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Citalopram, a selective serotonin reuptake inhibitor (SSRI), is often detected in aquatic ecosystems. In this investigation, developing zebrafish were continuously exposed to one nominal concentration of either 0, 10, or 1000 µg/L citalopram for 7 days. Ribonucleic acids were then extracted from zebrafish for RNA-sequencing using the NovoSeq 6000 (Illumina). Clean reads were obtained following the removal of both the adapter and poly-N sequences. Alignment and differential gene expression analysis was conducted using programs HISAT2 and StringTie assembler. Data were converted to FPKM to quantify differentially expressed transcripts. Significant clinical subnetworks enriched following citalopram exposure included sympathetic nerve activity, blood pressure, vascular tone, and arterial pressure. Regulated transcripts were related to diseases such as mechanical hyperalgesia, pain, inflammatory pain, obstructive hypertrophic cardiomyopathy, fatigue, Diamond-Blackfan anemia, and hypertrophic cardiomyopathy. Following exposure to 10 µg/L citalopram, several transcripts were linked to brain dysfunction like prostaglandin-endoperoxide synthase 2, microtubule associated protein tau, cathepsin B, and dystrophin. Genes related to cardiac dysfunction were altered in zebrafish following exposure to 1000 µg/L citalopram. Using literature and databases that describe gene interactions, molecular networks (clinical and disease networks) were constructed to understand effects of citalopram., (© 2024 The Author(s).)

Antineoplastic medications are present in aquatic environments and are measured at relatively high concentrations in hospital sewage effluent. Thus, it is important to characterize risk associated with waterborne exposures to anticancer drugs. The drug 5-fluorouracil (5-FU) is used to treat several types of cancers, acting to inhibit cell division and cellular metabolism. The objectives of this study were to determine the effects of 5-FU on developmental endpoints and lipid composition in zebrafish. 5-FU did not negatively affect development nor survival in developing zebrafish at concentrations up to 1000 μg/L. However, 5-FU increased neutral lipid content in zebrafish larvae, indicating potential for lipid dysregulation. To further discern effects on lipids, lipidomics was conducted and a total of 164 lipids belonging to 14 lipid classes were identified. Significant changes (false discovery rate < 0.05) in abundance were detected for 19 lipids including some ceramides, ether-linked phosphatidylethanolamines, and sphingomyelins among others. We also measured the expression levels of 14 lipid-related enzymes and transporters (e.g., acox3, dgat1, fads2, fasn, elovl2) using real-time PCR; however, mRNA abundance levels were not affected, suggesting transcriptional changes may not be a primary mechanism underlying lipid dysregulation. Locomotor activity was measured in zebrafish as lipids are needed for swimming activity in larvae. Exposure to 5-FU did not affect locomotor activity up to 1000 μg/L. We conclude that lipids accumulate in larval zebrafish with exposure to 5-FU, which can subsequently affect lipid composition. These data reveal potential lipid signatures of 5-FU exposure and contribute to risk assessments for antineoplastic exposure in aquatic environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pesticides are often used in agriculture and residential areas to mitigate pests and weeds. These chemicals can enter aquatic ecosystems via runoff and rain events, exerting negative effects on aquatic species. In rapidly developing fish embryos, metabolic disruption can alter the developmental trajectory and alter ATP levels. Therefore, it is important to quantify mitochondrial integrity in organisms following exposure to pesticides. To achieve this, a high throughput method to assess pesticide effects on oxidative phosphorylation and mitochondria has been optimized for fish embryos. Fish embryos are first exposed to pesticides for 24 or 48 h, and oxygen consumption rates are measured using the Seahorse XFe24/96 Flux Analyzer (formerly Seahorse Biosciences, now Agilent). The assay utilizes a single embryo and precisely measures oxygen consumption and extracellular acidification. Based upon these measurements, characteristics related to mitochondrial bioenergetics are calculated to provide information on mitochondrial integrity. Using this approach, one can identify pesticides affecting the electron transport chain and ultimately ATP production. In this chapter, we describe the mitochondrial stress test to understand mitochondrial dysfunction and metabolic shifts within the fish embryo., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Buspirone is a pharmaceutical used to treat general anxiety disorder by acting on the dopaminergic and serotoninergic system. Buspirone, like many human pharmaceuticals, has been detected in municipal wastewater; however, the environmental exposure risks are unknown for this psychoactive compound. We studied the effects of buspirone on the behavior of zebrafish, focusing on locomotor and anxiolytic behavior. We also measured transcripts associated with oxidative stress, neurotoxicity, and serotonin signaling to identify potential mechanisms underlying the behavioral changes. Concentrations ranged from environmentally relevant (nM) to physiologically active concentrations typical of human pharmaceuticals (μM). Buspirone treatment did not impact survival, nor did it induce deformities in zebrafish treated for 7 days up to 10 μM. There was a positive relationship between locomotor activity and buspirone concentration in dark periods of the visual motor response test. In the light-dark preference test, both the average time per visit to the dark zone and the percent cumulative duration in the dark zone were increased by 1 μM buspirone. Transcript levels of ache, manf, and mbp were decreased in larvae, while the expression of gap43 was increased following exposure to buspirone, indicating potential neurotoxic effects. There was also reduced expression of serotonin-related genes encoding receptors, transporters, and biosynthesis enzymes (i.e., 5ht1aa, sertb, and tph1a). These data increase understanding of the behavioral and molecular responses in zebrafish following waterborne exposure to neuroactive pharmaceuticals like buspirone., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Broflanilide is a novel insecticide that is classified as a non-competitive γ-aminobutyric acid (GABA) receptor antagonist. However, indiscriminate use can have negative effects on non-target species. The objective of this study was to determine the sub-lethal toxicity potential of broflanilide in early staged zebrafish. Embryos/larvae were assessed for multiple molecular and morphological endpoints following exposure to a range of concentrations of broflanilide. The insecticide did not affect hatch rate, the frequency of deformities, nor did it impact survival of zebrafish at exposure concentrations up to 500 μg/L over a 7-day period from hatch. There was also no effect on oxidative consumption rates in embryos, nor induction of reactive oxygen species in fish exposed up to 100 μg/L broflanilide. As oxidative stress was not prominent as a mechanism, we turned to RNA-seq to identify potential toxicity pathways. Gene networks related to neurotransmitter release and ion channels were altered in zebrafish, consistent with its mechanism of action of modulating GABA receptors, which regulate chloride channels. Noteworthy was that genes related to the circadian clock were induced by 1 μg/L broflanilide exposure. The locomotor activity of larval fish at 7 days was increased (i.e., hyperactivity) by broflanilide exposure based on a visual motor response test, corroborating expression data indicating neurotoxicity and motor dysfunction. This study improves the current understanding of the biological responses in fish to broflanilide exposure and contributes to risk assessment strategies for this novel pesticide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fipronil is a broad-spectrum pesticide presenting high acute toxicity to non-target organisms, particularly to aquatic species. Natural compounds stand out as promising alternatives to the use of synthetic pesticides such as fipronil. Thus, our study aimed to compare the toxicity of carvacrol (natural), acetylcarvacrol (semisynthetic), and fipronil (synthetic) to early staged zebrafish. We conducted a series of toxicity assays at concentrations ranging from 0.01 μM to 25 μM for fipronil and 0.01 μM to 200 μM for carvacrol and acetylcarvacrol, depending on the assay, after 7-days post-fertilization (dpf). The potency (EC 50 ) of fipronil was ∼1 μM for both deformities and mortality at 7 dpf, whereas EC 50 was >50 μM for carvacrol and >70 μM for acetylcarvacrol. Fipronil at 0.1 and 1 μM caused a decrease in body length and swim bladder area of larvae at 7dpf, but no difference was observed for either carvacrol or acetylcarvacrol. Based upon the visual motor response test, fipronil induced hypoactivity in larval zebrafish at 1 μM and acetylcarvacrol induced hyperactivity at 0.1 μM. Anxiolytic-type behaviors were not affected by any of these chemicals. All chemicals increased the production of reactive oxygen species at 7 dpf, but not at 2 dpf. Genes related to swim bladder inflation, oxidative stress, lipid metabolism, and mitochondrial activity were measured; only fipronil induced upregulation of atp5f1c. There were no changes were observed in oxygen consumption rates of fish and apoptosis. Taken together, our data suggest that carvacrol and its derivative may be safer replacements for fipronil due to their lower acute toxicity., Competing Interests: Declaration of competing interest The authors declare that there are no competing interests that could influence the work reported in this manuscript., (Copyright © 2023 Elsevier Inc. All rights reserved.)

"GenX" [ammonium perfluoro (2-methyl-3-oxahexanoate] was developed as a replacement chemical for toxic perfluorinated compounds to be used in product manufacturing. Here, we assessed developmental, mitochondrial, and behavioral toxicity endpoints in zebrafish embryos/larvae exposed to GenX. GenX exerted low toxicity to zebrafish embryos/larvae up to 20 mg/L. GenX did not affect mitochondrial oxidative phosphorylation nor ATP levels. ROS levels were reduced in larvae fish exposed to 10 and 100 µg/L, indicative of an antioxidant defense; however, ROS levels were elevated in fish exposed to 1000 µg/L. Increased expression of cox1 and sod2 in GenX exposed 7-day larvae was noted. GenX (0.1 or 1 µg/L) altered transcripts associated with neurotoxicity (elavl3, gfap, gap43, manf, and tubb). Locomotor activity of larvae was reduced by 100 µg/L GenX, but only in light periods. Perturbations of anxiety-related behaviors in larvae were not observed with GenX exposure. These data inform risk assessments for long-lived perfluorinated chemicals of concern., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Broflanilide is a novel pesticide that can antagonize ion channels and disrupt neurotransmitter systems in the brain. Zebrafish larvae were exposed to either 0, 1 or 10- µg/L broflanilide in the water for a period of 7 days during early development. RNA extraction was conducted on larval zebrafish for RNA-seq analysis using the Illumina NovoSeq 6000. Raw sequence data were processed through fastp and clean reads obtained by removing adapter and poly-N sequences. Alignment and differential gene expression analysis was conducted using HISAT2, StringTie assembler, and FPKM (Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced). Subnetwork enrichment analysis (SNEA) revealed that exposure to 1 µg/L broflanilide altered gene networks associated with axonal injury, depression, neuroinflammation, and traumatic brain injury while exposure to 10- µg/L broflanilide resulted in changes in gene networks associated with brain infarction and ischemia, excitotoxicity, and neurogenic inflammation. In addition, genes related to MPTP-induced neurotoxicity were altered by broflanilide which has relevance for Parkinson's disease. Several transcripts were identified as being associated with a disease network link to neurodegeneration and included phospholipase A2 activating protein, calpain 1, ATPase Na+/K+ transporting subunit alpha 2, glia maturation factor beta, sphingomyelin phosphodiesterase 1, leucine rich repeat kinase 2, glutamate ionotropic receptor NMDA type subunit 2C, lysosomal associated membrane protein, and calcium/calmodulin dependent protein kinase II alpha among others. Data presented here include disease biomarkers for a novel pesticide and can be reused to refine models that describe adverse outcome pathways for neurotoxicity., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Zebrafish are frequently used as a vertebrate model to elucidate toxicological and pharmacological mechanisms of action in the central nervous system. Pharmacological studies demonstrate that dopamine, signaling via several receptor subtypes, regulates zebrafish larval behavior. Quinpirole is a selective dopamine receptor agonist for D2 and D3 subtypes while ropinirole exhibits selectivity toward D2, D3, and D4 receptors. The main objective of this study was to determine the short-term actions of quinpirole and ropinirole on the locomotor activity and anxiolytic/anti-anxiolytic behaviors of zebrafish. Furthermore, dopamine signaling can cross talk with other neurotransmitter systems, including the GABAergic and glutamatergic system. As such, we measured transcriptional responses in these systems to determine whether dopamine receptor activation modulated GABAergic and glutaminergic systems. Ropinirole reduced locomotor activity of larval fish at concentrations of 1 μM and greater but quinpirole did not affect locomotor activity at all concentrations tested. Anxiolytic-related behaviors were also compared between the two pharmaceuticals. Noteworthy was that both dopamine receptor agonists at 1 μM increased the activity of zebrafish in the light phase of a light-dark preference test, which may be related to the activation of D2 and/or D3 receptors. In terms of interactions with other neurotransmitter systems, ropinirole up-regulated transcripts in larvae zebrafish related to both the GABAergic and glutamatergic systems (abat, gabra1, gabrb1, gad1b, gabra5, gabrg3, and grin1b). Conversely, quinpirole did not alter the abundance of any transcript measured, suggesting that dopamine-GABA interaction may involve D4-receptors, which has been noted in mammalian models. This study demonstrates pleiotropic actions of dopamine agonism on the GABA and glutamate system in larval zebrafish. This study has relevance for characterizing toxicants that act via dopamine receptors and for elucidating mechanisms of neurological disorders that involve motor circuits and multiple neurotransmitter systems, like Parkinson's disease., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Blood pressure medications are used to treat hypertension; however, low concentrations of beta-blockers in water systems can negatively impact aquatic wildlife. Here, we conducted a metabolic and behavioral study investigating atenolol, a beta-blocker frequently detected in global wastewater systems. The objectives were to determine the effects of low-level atenolol exposure on early stages of zebrafish. We measured survival, deformities, heartbeat, mitochondrial function, lipid and amino acid profiles, and locomotor activity to discern mechanisms of metabolic disruption. We hypothesized that atenolol disrupts lipid metabolism, which would negatively impact locomotor activity. Atenolol showed no overt toxicity to larval zebrafish up to 10 μg/L and deformities were infrequent (<5 %), and included cardiac edema and larvae with kinked tails. A hatch delay was observed at 2-day post-fertilization (dpf) for fish exposed to >5 μg/L atenolol. Heart rates were reduced in 2 and 3 dpf in fish treated with >500 ng/L atenolol. There was no change in oxygen consumption rates (basal and maximum respiration) of embryos when exposed to a range of atenolol concentrations, suggesting mitochondrial respiration was intact. Oil red staining for lipid content in larvae showed a global reduction in lipids with 10 μg/L exposure, prompting deeper investigation into the lipid profiles. Lipidomics quantified 86 lipids and revealed reduced abundance in Ceramide 18: 1 16:0 (Cer&#95;NS d18:1&#95;16:0), Ether linked Phosphatidylethanolamine 16:0 22:6 (EtherPE 16:0e&#95;22:6), and Ether linked Phosphatidylcholine 16:0 22:6 (EtherPC 16:0e&#95;22:6). We also quantified 12 amino acids and observed a subtle dose-dependent reduction in the levels of L-Histidine. Exposure to atenolol did not impact larval locomotor activity based on a Visual Motor Response test. Taken together, atenolol at environmentally relevant levels decreased heart rate of developing zebrafish and altered lipid content. As such, exposure to beta-blockers like atenolol may have negative consequences for developmental trajectories and growth of aquatic species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Sulfamethoxazole (SMX) is a broad-range bacteriostatic antibiotic widely used in animal and fish farming and is also employed in human medicine. These antibiotics can ultimately end up in the aquatic ecosystem and affect non-target organisms such as fish. To discern the effect of SMX on developing zebrafish embryos and larvae, we investigated a broad range of sub-lethal toxicity endpoints. Higher concentrations of SMX affected survivability, caused hatch delay, and induced malformations including edema of the yolk sac, pericardial effusion, bent tail, and curved spine in developing embryos. Lower levels of SMX provoked an inflammatory response in larvae at seven days post fertilization (dpf), as noted by up-regulation of interferon ( ifn-γ ) and interleukin 1β ( il-1β ). SMX also increased the expression of genes related to apoptosis, including BCL2-Associated Agonist of Cell Death ( bad ) and BCL2 Associated X, Apoptosis Regulator ( bax ) at 50 µg/L and decreased caspase 3 ( casp3 ) expression in a dose-dependent manner. SMX induced hyperactivity in larval fish at 500 and 2500 µg/L based upon the light/dark preference test. Collectively, this study revealed that exposure to SMX can disrupt the immune system by altering host defense mechanisms as well as transcripts related to apoptosis. These data improve understanding of antibiotic chemical toxicity in aquatic organisms and serves as a baseline for in-depth environmental risk assessment of SMX and antibiotics.

Butyrate, a short-chain fatty acid, is utilized by the gut epithelium as energy and it improves the gut epithelial barrier. More recently, it has been associated with beneficial effects on immune and cardiovascular homeostasis. Conversely, tumor necrosis factor alpha (TNFα) is a pro-inflammatory and pro-hypertensive cytokine. While butyrate and TNFα are both linked with hypertension, studies have not yet addressed their interaction in the colon. Here, we investigated the capacity of butyrate to modulate a host of effects of TNFα in primary rodent colonic cells in vitro. We measured ATP levels, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), mitochondrial oxidative phosphorylation, and glycolytic activity in colonocytes following exposure to either butyrate or TNFα, or both. To address the potential mechanisms, transcripts related to oxidative stress, cell fate, and cell metabolism (Pdk1, Pdk2, Pdk4, Spr, Slc16a1, Slc16a3, Ppargc1a, Cs, Lgr5, Casp3, Tnfr2, Bax, Bcl2, Sod1, Sod2, and Cat) were measured, and untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to profile the metabolic responses of colonocytes following exposure to butyrate and TNFα. We found that both butyrate and TNFα lowered cellular ATP levels towards a quiescent cell energy phenotype, characterized by decreased oxygen consumption and extracellular acidification. Co-treatment with butyrate ameliorated TNFα-induced cytotoxicity and the reduction in cell viability. Butyrate also opposed the TNFα-mediated decrease in MMP and mitochondrial-to-intracellular calcium ratios, suggesting that butyrate may protect colonocytes against TNFα-induced cytotoxicity by decreasing mitochondrial calcium flux. The relative expression levels of pyruvate dehydrogenase kinase 4 ( Pdk4 ) were increased via co-treatment of butyrate and TNFα, suggesting the synergistic inhibition of glycolysis. TNFα alone reduced the expression of monocarboxylate transporters slc16a1 and slc16a3, suggesting effects of TNFα on butyrate uptake into colonocytes. Of the 185 metabolites that were detected with LC-MS, the TNFα-induced increase in biopterin produced the only significant change, suggesting an alteration in mitochondrial biogenesis in colonocytes. Considering the reports of elevated colonic TNFα and reduced butyrate metabolism in many conditions, including in hypertension, the present work sheds light on cellular interactions between TNFα and butyrate in colonocytes that may be important in understanding conditions of the colon., Competing Interests: The authors declare no conflicts of interest.

The potential toxicity of several perfluoroalkyl and polyfluoroalkyl substances (PFASs) to aquatic species are not well understood. Here, we assessed the sub-lethal toxicity potential of perfluoroheptanoic acid (PFHpA) to developing zebrafish. PFHpA was not acutely toxic to fish up to 50 µM and there was > 96% survival in all treatments. Exposure to 200 µM PFHpA decreased ATP-linked respiration of embryos. There was no evidence for ROS induction in 7-day-old larvae fish exposed to 0.1 µM or 1 µM PFHpA. Twenty-four transcripts related to mitochondrial complexes I through V were measured and atp06, cox4i1, and cyc1 levels were decreased in larval zebrafish in a concentration-dependent manner by PFHpA exposure. Locomotor activity was reduced in fish exposed to 0.1 µM PFHpA based on a visual motor response test. Anxiolytic-type behaviors were not affected by PFHpA. This study contributes to environmental risk assessments for perfluorinated chemicals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Industrial and consumer products, such as pesticides, lubricants, and cosmetics, can contain perfluorinated compounds (PFCs). Although many short-chain PFCs have been linked to physiological and behavioral changes in fish, there are limited data on longer-chain PFCs. The objective of this study was to determine the potential impact of perfluorotetradecanoic acid (PFTeDA) exposure on zebrafish ( Danio rerio ) during early developmental stages. We measured several endpoints including gene expression, mitochondrial bioenergetics, and locomotor activity in zebrafish. Survival, timing of hatching, and deformity frequency were unaffected by PFTeDA at the concentrations tested (0.01, 0.1, 1, and 10 µM) over a 7-day exposure period. The expression levels of mitochondrial-related genes ( cox1 and mt-nd3 ) and oxidative stress-related genes ( cat, hsp70, and hsp90a ) were increased in larval fish with exposure to 10 µM PFTeDA; however, there was no change in oxidative respiration of embryos (i.e., basal respiration and oligomycin-induced ATP-linked respiration). Reactive oxygen species were reduced in larvae treated with 10 µM PFTeDA, coinciding with the increased transcription of antioxidant defense genes. Both the visual motor response test and light-dark preference test were conducted on 7 dpf larvae and yielded no significant findings. This study improves current knowledge regarding toxicity mechanisms for longer-chain PFCs such as PFTeDA.

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Strobilurin fungicides are quinone outside inhibitors (QoI) used to treat fungal pathogens for agricultural and residential use. Here, we compared the potential for neurotoxicity of the widely used strobilurins, azoxystrobin (AZS) and trifloxystrobin (TFS), in differentiated human SH-SY5Y cells. Fungicides did not include cytotoxicity up to 200 µM but both induced loss of cell viability at 48 h, with TFS showing slightly higher toxicity that AZS. Caspase 3/7 activity was induced in SH-SY5Y cells by both fungicides at 48 h (50 µM for AZS and 25 µM for TFS). ATP levels were reduced following a 24-hour exposure to > 25 µM AZS and > 6.25 µM TFS and both fungicides rapidly impaired oxidative respiration (~12.5 µM for AZS and ~3.125 µM TFS) and decreased oligomycin-induced ATP production, maximal respiration, and mitochondrial spare capacity. AZS at 100 µM showed a continual impairment of mitochondrial membrane potential (MMP) between 4 and 48 h while TFS at > 50 µM decreased MMP at 24 h. Taken together, TFS exerted higher mitochondrial toxicity at lower concentrations compared to AZS in SH-SY5Y cells. To discern toxicity mechanisms of strobilurin fungicides, lipidomics was conducted in SH-SY5Y cells following exposure to 6.25 µM and 25 µM AZS, and a total of 1595 lipids were detected, representing 49 different lipid classes. Lipid classes with the largest proportion of lipids detected in SH-SY5Y cells included triglycerides (17%), phosphatidylethanolamines (8%), ether-linked triglycerides (8%), phosphatidylcholines (7%), ether-linked phosphatidylethanolamines (6%), and diacylglycerols (5%). Together, these 5 lipid classes accounted for over 50% of the total lipids measured in SH-SY5Y cells. Lipids that were increased by AZS included acyl carnitine, which plays a role in long chain fatty acid utilization for mitochondrial β-oxidation, as well as non-modified, ether linked, and oxidized triacylglycerols, suggesting compensatory upregulation of triglyceride biosynthesis. The ceramide HexCer-NS, linked to neurodegenerative diseases, was decreased in abundance following AZS exposure. In summary, strobilurin fungicides rapidly inhibit mitochondrial oxidative respiration and alter the abundance of several lipids in neuronal cells, relevant for understanding environmental exposure risks related to their neurotoxicity., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The relative toxicity of glyphosate (GLY) and its metabolite aminomethylphosphonic acid (AMPA) to zebrafish were compared. Embryos/larvae were exposed to one dose of either GLY (0.1, 1, or 10 μM), AMPA (0.1, 1, or 10 μM), or a 1 μM mixture for 7-days post-fertilization. Survival, success of hatch, and deformity frequency were not different from controls. Neither chemical induced reactive oxygen species in larval fish. GLY increased superoxide dismutase 2 mRNA in larvae while AMPA increased catalase and superoxide dismutase 1 in a concentration-specific manner. GLY increased cytochrome c oxidase subunit 4 isoform 1 and citrate synthase mRNA in larvae while AMPA decreased cytochrome c oxidase I and increased 3-hydroxyacyl CoA dehydrogenase transcripts. Hyperactivity was noted in fish treated with GLY, but not AMPA nor the mixture. Anxiety-like behaviors were absent with exposure to GLY or AMPA. GLY and AMPA may exert different effects at the molecular and behavioral level., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The colonic epithelium is the site of production and transport of many vasoactive metabolites and neurotransmitters that can modulate the immune system, affect cellular metabolism, and subsequently regulate blood pressure. As an important interface between the microbiome and its host, the colon can contribute to the development of hypertension. In this critical review, we highlight the role of colonic inflammation and microbial metabolites on the gut brain axis in the pathology of hypertension, with special emphasis on the interaction between tumor necrosis factor α (TNFα) and short chain fatty acid (SCFA) metabolites. Here, we review the current literature and identify novel pathways in the colonic epithelium related to hypertension. A network analysis on transcriptome data previously generated in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats reveals differences in several pathways associated with inflammation involving TNFα (NF-κB and STAT Expression Targets) as well as oxidative stress. We also identify down-regulation of networks associated with gastrointestinal function, cardiovascular function, enteric nervous system function, and cholinergic and adrenergic transmission. The analysis also uncovered transcriptome responses related to glycolysis, butyrate oxidation, and mitochondrial function, in addition to gut neuropeptides that serve as modulators of blood pressure and metabolic function. We present a model for the role of TNFα in regulating bacterial metabolite transport and neuropeptide signaling in the gastrointestinal system, highlighting the complexity of host-microbiota interactions in hypertension., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

Antineoplastics treat cancers and enter aquatic ecosystems through wastewater and hospital effluent. Risks associated with antineoplastics are not well characterized in aquatic organisms. We conducted zebrafish embryo/larvae toxicity assays to evaluate responses to cyclophosphamide (0.01-50 µM). Zebrafish survival was affected by 5 µM cyclophosphamide and deformities were noted at > 1 µM. Oxidative respiration remained unchanged in embryos with exposure up to 200 µM. Reactive oxygen species were not increased by 50 µM cyclophosphamide exposure. More than 15 oxidative stress and immune-related transcripts were measured. Superoxide dismutase 2 and heat shock protein 70 and 90a were induced in larvae by cyclophosphamide. Immune-related transcripts were assessed due to immunosuppressive properties of cyclophosphamide, and mmp9 and myd88 levels were altered in expression. Hyperactivity of larvae was noted following 5 µM cyclophosphamide exposure. There was no change in anxiety-related endpoints (light-dark preference). Risks for larval fish exposed to cyclophosphamide in the environment may be low., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Human amylin forms structurally heterogeneous amyloids that have been linked to type-2 diabetes. Thus, understanding the molecular interactions governing amylin aggregation can provide mechanistic insights in its pathogenic formation. Here, we demonstrate that fibril formation of amylin is altered by synthetic amphipathic copolymer derivatives of the styrene-maleic-acid (SMAQA and SMAEA). High-speed AFM is used to follow the real-time aggregation of amylin by observing the rapid formation of de novo globular oligomers and arrestment of fibrillation by the positively-charged SMAQA. We also observed an accelerated fibril formation in the presence of the negatively-charged SMAEA. These findings were further validated by fluorescence, SOFAST-HMQC, DOSY and STD NMR experiments. Conformational analysis by CD and FT-IR revealed that the SMA copolymers modulate the conformation of amylin aggregates. While the species formed with SMAQA are α-helical, the ones formed with SMAEA are rich in β-sheet structure. The interacting interfaces between SMAEA or SMAQA and amylin are mapped by NMR and microseconds all-atom MD simulation. SMAEA displayed π-π interaction with Phe23, electrostatic π-cation interaction with His18 and hydrophobic packing with Ala13 and Val17; whereas SMAQA showed a selective interaction with amylin's C terminus (residues 31-37) that belongs to one of the two β-sheet regions (residues 14-19 and 31-36) involved in amylin fibrillation. Toxicity analysis showed both SMA copolymers to be non-toxic in vitro and the amylin species formed with the copolymers showed minimal deformity to zebrafish embryos. Together, this study demonstrates that chemical tools, such as copolymers, can be used to modulate amylin aggregation, alter the conformation of species., Competing Interests: Declaration of interests The polymers used in this study SMAEA and SMAQA are produced in the Ramamoorthy lab at Michigan. They are US patented., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pendimethalin is a dinitroaniline herbicide used to control broadleaf weeds by inhibiting the formation of microtubules during cell division. Its use on a variety of crops leads to its potential entry into aquatic environments, but little is known about its sub-lethal toxicity to early developmental stages of aquatic vertebrates. To address this knowledge gap, we assessed the toxicity of pendimethalin to zebrafish embryos and larvae by measuring mortality, developmental abnormalities, oxidative respiration, reactive oxygen species, gene expression, and locomotor activity following exposure to the herbicide throughout early development. Embryos at ~6 h post-fertilization (hpf) were exposed to either a solvent control (0.1% DMSO, v/v), embryo rearing medium (ERM), or one dose of either 1, 2.5, 5, or 25 μM pendimethalin for up to 7-days post fertilization depending on the bioassay. Exposure to 25 μM pendimethalin resulted in high prevalence of spinal curvature, tail malformations, pericardial edema, and yolk sac edema at 4 dpf, while exposure to 5 μM pendimethalin induced pericardial edema and lordosis in the fish exposed over 7 dpf. Exposure to pendimethalin up to 5 μM did not negatively impact oxidative respiration (e.g., basal respiration, oligomycin-induced ATP production) in embryos following a 24-h exposure. Pendimethalin did not induce reactive oxygen species at concentrations of 1-5 μM. Levels of transcripts associated with oxidative respiration and damage response were altered in 7d-larvae; cox1 mRNA was increased in larvae fish exposed to 1 μM while cox5a1 and sod2 mRNA were decreased with 2.5 μM exposure. The Visual Motor Response (VMR) test for light-dark response revealed that larval activity in the dark period was reduced for zebrafish exposed to >1 μM pendimethalin compared to ERM and DMSO solvent control groups. These data inform on the sub-lethal toxicity of pendimethalin to early stages of fish embryos and larvae., (Copyright © 2021. Published by Elsevier Inc.)

Acetochlor is one of the most widely used herbicides in the world, however, there are few data on the sub-lethal effects of acetochlor on early developmental stages of fish. To address this, we measured survival, deformity, swim bladder formation, embryo oxygen consumption rates, reactive oxygen species (ROS) levels, transcripts (related to swim bladder formation, oxidative damage response, and apoptosis) and behavior responses following exposure to acetochlor (0.001 µM up to 125 µM). Exposure to acetochlor at concentrations 50 µM and above exerted 100% mortality after 3 dpf, and significantly reduced the size of the swim bladder (25 µM). In embryos, basal respiration, oligomycin-induced ATP production, and maximal respiration were decreased 30-60% following a 24 h exposure to 125 μM acetochlor. Acetochlor did not affect ROS levels up to 25 µM in larvae with acute exposure. Acetochlor at 25 µM increased mRNA levels of bax1, hsp70, and hsp90a by ~4-fold in larval zebrafish. In both the visual motor response and light-dark preference test, 25 µM acetochlor increased locomotor activity of larval fish. At lower exposure concentrations, 100 and 1000 nM acetochlor increased the mean time spent in the dark zone, suggesting promotion of anxiolytic behavior. This study presents a comprehensive evaluation of sublethal effects of acetochlor, spanning molecular responses to behavior, which can be used to refine risk assessment decisions for aquatic environments., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Strobilurin fungicides are among the most widely used in the world and have characteristics that include high water solubility and toxicity to aquatic organisms. While several studies report on mechanisms of toxicity of strobilurins in fish, there are no data on the sub-lethal toxicity of fish to the fungicide fenamidone. To address this gap, survival and hatch rate, deformities, mitochondrial bioenergetics, expression of oxidative stress and apoptotic genes, and behavior (locomotor activity and anxiolytic-related behaviors) were assessed in zebrafish embryos and larvae following exposure to fenamidone. Fenamidone negatively affected development of zebrafish embryos, causing a delay of hatching time at concentrations of 2.5 and 5 μM. Fenamidone caused morphological deformities in zebrafish, including pericardial edema, yolk sac edema, tail deformities, and spinal curvature. Exposure to 1.5 μM fenamidone reduced surface area of swim bladder in larvae at 6 dpf. Fenamidone significantly reduced oxygen consumption rates of embryos; 5 μM fenamidone decreased basal respiration (~85%), oligomycin induced ATP-linked respiration (~70%), FCCP-induced maximal respiration (~75%) and non-mitochondrial respiration (~90%) compared to controls. Sod2 mRNA levels were decreased by fenamidone in larval fish. Locomotor activity was significantly decreased in zebrafish larvae following exposure to 2 μM fenamidone but there was no evidence for anxiolytic nor anxiety-related behaviors (exposures of 100 nM up to 1.5 µM). This study addresses a data gap for potential risks associated with fenamidone exposure in developing fish., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The phenylpyrazole fipronil is an insecticide that inhibits γ -amino-butyric acid (GABA) ionotropic receptors in the central nervous system. Experimental evidence suggests that fipronil acts as a neurotoxin and it is implicated in neurodegenerative diseases; however, the mechanisms of neurotoxicity are not fully elucidated. The objective of this study was to quantify mechanisms of fipronil-induced neurotoxicity in dopamine cells. Rat primary immortalized mesencephalic dopaminergic cells (N27) were treated with fipronil (0.25 up to 500 μM depending on the assay). We measured endpoints related to mitochondrial bioenergetics, mitophagy, mitochondrial membrane potential, and ATP production in addition to discerning transcriptome responses to the pesticide. Fipronil reduced cell viability at 500 μM after 24 h exposure and caspase 3/7 activity was significant increased after 6 and 12 h by 250 and 500 μM fipronil. Subsequent endpoints were thus assessed at concentrations that were below cytotoxicity. We measured oxidative respiration of N27 cells following a 24 h exposure to one dose of either 0.25, 2.5, 25, or 50 μM fipronil. Oxygen consumption rates (OCR) were not different between vehicle-control and 0.25 or 2.5 μM fipronil treatments, but there was a ∼40-60 % reduction in basal respiration, as well as reduced oligomycin-induced ATP production at 50 μM. The reduction in OCR is hypothesized to be related to lower mitochondrial mass due to mitophagy. Mitochondrial membrane potential was also sensitive to fipronil, and it was compromised at concentrations of 2.5 μM and above. To further elucidate the mechanisms linked to neurotoxicity, we conducted transcriptomics in dopamine cells following treatment with 25 μM fipronil. Fipronil suppressed transcriptional networks associated with mitochondria (damage, depolarization, permeability, and fission), consistent with its effects on mitochondrial membrane potential. Altered gene networks also included those related to Alzheimer disease, inflammatory disease, nerve fiber degeneration, and neurofibrillary tangles. This study clarifies molecular targets of fipronil-induced neurotoxicity and supports, through multiple lines of evidence, that fipronil acts as a mitochondrial toxicant in dopamine cells. This is relevant to neurodegenerative diseases like Parkinson's disease as exposure to fipronil is associated with the progressive loss of nigrostriatal dopaminergic neurons in rodents., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Perfluorooctanoic acid (PFOA) is an abundant per- and polyfluoroalkyl substance (PFAS) detected in both indoor and outdoor environments. While studies suggest exposure concerns for humans, studies investigating PFOA-induced neurotoxicity are lacking. To address this gap, we exposed differentiated human SH-SY5Y cells to PFOA (0.1 μM up to 500 μM) at different time points (4, 24, 48, and 72 h) and measured cell viability, Casp3/7 activity, ATP levels, ATP synthase enzyme activity, mitochondrial membrane potential, reactive oxygen species (ROS), oxygen consumption rates for mitochondrial stress test (XFe24 Flux analyzer), glucose utilization, and global metabolome profiles to assess the potential for PFOA-induced neurotoxicity. Treatment with 10 or 100 μM PFOA did not compromise cell viability nor induce cytotoxicity to SH-SY5Y cells over a 48-hour exposure period. However, >250 μM PFOA compromised cell viability, induced cytotoxicity, and induced caspase 3/7 activity at 48 h. ATP levels were reduced in cells treated with 400 μM PFOA for 24 and 48 h, and with 100 μM PFOA and higher at 72 h. ATP synthase activity was inhibited by 250 μM PFOA but was unchanged by PFOA treatment at 200 μM or less. Conversely, mitochondrial membrane potential was reduced by >10 μM PFOA after 24 h. Total ROS was increased with 100 μM PFOA and higher after 4 h of exposure. Several mitochondria-related endpoints (basal respiration, ATP production, maximum respiration) were negatively affected at 250 μM PFOA at both 24- and 48-hour exposure, but were unaltered at concentrations of 100 μM PFOA or less. One exception was mitochondrial spare capacity, which was reduced by 100 μM PFOA after 24-hour exposure. Similarly, glycolysis, glycolytic capacity, and glycolytic reserve of SH-SY5Y cells were not altered by 10 nor 100 μM PFOA. Nontargeted metabolomics was conducted in cells treated with either 10 or 100 μM PFOA for 48 h, as these two concentrations were not cytotoxic and 28 metabolites differed among treatments. Notable was that 10 μM PFOA had little effect on the SH-SY5Y metabolome, and the metabolic profile was not statistically different from media nor solvent controls. On the other hand, 100 μM PFOA shifted the metabolic signature of the neuronal cells, leading to reduced abundance of ATP-related metabolites (adenine, nicotinamide), neurotransmitter precursors (DL-tryptophan, l-tyrosine), and metabolites that protect mitochondria during oxidative stress (betaine, orotic acid, and l-acetyl carnitine). We hypothesize that this metabolic signature may be associated with the reduced mitochondrial membrane potential observed at lower PFOA concentrations. Metabolic shifts appear to precede compromised cell viability, cytotoxicity, and apoptosis. This study generates mechanistic knowledge regarding PFOA-induced neurotoxicity, focusing on mitochondrial oxidative respiration and the neuronal metabolome., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In teleost fish, radial glial cells (RGCs) are progenitor cells for neurons and the major cell type synthesizing neuroestrogens. We hypothesized that chemical exposure impairs mitochondrial bioenergetics of RGCs, which then may lead to downstream consequences for neuroestrogen production. Here we provide proof of concept that mitochondria of RGCs can be perturbed by fungicides. We isolated RGCs from a mixed sex population of goldfish (Carassius auratus) and measured metabolic capacity of primary cells to a model mitotoxin fluazinam, a broad-spectrum fungicide that inhibits mitochondria electron transport chain (or ETC) Complex I. Using immunocytochemistry and real-time PCR, we demonstrate that the goldfish primary cell cultures are highly enriched for glia after multiple passages. Cytotoxicity assays revealed that glia treated with >25 μM fluazinam for 24 and 48-h showed reduced viability. As such, metabolic assays were conducted with non-cytotoxic concentrations (0.25-12.5 μM). Fluazinam did not affect oxygen consumption rates of RGCs at 24 h, but after 48 h, oligomycin induced ATP-linked respiration was decreased by both 6.25 and 12.5 μM fluazinam. Moreover, concentrations as low as 0.25 μM disrupted the mitochondrial membrane potential of RGCs, reflecting strong uncoupling effects of the fungicide on mitochondria. Here we provide proof of concept that mitochondrial bioenergetics of teleostean RGCs can be responsive to agrochemicals. Additional studies are required to address low-dose exposures in vivo and to determine if metabolic disruption impairs neuroendocrine functions of RGCs. We propose this mechanism constitutes a novel aspect of neuroendocrine disruption, significant because dysregulation of neuron-glia communication is expected to contribute to neuroendocrine disruption., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Strobilurin fungicides have been frequently detected in aquatic environments and can induce mitochondrial toxicity to non-target aquatic organisms. However, the derived toxicity and subsequent mechanisms related to their adverse effects are not fully elucidated. In the present study, we compared the mitochondrial and developmental toxicity of azoxystrobin, pyraclostrobin, and trifloxystrobin using zebrafish embryo/larvae. The results showed that all three strobilurins inhibited mitochondrial and non-mitochondrial respiration (the potency is pyraclostrobin ≈ trifloxystrobin > azoxystrobin). Behavioral changes indicated that sublethal doses of pyraclostrobin and azoxystrobin caused hyperactivity of zebrafish larvae in dark cycles, whereas trifloxystrobin resulted in hypoactivity of zebrafish larvae. In addition, pyraclostrobin exposure impaired the inflation of swim bladder, and caused down-regulation of annexin A5 (anxa5) mRNA levels, and up-regulated transcript levels of pre-B-cell leukemia homeobox 1a (pbx1a); conversely, azoxystrobin and trifloxystrobin did not cause detectable effects with swim bladder inflation. Molecular docking results indicated that azoxystrobin had higher interacting potency with iodotyrosine deiodinase (IYD), prolactin receptor (PRLR), antagonistic conformation of thyroid hormone receptor β (TRβ) and glucocorticoid receptor (GR) compared to pyraclostrobin and trifloxystrobin; pyraclostrobin and azoxystrobin were more likely to interact with the antagonistic conformation of TRβ and GR, respectively. These results may partially explain the different effects observed in behavior and swim bladder inflation, and also point to potential endocrine disruption induced by these strobilurins. Taken together, our study revealed that all three strobilurins alter mitochondrial bioenergetics and cause developmental toxicity. However, the toxic phenotypes and underlying mechanisms of each chemical may differ, and this requires further investigation. Pyraclostrobin showed higher mitochondrial toxicity at lethal doses and higher developmental toxicity at sublethal doses compared to the two other strobilurins tested. These results provide novel information for toxicological study as well as risk assessment of strobilurin fungicides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Metolachlor herbicides are derived from the chloroacetamide chemical family of which there are the S- and R-metolachlor isomers. S-metolachlor is a selective herbicide that inhibits cell division and mitosis via enzyme interference. The herbicide is used globally in agriculture and studies report adverse effects in aquatic organisms; however, there are no studies investigating sub-lethal effects of S-metolachlor on swim bladder formation, mitochondrial ATP production, nor light-dark preference behaviors in fish. These endpoints are relevant for larval locomotor activity and metabolism. To address these knowledge gaps, we exposed zebrafish embryos/larvae to various concentrations of S-metolachlor (0.5-50 µM) over early development. S-metolachlor affected survival, hatching percentage, and increased developmental deformities at concentrations of 50 µM and above. Exposure levels as high as 200 µM for 24 and 48 h did not alter oxygen consumption rates in zebrafish, and there were no changes detected in endpoints related to mitochondrial oxidative phosphorylation. We observed impairment of swim bladder inflation at 50 µM in 6 dpf larvae. To elucidate mechanisms related to this, we measured relative transcript abundance for genes associated with the swim bladder (smooth muscle alpha (α)-2 actin, annexin A5, pre-B-cell leukemia homeobox 1a). Smooth muscle alpha (α)-2 actin mRNA levels were reduced in fish exposed to 50 µM while annexin A5 mRNA levels were increased in abundance, corresponding to reduced swim bladder size in larvae. A visual motor response test revealed that larval zebrafish exhibited some hyperactivity in the light with exposure to the herbicide and only the highest dose tested (50 µM) resulted in hypoactivity in the dark cycle. Regression analysis indicated that there was a positive relationship between surface area of the swim bladder and distance traveled, and the size of the swim bladder explained ~10-14% in the variation for total distance moved. Lastly, we tested larvae in a light dark preference test, and we did not detect any altered behavioral response to any concentration tested. Here we present new data on sublethal endpoints associated with exposure to the herbicide S-metolachlor and demonstrate that this chemical may disrupt transcripts associated with swim bladder formation and morphology, which could ultimately affect larval zebrafish activity. These data are expected to contribute to further risk assessment guidelines for S-metolachlor in aquatic ecosystems., (Copyright © 2020. Published by Elsevier Inc.)

Pesticides are typically applied to crops as acute applications, and residual effects of such intermittent exposures are not often characterized in developing fish. Fipronil is an agricultural pesticide that inhibits γ-amino-butyric acid (GABA) gated chloride channels. In this study, zebrafish (Danio rerio) embryos were exposed for 48 h (starting at ~3 h post fertilization, hpf) to various concentrations of fipronil (0.02 μg/L up to 4000 μg/L). Following this acute exposure, a subset of fish was transferred to clean water for a 7-day depuration phase. We hypothesized that a pulse exposure to fipronil during critical periods of central nervous system development would adversely affect fish later in life. After a 48 hour pulse exposure, survival was reduced in embryos exposed to 2 μg fipronil/L or greater. However, there was no further mortality during the depuration phase, nor were there changes in body length nor notochord length in larvae 9 dpf (days post-fertilization) compared to controls. Additional experiments were carried out at higher concentrations over 96 h (up to 4 dpf) to also elucidate developmental effects and teratogenicity of fipronil (43.7 μg/L up to 4370 μg/L). Fipronil at these higher concentrations significantly impacted the development of zebrafish, and the following morphometric and teratogenic effects were observed in 4 dpf fish; reduced body length, yolk sac and pericardial edema, reduced midbrain length, reduced optic and otic diameter, and truncation of the lower jaw. In depurated fish, we hypothesized that there would exist residual effects of exposure at the molecular level. Transcriptome profiling was therefore conducted on 9 dpf depurated larvae exposed initially for 48 h to one dose of either 0.2 μg/L, 200 μg/L or 2000 μg/L fipronil. The expression of gene networks associated with glycogen and omega-3-fatty acid metabolism were decreased in larvae exposed to each of the three concentrations of fipronil, suggesting metabolic disruption. Moreover, transcriptomics revealed that fipronil suppressed gene networks related to light-dark adaptation, photoperiod sensing, and circadian rhythm. Based on these data, we tested fish for altered behavioral responses in a Light-Dark preference test. Larvae exposed to >200 μg fipronil/L as embryos showed fewer number of visits (20-30% less) to the dark zone compared to controls. Larvae also spent a lower amount of time in the dark zone compared to controls, suggesting that fipronil strengthened dark avoidance behavior which is indicative of anxiety. This study demonstrates that a short pulse exposure to fipronil can affect transcriptome networks for metabolism, circadian rhythm, and response to light in fish after depuration, and these molecular responses are hypothesized to be related to aberrant behavioral effects observed in the light-dark preference test., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Fipronil is a phenylpyrazole pesticide that is used in both residential and agricultural applications. Fipronil is detected in run-off and water systems that are near areas in which the pesticide has been applied. The pesticide acts to antagonize gamma aminobutyric acid receptors, leading to over-excitation in the central nervous system. Fipronil has relatively high toxicity to fish, but the mechanisms underlying the toxicity are not well understood in embryonic stages. Zebrafish embryos were exposed to a single concentration of fipronil for 48 h at ∼3-4 h-post-fertilization. Following a 7-day depuration phase, transcriptome and behavioral analyses were conducted. Transcriptomics identified neural processes as those differentially expressed with different doses of fipronil (0.2 µg, 200 µg and 2 mg fipronil/L). Gene networks associated with astrocyte differentiation, myelination, neural tube development, brain stem response, innervation, nerve regeneration, astrocyte differentiation, among other pathways were altered with exposure. In addition, miRNA-related events are disrupted by fipronil exposure and genes associated with primary or pri-miRNA processing were increased in larval fish exposed to the pesticide. These data present putative mechanisms associated with neurological impacts at later ages of zebrafish. This is important because it is not clear how early exposure to pesticides like fipronil affect central nervous system function and organisms later in life., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article., (© 2020 The Author(s). Published by Elsevier Inc.)

Indiscriminate overuse or occupational exposure to agricultural chemicals can lead to neurotoxicity. Many pesticides act to impair mitochondrial function which can lead to exacerbation of neurodegeneration. Triazole fungicides are applied to grain, fruit, and vegetable crops to combat mold and fungi and their use is increasing worldwide. Here, we assessed the in vitro toxicity of two widely used triazole fungicides, propiconazole and tebuconazole, to mitochondria using differentiated SH-SY5Y neuroblastoma cells as an in vitro cell model used in Parkinson's disease research. Cell viability (based on ATP levels), mitochondrial membrane potential, oxidative respiration, and reactive oxygen species (ROS) were measured following fungicide treatments. Cell viability was decreased with 100 μM propiconazole after 24 and 48 h, while tebuconazole required higher doses to affect viability (-200 μM at 24 h). Mitochondrial membrane potential (MMP) was reduced with 50 μM propiconazole after 24 h while 200 μM tebuconazole reduced MMP. Oxidative respiration of SH-SY5Y cells was then measured using a XFe24 Flux analyzer and 100 μM propiconazole reduced basal respiration, oligomycin-induced ATP production, and FCCP-induced maximum respiration by -40-50%, while tebuconazole did not affect mitochondrial bioenergetics at the concentrations tested. Acute exposure to 100 μM propiconazole over 4 h did not immediately affect oxidative respiration in SH-SY5Y cells. ROS were not induced by propiconazole and tebuconazole up to 100 and 300 μM respectively. Based on these results, we focused our lipidomics investigations on SH-SY5Y exposed only to propiconazole, as lipid dysregulation is associated with mitochondrial dysfunction. Both 50 and 100 μM propiconazole altered the abundance of some ceramides, specifically reducing glucosylceramide non-hydroxyfatty acid-sphingosine (HexCer-NS) and increasing N-stearoyl-phytosphingosine (CerNP). Moreover, a recently discovered bioactive lipid called fatty acid ester of hydroxy fatty acid (FAHFA) was increased 5-fold, hypothesized to be a neuroprotective mechanism that has been demonstrated in other studies of human diseases. Additional lipids reduced in abundance included oxidized phosphatidylcholine (OxPC) and oxidized phosphatidylethanolamine (OxPE). There were no changes in cellular triacylglycerols nor total lipids with exposure to propiconazole. Taken together, this study provides insight into the toxicity of triazole fungicides in neuronal cells, which has implications for neurodegenerative diseases that involve the mitochondria such as Parkinson's disease., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Due to run-off and rain events, agrochemicals can enter water catchments, exerting endocrine disruption effects and toxicity to aquatic organisms. Linuron is a phenylurea herbicide used to control a wide variety of vegetative weeds in agriculture in addition to residential applications. However, there are few studies that quantify its toxicity to early developmental stages of fish. The objectives of this study were to assess the acute toxicity of linuron to zebrafish embryos/larvae by measuring mortality, morphological deformities, oxidative respiration, gene expression, and locomotor activity via the Visual Motor Response test. Zebrafish embryos at ~6-h post-fertilization (hpf) were exposed to either embryo rearing medium (ERM), or one dose of 0.625, 1.25, 2.5, 5, and 10 μM linuron for up to 7 days post-fertilization (dpf) depending on the assay. Zebrafish larvae exposed to linuron displayed pericardial edema, yolk sac edema, and spinal curvature. Oxidative respiration assessments in embryos using the Agilent XF e 24 Flux Analyzer revealed that linuron decreased mean basal respiration and oligomycin-induced ATP-linked respiration in 30 hpf embryos at 20 μM after a 24-hour exposure. In 7 dpf larvae, transcript abundance was determined for 6 transcripts that have a role in oxidative respiration (atp06, cox1, cox4-1, cox5a1, cytb, and nd1); the relative abundance of these transcripts was not altered with linuron treatment. A Visual Motor Response test was conducted on 7 dpf larvae to determine whether linuron (0.625 to 5 μM) impaired locomotor activity. Larval activity in the dark period decreased in a dose dependent manner and there were indications of hypoactivity as low as 1.25 μM. Transcript abundance was thus determined for tyrosine hydroxylase (th1) and glutamic acid decarboxylase 67 (gad1b), two rate limiting enzymes that control the production of dopamine and gamma-aminobutyric acid respectively. The mRNA levels of gad1b (p = 0.019) were reduced with increasing concentrations of linuron while th1 (p = 0.056) showed a similar decreasing trend, suggesting that neurotransmitter biosynthesis may be altered with exposure to linuron. This study improves knowledge related to the toxicity mechanisms for linuron and is the first to demonstrate that this anti-androgenic chemical impairs oxidative respiration and exerts neurotoxic effects associated with neurotransmitter biosynthesis during early development. These data are significant for environmental risk assessment of agrochemicals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Esfenvalerate is a pyrethroid insecticide used widely for agricultural and residential applications. This insecticide has been detected in aquatic environments at concentrations that can induce sub-lethal effects in organisms. In this study, zebrafish embryos were used to examine the effects of environmentally-relevant concentrations of esfenvalerate on development and behavior. It was hypothesized that esfenvalerate exposure would impair locomotion due to its effects on the central nervous system. We also measured mitochondrial bioenergetics and the expression of genes (dopamine system) as putative mechanisms of locomotor impairment. Concentrations of 0.02, 0.2 and 2 μg/L esfenvalerate did not induce significant mortality nor deformity in zebrafish, but there was an acceleration in hatching time for zebrafish exposed to 2 μg/L esfenvalerate. As an indicator of neurotoxicity, the Visual Motor Response (VMR) test was conducted with 5, 6, and 7 dpf zebrafish after continuous exposure, and higher concentrations were used (4 and 8 μg/L esfenvalerate) to better discern age-and dose dependent responses in behavior. Experiments revealed that, unlike the other stages, 6 dpf larvae showed evidence for hypo-activity with esfenvalerate, suggesting that different stages of larval development may show increased sensitivity to pyrethroid exposure. This may be related to age-dependent maturation of the central nervous system. We hypothesized that reduced larval activity may be associated with impaired production of ATP and the function of mitochondria at earlier life stages, however dramatic alterations in oxidative phosphorylation were not observed. Based on evidence that dopamine regulates behavior and studies showing that other pyrethroids affect dopamine system, we measured transcripts involved in dopaminergic signaling. We found that dopamine active transporter was down-regulated with 0.2 μg/L esfenvalerate. Lastly, we comprehensively summarize the current literature (>20 studies) regarding the toxicity of pyrethroids in zebrafish, which is a valuable resource to those studying these pesticides. This study demonstrates that esfenvalerate at environmentally-relevant levels induces hypoactivity that are dependent upon the age of the zebrafish, and these behavioral changes are hypothesized to be related to impaired dopamine signaling., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Butylated hydroxytoluene (BHT) is one of the most frequently used synthetic phenolic antioxidants added to food and consumer products such as plastics as a preservative. Due to its high production volume, BHT has been detected in aquatic environments, raising concerns about sub-lethal toxicity. However, there are limited toxicological data for BHT, especially in fish. In this study, zebrafish embryos were exposed to BHT at concentrations ranging 0.01-100 μM for up to 6 days post fertilization (dpf). Acute toxicity was assessed, and experiments revealed that BHT had a 96 h LC50 value of 57.61 μM. At sub-lethal doses (0.1-60 μM), BHT markedly decreased heart rates of zebrafish embryos at 48 h and 72 h by ∼25-30%. Basal and maximal respiration of zebrafish embryos at 24 hpf were decreased by 59.3% and 41.4% respectively following exposure to 100 μM BHT. Behavior in zebrafish was measured at 6 dpf following exposures to 0.01-10 μM BHT. Locomotor behaviors (e.g. total distance moved and velocity) were significantly increased in larvae at doses higher than 0.1 μM BHT. In addition, dark-avoidance behavior was decreased following exposure to 0.01 μM BHT, while conversely, it was increased in zebrafish exposed to 0.1 μM BHT. To investigate potential underlying mechanisms that could explain behavioral changes, transcripts involved in dopamine signaling were measured. Relative expression of dat mRNA was increased in larval fish from the 0.01 μM BHT treatment, while there were no effects on dat mRNA levels at higher concentrations. The mRNA levels of drd3 were decreased in zebrafish from the 1 μM BHT treatment. Taken together, BHT can affect the expression of the dopamine system, which is hypothesized to be related to the abnormal anxiety-associated behavior of larval zebrafish., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 XF e 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., (© 2019 Wiley Periodicals, Inc.)

Introduction: Pyrovalerone (4-methyl-β-keto-prolintane) is a synthetic cathinone (beta-keto-amphetamine) derivative. Cathinones are a concern as drugs of abuse, as related street drugs such as methylenedioxypyrovalerone have garnered significant attention. The primary mechanism of action of cathinones is to inhibit reuptake transporters (dopamine and norepinephrine) in reward centers of the central nervous system., Methods: We measured bioenergetic, behavioral, and molecular responses to pyrovalerone (nM-µM) in zebrafish to evaluate its potential for neurotoxicity and neurological impairment., Results: Pyrovalerone did not induce any mortality in zebrafish larvae over a 3- and 24-hr period; however, seizures were prevalent at the highest dose tested (100 µM). Oxidative phosphorylation was not affected in the embryos, and there was no change in superoxide dismutase 1 expression. Following a 3-hr treatment to pyrovalerone (1-100 µM), larval zebrafish (6d) showed a dose-dependent decrease (70%-90%) in total distance moved in a visual motor response (VMR) test. We interrogated potential mechanisms related to the hypoactivity, focusing on the expression of dopamine-related transcripts as cathinones can modulate the dopamine system. Pyrovalerone decreased the expression levels of dopamine receptor D1 (~60%) in larval zebrafish but did not affect the expression of tyrosine hydroxylase, dopamine active transporter, or any other dopamine receptor subunit examined, suggesting that pyrovalerone may regulate the expression of dopamine receptors in a specific manner., Discussion: Further studies using zebrafish are expected to reveal new insight into molecular mechanisms and behavioral responses to cathinone derivates, and zebrafish may be a useful model for understanding the relationship between the dopamine system and bath salts., (© 2019 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Broad applications and exposure to the fungicide maneb can lead to toxicity in non-target organisms. Maneb is also associated with neurogenerative diseases such as Parkinson's disease (PD). The objectives of this study were to determine the acute toxicity of maneb to zebrafish by measuring mitochondrial bioenergetics, locomotor activity, and the expression of genes related to the oxidative damage response, as well as those related to dopamine signaling due to its association with PD. Zebrafish embryos at 6 h post-fertilization (hpf) were exposed to either solvent control (0.1% DMSO, v/v), or one dose of 0.1, 0.5, 1.0 and 10.0 µM maneb for 96 h. Maneb was moderately toxic to zebrafish embryos, and had a 96-h LC 50 value of 4.29 μM (~ 1.14 mg/L). Maneb induced a dose-dependent increase in mortality, decreased hatching rate, and increased notochord deformity rate at both 1.0 and 10.0 µM after 72 and 96 h. Total body length was also significantly reduced with 1.0 µM maneb. A 50-60% decrease in mean basal oxygen consumption rate was also observed in embryos following a 24 hpf exposure to 10.0 µM maneb but oligomycin-induced ATP production and FCCP-induced maximum respiration remained unaffected. No change was detected in the expression levels of genes associated with oxidative stress (sod1 and sod2), nor those related to dopamine synthesis (th1), dopamine transporter (dat), dopamine receptors (drd1, drd2a, drd3, and drd4b). Thus, modifying the expression of these transcripts may not be a mechanism for maneb-induced developmental toxicity in zebrafish. To assess the potential for neurotoxicity, a dark photokinesis assay was conducted in larvae following 7 d exposure to 0.1, 0.5 and 1.0 μM maneb. Larvae exposed to 0.5 and 1.0 μM maneb showed signs related to hypoactivity, and this reduced activity is hypothesized to be associated with notochord defects as this deformity was prevalent at higher concentrations of maneb. Overall, these data demonstrate that maneb negatively affects embryonic development (i.e. notochord development), affects basal oxygen consumption rates of embryos, and induces hypoactivity in larval fish. This study improves understanding regarding the developmental neurotoxicity of the fungicide maneb to zebrafish., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Among the benzotriazole ultraviolet stabilizers (BUVSs), UV-234 and UV-320 are frequently detected in aquatic ecosystem. Despite the fact that these chemicals are present in low ng/L levels in surface water, they show high bio-accumulation potential and pose exposure risks to aquatic organisms. However, there are limited toxicological data available in fish. In this study, zebrafish embryos were exposed to 0.01, 0.1 and 1 μM UV-234 or UV-320 for up to 6 days. Developmental toxicity as well as effects on mitochondrial bioenergetics, immune system responses, and locomotor activity in zebrafish were measured. After UV-234 treatment (0.1-1 μM), hatching time of embryos was increased compared to controls. There was also a ∼20-40% reduction in non-mitochondrial respiration and oligomycin-dependent mitochondrial respiration in embryos treated with 1 μM UV-234 for 24 and 48 h respectively; conversely basal respiration and non-mitochondrial respiration were increased ∼20-30% in embryos treated with 1 μM UV-320 at 48 h. Transcript levels of sod1 were down-regulated with BUVSs while sod2 mRNA was highly up-regulated with both UV-234 and UV-320, suggesting an oxidative damage response. Considering that mitochondrial signaling regulates innate immune pathways, we measured the expression of immune related transcripts (tlr5a, tlr5b, mmp9, il8, tnfa, cxcl-C1c, nfkb1, and ifng). Of these, only il8 and cxcl-C1c mRNA were decreased in response to 0.1 μM UV-320. To associate early molecular events with behavior, locomotor activity was assessed. UV-234 reduced larval activity in a dark photokinesis assay by ∼15%, however behavioral responses at environmentally-relevant concentrations of BUVSs were not consistent across experiments nor BUVSs. These data suggest that BUVSs can perturb mitochondrial bioenergetics, embryonic development, and locomotor activity of zebrafish, but these responses appear to be dose-, time- and BUVSs dependent, suggesting these chemicals may have unique modes of action., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cyproconazole is a triazole fungicide used to protect a diverse range of fruits, vegetables, and grain crops. As such, it has the potential to enter aquatic environments and affect non-target organisms. The objective of this study was to assess the acute toxicity of the triazole fungicide cyproconazole to zebrafish embryos by assessing mortality, developmental defects, morphological abnormality, oxidative respiration, and locomotor activity following a 96-h exposure. Zebrafish embryos at 6-h post-fertilization (hpf) were exposed to either a solvent control (0.1% DMSO, v/v), or one dose of 10, 25, 50, 100, 250, and 500 μM cyproconazole for 96 h. Data indicated that cyproconazole exhibited low toxicity to zebrafish embryos, with a 96-h LC 50 value of 90.6 μM (~ 26.4 mg/L). Zebrafish embryos/larvae displayed a significant decrease in spontaneous movement, hatching rate, and heartbeats/20 s with 50, 100, and 250 μM cyproconazole exposure. Malformations (i.e., pericardial edema, yolk sac edema, tail deformation, and spine deformation) were also detected in zebrafish exposed to ≥ 50 μM cyproconazole, with significant increases in cumulative deformity rate at 48, 72, and 96 hpf. In addition, a 20-30% decrease in basal and oligomycin-induced ATP respiration was observed after 24-h exposure to 500 μM cyproconazole in embryos. To determine if cyproconazole affected locomotor activity, a dark photokinesis assay was conducted in larvae following 7-day exposure to 1, 10, and 25 μM cyproconazole in two independent trials. Activity in the dark period was decreased for zebrafish exposed to 25 μM cyproconazole in the first trial, and hypoactivity was also observed in zebrafish exposed to 1 μM cyproconazole in a second trial, suggesting that cyproconazole can affect locomotor activity. These data improve understanding of the toxicity of cyproconazole in developing zebrafish and contribute to environmental risk assessments for the triazole fungicides on aquatic organisms. We report that, based on the overall endpoints assessed, cyproconazole exhibits low risk for developing fish embryos, as many effects were observed above environmentally-relevant levels.

Ziram is a broad spectrum pesticide that belongs to the class of dimethyl-dithiocarbamate (DTC) fungicides. The objectives of this study were to assess the effects of ziram in developing zebrafish. Ziram was highly toxic to zebrafish embryos, with a 96-h LC 50 value of 1082.54 nM (∼0.33 mg/L). Zebrafish embryos at 6 h post-fertilization (hpf) were exposed to solvent control (0.1% DMSO), or one dose of 1, 10, 100, and 1000 nM ziram for 96 h. Ziram induced lethality in a dose-dependent manner, decreased hatching rate and heartbeat, and caused wavy deformities at 72 and 96 hpf at 100 and 1000 nM. Basal oxygen consumption rates of zebrafish at 24 hpf were decreased with 1000 nM, suggesting that ziram affects oxidative phosphorylation. We also measured the expression of transcripts associated with the oxidative stress response (sod1 and sod2) and dopamine receptor signaling at ∼96 h of exposure. There was no difference in the expression of genes related to oxidative stress, nor those related to the dopamine system. Locomotor activity was also assessed in larval zebrafish (7 dpf), and ziram increased total activity, the velocity in light zone, and total distance moved at 10 nM, while it decreased the mean time spent in the dark zone at 1 and 10 nM. Behavioral responses were dependent upon the time point and clutch examined. These data demonstrate that ziram negatively impacts embryonic development (i.e. mortality, hatching, heartbeat and notochord development) of zebrafish, decreases basal respiration of embryos, and alters behavioral responses in larvae., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Fluazinam is a pyridinamine fungicide that induces oxidative stress and mitochondrial damage in cells, and it has been reported to be neurotoxic. To characterize the biological effects of fluazinam, we assessed mitochondrial bioenergetics, dopamine system expression, and behavior of early life staged zebrafish (0.01 μM-0.5 μM). Fluazinam at environmentally-relevant levels did not induce sub-lethal effects in larvae, but at the LC 50 (0.5 μM), fluazinam decreased basal and ATP-linked respiration significantly in embryos. As mitochondria are directly related to redox homeostasis and apoptosis, the expression of genes related to oxidative stress and apoptosis were measured. Superoxide dismutase 2 (sod2), heat stock protein 70 (hsp70), bcl2-associated X protein (bax), and caspase 9 (casp9) mRNA levels were up-regulated by 0.5 μM fluazinam. Taken together, there was evidence for mitochondrial dysfunction and oxidative damage at the highest concentration of fluazinam (0.5 μM) tested. As there are reports for fluazinam-induced neurotoxicity in dopamine synthesizing cells, transcriptional targets in the dopamine system were assessed in the zebrafish. Tyrosine hydroxylase 1 (th1) and dopamine receptor 2a (drd2a) mRNA levels were decreased by 0.5 μM fluazinam, suggesting that this fungicide may affect the dopaminergic system. To further assess the potential for fluazinam-mediated neuromodulation, the dark photokinesis response was assessed in larvae following exposure. Larvae exposed to 0.1 μM fluazinam showed hyperactivity, while larvae exposed to 0.2 and 0.3 μM showed hypo-activity. This study demonstrates that fluazinam disrupts mitochondrial bioenergetics in zebrafish, inducing an oxidative stress response, and aberrant behaviors in larvae that are dose dependent., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The objectives of this study were to compare the biological responses in developing zebrafish to two organophosphate insecticides, chlorpyrifos (CPF) and diazinon (DZN). Zebrafish embryos were exposed to either solvent control (0.1% DMSO, v/v), or one dose of 0.1, 1.0, 10.0 and 25.0 μM CPF, as well as one dose of 0.1, 1.0, 10.0 and 100.0 μM DZN for 96 h. CPF at 10.0 and 25.0 μM caused 70-80% and 100% mortality in embryos after 96 h exposure, whereas embryos treated with 10.0 and 100.0 μM DZN showed 30-40% and 70-80% lethality. CPF at 10.0 μM significantly decreased cumulative hatching rate, whereas hatching rate was significantly reduced in embryos treated with 100.0 μM DZN. Spinal lordosis was primarily observed in larvae exposed to 1.0 and 10.0 μM CPF, whereas pericardial edema was mainly detected with 10.0 and 100.0 μM DZN exposure. Embryo exposed to 1.0, 10.0 and 25.0 μM CPF exhibited no mitochondrial dysfunction; exposure to 100.0 μM DZN significantly inhibited mitochondrial bioenergetics. To determine if CPF and DZN affected larval activity, dark photokinesis response was assessed in larvae following 7 days exposure to 0.1 and 1.0 μM CPF, as well as to 0.1 1.0, and 10.0 μM DZN. Larvae exposed to 1.0 μM CPF showed hypoactivity, whereas the activity in the dark was not overtly changed in larvae exposed to DZN. In summary, CPF showed higher developmental toxicity compared to DZN. Moreover, based on the types of morphological deformities noted, as well as differences in locomotor activity, we conclude that OPs have unique chemical-specific modes of action that can result in varied biological responses during early development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mitochondrial dysfunction is a prevalent molecular event that can result in multiple adverse outcomes. Recently, a novel high throughput method to assess metabolic capacity in fish embryos following exposure to chemicals has been adapted for environmental toxicology. Assessments of oxygen consumption rates using the Seahorse XF(e) 24/96 Extracellular Flux Analyzer (Agilent Technologies) can be used to garner insight into toxicant effects at early stages of development. Here we synthesize the current state of the science using high throughput metabolic profiling in zebrafish embryos, and present considerations for those wishing to adopt high throughput methods for mitochondrial bioenergetics into their research. Chemicals that have been investigated in zebrafish using this metabolic platform include herbicides (e.g. paraquat, diquat), industrial compounds (e.g. benzo-[a]-pyrene, tributyltin), natural products (e.g. quercetin), and anti-bacterial chemicals (i.e. triclosan). Some of these chemicals inhibit mitochondrial endpoints in the μM-mM range, and reduce basal respiration, maximum respiration, and spare capacity. We present a theoretical framework for how one can use mitochondrial performance data in zebrafish to categorize chemicals of concern and prioritize mitochondrial toxicants. Noteworthy is that our studies demonstrate that there can be considerable variation in basal respiration of untreated zebrafish embryos due to clutch-specific effects as well as individual variability, and basal oxygen consumption rates (OCR) can vary on average between 100 and 300 pmol/min/embryo. We also compare OCR between chorionated and dechorionated embryos, as both models are employed to test chemicals. After 24 h, dechorionated embryos remain responsive to mitochondrial toxicants, although they show a blunted response to the uncoupling agent carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP); dechorionated embryos are therefore a viable option for investigations into mitochondrial bioenergetics. We present an adverse outcome pathway framework that incorporates endpoints related to mitochondrial bioenergetics. High throughput bioenergetics assays conducted using whole embryos are expected to support adverse outcome pathways for mitochondrial dysfunction., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Dopamine (DA) plays a significant role in cognition, motor function and social behavior. The objectives of this study were to (1) quantify the temporal expression of transcripts (DA receptors, transporters and tyrosine hydroxylase) associated with DA signaling during early stages of zebrafish development and (2) determine their expression profiles following treatment with a D 2 receptor antagonist domperidone (DMP). We also assessed locomotor behavior following treatment with DMP using alternating periods of light and dark (ie, dark photokinesis), as DA plays a key role in behavior. Relative expression levels of transcripts that were investigated and related to the DA system were detected after the first 24 hours postfertilization (hpf). Some DA receptor transcripts (eg, drd4c) increased in abundance earlier in the embryo compared with other receptors (eg, drd3), suggesting that DA receptor paralogs may have unique roles in development. Treatment of larvae with DMP resulted in the upregulation of DA receptor transcripts (ie, drd1, drd7, drd4b, drd4c) and DA transporter 1 (ie, slc6a3), and it is hypothesized that upregulation of genes related to the DA system is a compensatory neurophysiological response to DA receptor antagonism. Larval activity during dark photokinesis (measured by distance traveled) was also elevated by DMP. We hypothesize that behavioral responses observed with DMP may be related to the regulation of deep brain photoreception in zebrafish (Danio rerio) (ZF) larvae by DA., (© 2018 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

Diquat is a non-selective bipyridylium herbicide which has replaced its sister compound paraquat, as paraquat is associated to an increased risk for Parkinson's disease. However, the propensity of diquat to propagate reactive oxygen species ensures that diquat remains an exposure risk in non-target organisms. In this study, zebrafish (Danio rerio) embryos were exposed to diquat (1, 10, 100μM) beginning at ∼6h post fertilization for up to 7days to learn more about the mechanisms underlying diquat toxicity during vertebrate development. Zebrafish embryos exposed to diquat for 96h did not show any significant mortality nor deformity compared to controls. Moreover, there was no difference in the timing of hatch, an indicator of stress, for fish exposed to diquat. To determine whether changes in mitochondrial bioenergetics occurred in early development as a response to diquat exposure, oxygen consumption rate was measured in whole embryos. Basal respiration and ATP production were decreased following a 24h diquat exposure at 100μM, suggesting that diquat negatively affects oxidative phosphorylation. We also assessed locomotor behavior as a sensitive endpoint for impaired activity and neurotoxicity. Seven day old (7 dpf) zebrafish treated with diquat at the highest doses tested (10-100μM) showed an increase (hyper-activity) in total distance travelled, velocity, movement cumulative duration, and overall activity compared to unexposed fish. Lastly, in 7d fish, we measured transcripts related to redox balance and apoptosis as diquat has been reported to induce oxidative stress and can affect mitochondrial bioenergetics. Larvae exposed to 10μM diquat showed higher transcript levels of catalase compared to control fish, implying that reactive oxygen species are produced following diquat exposure. Transcript levels of sod1, sod2, bcl2, bax and caspase 3 however did not vary in abundance among treatments with diquat. This study improves mechanistic understanding of diquat in fish at early stages of development and presents evidence that diquat disrupts mitochondrial bioenergetics and behavior., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The dipyridyl herbicide paraquat induces oxidative stress in cells and is implicated in adult neurodegenerative diseases. However, less is known about paraquat toxicity in early stages of vertebrate development. To address this gap, zebrafish (Danio rerio) embryos were exposed to 1, 10 and 100 μM paraquat for 96 h. Paraquat did not induce significant mortality nor deformity in embryos and larvae, but it did accelerate time to hatch. To evaluate whether mitochondrial respiration was related to earlier hatch times, oxygen consumption rate was measured in whole embryos. Maximal respiration of embryos exposed to 100 μM paraquat for 24 h was reduced by more than 70%, suggesting that paraquat negatively impacts mitochondrial bioenergetics in early development. Based upon this evidence for mitochondrial dysfunction, transcriptional responses of oxidative stress- and apoptosis-related genes were measured. Fish exposed to 1 μM paraquat showed higher expression levels of superoxide dismutase 2, heat shock protein 70, Bcl-2-associated X protein, and B-cell CLL/lymphoma 2a compared to control fish. No differences among groups were detected in larvae exposed to 10 and 100 μM paraquat, suggesting a non-monotonic response. We also measured endpoints related to larval behavior and dopaminergic signaling as paraquat is associated with degeneration of dopamine neurons. Locomotor activity was stimulated with 100 μM paraquat and dopamine transporter and dopamine receptor 3 mRNA levels were increased in larvae exposed to 1 μM paraquat, interpreted to be a compensatory response at lower concentrations. This study improves mechanistic understanding into the toxic actions of paraquat on early developmental stages., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mitochondria are sensitive targets of environmental chemicals. Dieldrin (DLD) is an organochlorine pesticide that remains a human health concern due to high lipid bioaccumulation, and it has been epidemiologically associated to an increased risk for Parkinson's disease (PD). As mitochondrial dysfunction is involved in the etiology of PD, this study aimed to determine whether DLD impaired mitochondrial bioenergetics in dopaminergic cells. Rat immortalized dopaminergic N27 cells were treated for 24 or 48h with one dose of either a solvent control, 2.5, 25, or 250μM DLD. Dopaminergic cells treated with 250μM DLD showed increased Casp3/7 activity at 24 and 48h. DLD also caused a dose dependent reduction in cell viability of ∼25-30% over 24h. No significant effects on cell viability, apoptosis, nor cytotoxicity were detected at 24 or 48h with 2.5μM DLD. Following a 24h exposure to 2.5 and 25μM DLD, viable cells were subjected to a mitochondrial stress test using the Seahorse XFe24 Extracellular Flux Analyzer. Following three independent experiments conducted for rigor, dopaminergic cells that were treated with 2.5 and 25μM DLD consistently showed a reduction in maximum respiration and spare capacity compared to the control group. Molecular responses were measured to determine mechanisms of DLD-induced mitochondrial dysfunction. There were no changes in transcripts associated with mitochondrial membrane potential and permeability (e.g. Ant, Hk1, Tspo, Vdac), nor PI3 K/Akt/mTor signaling or mitochondrial-associated apoptotic factors (Bax, Bcl2, Casp3). However, transcript levels for Chop/Gadd153 (DNA Damage Inducible Transcript 3), an apoptotic gene activated following endoplasmic reticulum (ER) stress, were 3-fold higher in N27 cells treated with DLD, suggesting that DLD-induced mitochondrial dysfunction is related to ER stress. Dopamine cells were also assessed for changes in tyrosine hydroxylase (TH) protein, which did not differ among treatments. This study demonstrates that DLD impairs oxidative respiration in dopamine cells, and ER stress is hypothesized to be associated with the DLD-induced mitochondrial dysfunction. This is important as ER stress is also linked to PD. This study presents mechanistic insight into pesticide-induced mitochondrial dysfunction using a chemical that is reported to be associated to a higher risk for neurodegenerative disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)