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.

Exposure to the antineoplastic ifosfamide alters molecular pathways related to cardiovascular function, increases heart rate, and induces hyperactivity in zebrafish (Danio rerio).

Ifosfamide is an alkylating antineoplastic drug used in chemotherapy, but it is also detected in wastewater. Here, the objectives were to (1) determine teratogenic, cardiotoxic, and mitochondrial toxicity potential of ifosfamide exposure; (2) elucidate mechanisms of toxicity; (3) characterize exposure effects on larval behavior. Survival rate, hatch rate, and morphological deformity incidence were not different amongst treatments following exposure levels up to 1000 µg/L ifosfamide over 7 days. RNA-seq reveled 231 and 93 differentially expressed transcripts in larvae exposed to 1 µg/L and 100 µg/L ifosfamide, respectively. Several gene networks related to vascular resistance, cardiovascular response, and heart rate were affected, consistent with tachycardia observed in exposed embryonic fish. Hyperactivity in larval zebrafish was observed with ifosfamide exposure, potentially associated with dopamine-related gene networks. This study improves ecological risk assessment of antineoplastics by elucidating molecular mechanisms related to ifosfamide toxicity, and to alkylating agents in general.

A comparative review of the toxicity mechanisms of perfluorohexanoic acid (PFHxA) and perfluorohexanesulphonic acid (PFHxS) in fish.

Industrial and consumer goods contain diverse perfluoroalkyl substances (PFAS). These substances, like perfluorohexanoic acid (PFHxA) and perfluorohexanesulphonic acid (PFHxS), are under increased scrutiny due to their potential toxicity to aquatic organisms. However, our understanding of their biological impacts and mechanisms of action remains limited. The objectives of this review were to compare data for levels of PFHxA and PFHxS in aquatic environments and fish tissues, as well as toxicity mechanisms related to morphological, endocrine, metabolic, and behavioral endpoints. A computational assessment was also performed to identify putative mechanisms of toxicity and to characterize exposure biomarkers. Studies have shown that both PFHxA and PFHxS residues are present in diverse marine and freshwater fish tissues, suggesting the importance of monitoring these PFAS in aquatic organisms. In fish tissues, these chemicals have been reported to be as high as 37.5 ng/g for PFHxA and 1290 ng/g for PFHxS, but their persistence in aquatic environments and degradation in tissues requires further study. In terms of mechanisms of toxicity, both oxidative stress and endocrine disruption have been reported. Based on evidence for endocrine disruption, we modeled interactions of estrogen and androgen receptors of several fish species with PFHxA and PFHxS. Molecular docking revealed that PFHxS has a stronger affinity for interacting with the estrogen and androgen receptors of fish compared to PFHxA and that estrogen and androgen receptors of fathead minnow, zebrafish, Atlantic salmon, and largemouth bass show comparable binding affinities for each chemical except for salmon Esr2b, which was predicted to have lower affinity for PFHxA relative to Esr2a. While mechanistic data are lacking in fish in general for these chemicals, a computational approach revealed that PFHxA can perturb the endocrine system, nervous system, and is linked to changes in kidney and liver weight. Proteins associated with PFHxA and PFHxS exposures in fish include those related to lipid and glucose regulation, reproductive proteins like KISS metastasis suppressor, and proteins associated with the immune system (specifically RAG1, RAG2), all of which are potential biomarkers of exposure. Taken together, we synthesize current knowledge regarding the environmental fate and ecotoxicology of PFHxA/PFHxS in fish species.

Neurotoxicity of Benzotriazole Ultraviolet Stabilizers in Teleost Fishes: A Review.

Plastic additives that maintain integrity have been extensively studied for potential toxicity to fish; however, chemicals that protect polymers from (artificial) UV degradation are less studied. Benzotriazole UV stabilizers (BUVSs) are the most widely used UV stabilizers in plastics and are often used in sunscreens, cosmetics, paint, and food packaging. BUVSs can negatively affect aquatic wildlife when released into the environment via plastic degradation. In this review, we summarize the distribution of BUVSs globally and discuss neurotoxicological endpoints measured in fish to understand how these plastic additives can affect the neurological health of teleost fishes. BUVSs have been detected in aquatic environments at concentrations ranging from 0.05 up to 99,200 ng/L. Studies show that BUVSs affect behavioral responses and acetylcholinesterase activity, indicators of neurotoxicity. Our computational analysis using transcriptome data suggests certain pathways associated with neurodegeneration are responsive to exposure to BUVSs, like "Complement Activation in Alzheimer's Disease". Based on our review, we identify some research needs for future investigations: (1) molecular studies in the central nervous system to define precise mechanisms of neurotoxicity; (2) a wider range of tests for assessing aberrant behaviors given that BUVSs can affect the activity of larval zebrafish; and (3) histopathology of the nervous system to accompany biochemical analyses. These data are expected to enhance understanding of the neurotoxicity potential of benzotriazoles and other plastic additives.

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.

Occurrence and toxicity mechanisms of perfluorobutanoic acid (PFBA) and perfluorobutane sulfonic acid (PFBS) in fish.

Perfluorobutanoic acid (PFBA) and perfluorobutane sulfonic acid (PFBS) are short-chain perfluoroalkyl substances (PFAS) ubiquitous in the environment. Here we review data on the presence and toxicity mechanisms of PFBA and PFBS in fish. We aimed to (1) synthesize data on physiological systems perturbed by PFBA or PFBS; (2) determine whether toxicity studies use concentrations reported in aquatic ecosystems and fish tissues; (3) conduct a computational toxicity assessment to elucidate putative mechanisms of PFBA and PFBS-induced toxicity. PFBA and PFBS are reported in the low ng/L in aquatic systems, and both substances are present in tissues of several fish including carp, bass, tilapia, and drum species. Evidence supports toxicity effects on several organ systems, including the cardiac, immune, hepatic, and reproductive system. Multigenerational effects in fish have also been documented for these smaller chain PFAS. To further elucidate mechanisms of reproductive impairment, we conducted in silico molecular docking to evaluate chemical interactions with several fish estrogen receptors, specifically zebrafish, fathead minnow, and Atlantic salmon. PFBS showed higher binding affinity for fish estrogen receptors relative to PFBA. Computational analysis also pointed to effects on lipids "Adipocyte Hypertrophy and Hyperplasia", "Lipogenesis Regulation in Adipocyte", and estrogen-related processes. Based on our review, most data for PFBA and PFBS are gathered for concentrations outside environmental relevance, limiting our understanding of their environment impacts. At the time of this review, there is relatively more toxicity data available for PFBS relative to PFBA in fish. This review synthesizes data on environmental levels and toxicology endpoints for PFBA and PFBS in fish to guide future investigations and endpoint assessments.

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.

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.

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.

A synthesis on the sub-lethal toxicity of atenolol, a beta-blocker, in teleost fish.

Blood pressure medications like atenolol are detected in aquatic ecosystems. The objectives here were to (1) map the global presence of atenolol in surface water and sewage; (2) present current knowledge regarding removal efficiency and degradation of atenolol; (3) identify biological endpoints sensitive to exposure; (4) reveal molecular biomarkers that may be useful for exposure studies in fish; (5) determine whether toxicology studies are within environmental relevance. In fish, atenolol exposure affects endocrine and immune systems, metabolism, and epigenetics. Fewer than half of all studies measuring biological responses use environmentally-relevant concentrations. Heart rate appeared most sensitive to atenolol exposure relative to other endpoints. Data are lacking for behavioral responses to atenolol. Molecular biomarkers for atenolol may include those associated with acute kidney injury, cholestasis, and hypertriglyceridemia. Head kidney and liver may therefore be useful for detecting atenolol-induced effects. This review synthesizes knowledge regarding atenolol-induced toxicity in fish.

A comparative review and computational assessment of acetochlor toxicity in fish: A novel endocrine disruptor?

Acetochlor is a chloroacetamide herbicide applied to various crops worldwide and is one of the top selling herbicides on the global market. Due to rain events and run-off, the potential for acetochlor-induced toxicity is a concern for aquatic species. Here we review the current state of knowledge regarding the concentrations of acetochlor in aquatic ecosystems globally and synthesize the biological impacts of acetochlor exposure to fish. We compile toxicity effects of acetochlor, outlining evidence for morphological defects, developmental toxicity, endocrine and immune system disruption, cardiotoxicity, oxidative stress, and altered behavior. To identify mechanisms of toxicity, we utilized computational toxicology and molecular docking approaches to uncover putative toxicity pathways. Using the comparative toxicogenomics database (CTD), transcripts responsive to acetochlor were captured and graphically depicted using String-DB. Gene-ontology analysis revealed that acetochlor may disrupt protein synthesis, blood coagulation, signaling pathways, and receptor activity in zebrafish. Further pathway analysis revealed potential novel targets for acetochlor disruption at the molecular level (e.g., TNF alpha, heat shock proteins), highlighting cancer, reproduction, and the immune system as biological processes associated with exposure. Highly interacting proteins in these gene networks (e.g., nuclear receptors) were selected to model binding potential of acetochlor using SWISS-MODEL. The models were used in molecular docking to strengthen evidence for the hypothesis that acetochlor acts as an endocrine disruptor, and results suggest estrogen receptor alpha and thyroid hormone receptor beta may be preferential targets for disruption. Lastly, this comprehensive review reveals that, unlike other herbicides, neither immunotoxicity nor behavioral toxicity have been fully investigated as sub-lethal endpoints for acetochlor, and such mechanisms of toxicity should be emphasized in future research investigating biological responses of fish to the herbicide.

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.

In silico microRNA network data in zebrafish after antineoplastic ifosfamide exposure.

Ifosfamide is a cancer-fighting chemotherapeutic that has been detected in aquatic ecosystems. Zebrafish larvae were exposed to either 0, 1 or 100 µg/L ifosfamide in the water for 7 days, and fish were subjected to total RNA extraction and RNA-seq analysis with the Illumina NovoSeq 6000 instrument. Raw sequence data were processed through fastp and clean reads obtained by removing adapter and poly-N sequences, as well as low quality reads. Differential gene expression was performed using the abundance of transcripts that mapped to the zebrafish genome. To uncover putative targets regulated by microRNAs, Pathway Studio 12.0 was used to conduct a subnetwork enrichment analysis. Expression data were used to predict which microRNAs were important for the response to ifosfamide exposure. There were 21 common microRNAs identified in both the "IFOS1" and "IFOS100" datasets. These were MIR150, MIR6515, MIR657, MIR216A, m_Mir741, MIRLET7E, miR-let-7, MIR2392, r_Mir3551, MIR181B1, MIR33A, MIR502, MIR193B, MIR146A, MIR431, MIR647, m_Mir1192, MIR297, MIR328, and MIR4717. Data can be re-used to advance adverse outcome pathways in regulatory toxicology and to refine biomarker discovery for antineoplastics 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.

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.

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.

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.

Perfluorotetradecanoic Acid (PFTeDA) Induces Mitochondrial Damage and Oxidative Stress in Zebrafish (Danio rerio) Embryos/Larvae.

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.

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

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