been gained about transcriptional and proteomic networks, and the metabolic pathways perturbed by pesticides. These networks and cell signaling pathways constitute potential therapeutic targets for intervention to slow or mitigate neurodegenerative diseases. Here we review the epidemiological evidence that supports a role for specific pesticides in the etiology of PD and identify molecular profiles amongst these pesticides that may contribute to the disease. Using the Comparative Toxicogenomics Database, these transcripts were compared to those regulated by the PD-associated neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). While many transcripts are already established as those related to PD (alpha-synuclein, caspases, leucine rich repeat kinase 2, and parkin2), lesser studied targets have emerged as "pesticide/PD-associated transcripts" [e.g., phosphatidylinositol glycan anchor biosynthesis class C (Pigc), allograft inflammatory factor 1 (Aif1), TIMP metallopeptidase inhibitor 3, and DNA damage inducible transcript 4]. We also compared pesticide-regulated genes to a recent meta-analysis of genome-wide association studies in PD which revealed new genetic mutant alleles; the pesticides under review regulated the expression of many of these genes (e.g., ELOVL fatty acid elongase 7, ATPase H+ transporting V0 subunit a1, and bridging integrator 3). The significance is that these proteins may contribute to pesticide-related increases in PD risk. This review collates information on transcriptome responses to PD-associated pesticides to develop a mechanistic framework for quantifying PD risk with exposures.