Characterization of bioenergetic and kinematic plasticity of bovine sperm reveals novel and dynamic temporal traits.

In brief: Evaluation of sperm samples with similar motility after thawing has limited value to identify differences in sperm bioenergetic capacity. Maintaining sperm for 24 hr at room temperature is sufficient to detect bioenergetic and kinematics divergences. Abstract: Sperm transport through the female reproductive tract requires energy for motility and fertilization. Sperm kinematic assessment is conducted as an industry standard to estimate semen quality prior to bovine insemination. However, individual samples with similar post-thaw motility result in different pregnancy outcomes, suggesting that differences in bioenergetics may be important for sperm function. Thus, characterization of bioenergetic and kinematic parameters of sperm over time may reveal novel metabolic requirements for sperm function. Post-thawed sperm from five samples of individual (A, B, C) and pooled bulls (AB, AC) were assessed at 0 and 24 h after thawing. Sperm were evaluated for kinematics via computer-assisted sperm analyses and bioenergetic profiles using a Seahorse Analyzer for basal respiration (BR), mitochondrial stress test (MST), and energy map (EM). Motility was nearly identical among samples after thawing and no differences in bioenergetics were detected. However, after 24 h of sperm storage, pooled sperm samples (AC) presented with higher BR and proton leakage compared to other samples. Sperm kinematic variability among samples was higher after 24 h, suggesting difference in sperm quality may manifest over time. Despite a reduction in motility and mitochondrial membrane potential, BR was higher at 24 h compared to 0 h for nearly all samples. A metabolic divergence between samples was detected by EM, indicating a shift in bioenergetic profiles over time that was undetected after thawing. These new bioenergetic profiles elucidate a novel dynamic plasticity of sperm metabolism over time while suggesting an influence of heterospermic interactions for further investigation.

Tumor Necrosis Factor Alpha and the Gastrointestinal Epithelium: Implications for the Gut-Brain Axis and Hypertension.

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.

The agrochemical S-metolachlor disrupts molecular mediators and morphology of the swim bladder: Implications for locomotor activity in zebrafish (Danio rerio).

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.

Behavioral and developmental toxicity assessment of the strobilurin fungicide fenamidone in zebrafish embryos/larvae (Danio rerio).

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.

Exposure to acetochlor impairs swim bladder formation, induces heat shock protein expression, and promotes locomotor activity in zebrafish (Danio rerio) larvae.

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.

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

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

Developmental neurotoxicity of maneb: Notochord defects, mitochondrial dysfunction and hypoactivity in zebrafish (Danio rerio) embryos and larvae.

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 LC50 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.

High-Resolution Respirometry for the Assessment of Teratogenic Chemicals.

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.

Exposure to the pharmaceutical buspirone alters locomotor activity, anxiety-related behaviors, and transcripts related to serotonin signaling in larval zebrafish (Danio rerio).

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.

Waterborne exposure to the antineoplastic 5-fluorouracil alters lipid composition in larval zebrafish (Danio rerio).

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.

Sulfamethoxazole (SMX) Alters Immune and Apoptotic Endpoints in Developing Zebrafish (Danio rerio).

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 1ß (il-1ß). 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.

Developmental and mitochondrial toxicity assessment of perfluoroheptanoic acid (PFHpA) in zebrafish (Danio rerio).

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.

Lipidomic, metabolomic, and behavior responses of zebrafish (Danio rerio) exposed to environmental levels of the beta blocker atenolol.

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_NS d18:1_16:0), Ether linked Phosphatidylethanolamine 16:0 22:6 (EtherPE 16:0e_22:6), and Ether linked Phosphatidylcholine 16:0 22:6 (EtherPC 16:0e_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.

Differential effects of dopamine receptor agonists ropinirole and quinpirole on locomotor and anxiolytic behaviors in larval zebrafish (Danio rerio): A role for the GABAergic and glutamate system?

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.

Interaction between Butyrate and Tumor Necrosis Factor α in Primary Rat Colonocytes.

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.

Molecular and behavioral toxicity assessment of tiafenacil, a novel PPO-inhibiting herbicide, in zebrafish embryos/larvae.

Tiafenacil is a newly registered herbicide and a protoporphyrinogen IX oxidase inhibitor. However, sub-lethal effects of PPO-inhibitors in aquatic species are unknown. Embryos or larvae were exposed to 0.1 µg/L up to 10 mg/L tiafenacil for 7-days post-fertilization. Decreased survival (> 50%) and deformities were noted at concentrations > 1 mg/L. Potency (EC50) of tiafenacil for 5- and 7-day larvae were 818.1 µg/L and 821.7 µg/L, respectively. Pericardial and yolk sac edema were the most frequent deformities observed. Heartbeat frequency at 3 dpf was decreased in zebrafish exposed to > 10 µg/L tiafenacil, coinciding with increased reactive oxygen species. Oxygen consumption rates were not affected by tiafenacil, nor did we detect differences in indicators of apoptosis. The abundance of eighteen transcripts related to oxidative stress and mitochondrial complexes I through V were unchanged. Larval activity was decreased with exposure to 1000 µg/L tiafenacil. These data contribute to risk assessment for a new class of herbicide.

The novel insecticide broflanilide dysregulates transcriptional networks associated with ion channels and induces hyperactivity in zebrafish (Danio rerio) larvae.

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.

Dataset for diseases associated with exposure to broflanilide, a novel pesticide, in larval zebrafish (Danio rerio).

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.

Toxicity assessment of carvacrol and its acetylated derivative in early staged zebrafish (Danio rerio): Safer alternatives to fipronil-based pesticides?

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 (EC50) of fipronil was ∼1 µM for both deformities and mortality at 7 dpf, whereas EC50 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.

Evidence for neurotoxicity and oxidative stress in zebrafish embryos/larvae treated with HFPO-DA ammonium salt (GenX).

"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.