Adverse effects of fenpropathrin on the intestine of common carp (Cyprinus carpio L.) and the mechanism involved.

Fenpropathrin (FEN), a pyrethroid pesticide, is frequently detected in natural water bodies, unavoidable pose adverse effects to aquatic organisms. However, the harmful effects and potential mechanisms of FEN on aquatic species are poorly understood. In this study, common carp were treatment with FEN at 0.45 and 1.35 µg/L for 14 d, and the toxic effects and underlying mechanisms of FEN on the intestine of carp were revealed. RNA-seq results showed that FEN exposure cause a wide range of transcriptional alterations in the intestine and the differentially expressed genes were mainly enrichment in the pathways related to immune and metabolism. Specifically, FEN exposure induced pathological damage and altered submicroscopic structure of the intestine, elevated the levels of Bacteroides fragilis enterotoxin, altered the contents of claudin-1, occludin, and zonula occluden-1 (ZO-1), and causing injury to the intestinal barrier. In addition, inflammation-related index TNF-α in the serum and IL-6 in the intestinal tissues were generally increased after FEN exposure. Moreover, FEN exposure promoted an increase in reactive oxygen species (ROS), altered the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), upregulated the contents of malondialdehyde (MDA) in the intestines. The apoptosis-related parameter cytochrome c, caspase-9, and caspase-3 were significantly altered, indicating that inflammation reaction, oxidative stress, and apoptosis may be involved in the toxic mechanism of FEN on carp. Moreover, FEN treatment also altered the intestinal flora community significantly, which may affect the intestinal normal physiological function and thus affect the growth of fish. Overall, the present study help to clarify the intestinal reaction mechanisms after FEN treatment, and provide a basis for the risk assessment of FEN.

Fenpropathrin induces neurotoxic effects in common carp (Cyprinus carpio L.).

Fenpropathrin (FEN) is a synthetic pyrethroid that has been frequently detected in aquatic environments, yet the neurotoxic impacts and underlying mechanisms on nontarget organisms are lacking. In this experiment, common carp were exposed to 0.45 and 1.35 µg/L FEN for 14 d and exhibited abnormal locomotor behaviour. Biochemical and molecular analysis results indicated that FEN altered the contents of tight junction proteins (claudin-1, occludin, and ZO-1), disturbed Na+-K+-ATPase and AChE activities, caused abnormal expression of neurotransmitters (ACh, DA, GABA, 5-HT, and glutamate) and caused histological damage in the brain, suggesting that FEN may damage the blood-brain barrier and induce neurotoxicity in carp. Furthermore, FEN also promoted an increase in ROS, changed SOD and CAT activities, and generally upregulated the contents of MDA, 8-OHdG, and protein carbonyl in the brain, indicating that FEN can induce oxidative stress and cause damage to lipids, DNA, and proteins. Moreover, inflammation-related indicators (TNF-α, IL-1ß, IL-6, and IL-10), mitophagy-related genes (PINK1, parkin, ulk1, beclin1, LC3, p62, tfeb, and atg5), and apoptosis-related parameters (p53, bax, bcl-2, caspase-3, caspase-8, and caspase-9) were also significantly changed, suggesting that inflammation, mitophagy, and apoptosis may participate in FEN-induced neurotoxicity in carp. This study refines the understanding of the toxicity mechanism of FEN and thus provides data support for the risk assessment of FEN.

Single and Combined Effects of Chlorpyrifos and Glyphosate on the Brain of Common Carp: Based on Biochemical and Molecular Perspective.

Chlorpyrifos (CPF) and glyphosate (GLY) are the most widely used organophosphate insecticide and herbicide worldwide, respectively; co-occurrence of CPF and GLY in aquatic environments occurs where they inevitably have potential hazards to fish. However, the potential mechanisms of CPF and GLY to induce toxicity have not been fully explored. To identify the adverse impacts of CPF and GLY on fish, either alone or in combination (MIX), CPF (25 µg/L) and GLY (3.5 mg/L) were set up according to an environmentally relevant concentration to expose to common carp for 21 days. After exposure, CPF and GLY decreased the activities of acetylcholinesterase and Na+/K+-ATPase, altered monoamine oxidase levels, decreased antioxidant enzyme activities (superoxide dismutase, catalase, glutathione S-transferase and glutamic reductase), and induced the accumulation of malondialdehyde in the carp brain. The parameters in the MIX groups had a greater impact compared to that in the CPF or GLY group, suggesting that both single and combined exposure could affect neurological signaling systems and cause oxidative stress and lipid peroxidation damage in carp brains, and that MIX exposure increases the impact of each pollutant. RNA-seq results showed that single or combined exposure to CPF and GLY induced global transcriptomic changes in fish brains, and the number of differentially expressed genes in MIX-treated carp brains were globally increased compared to either the CPF or GLY groups, suggesting that the effects of co-exposure were greater than single exposure. Further analysis results revealed that the global transcriptomic changes participated in oxidative stress, immune dysfunction, and apoptosis of fish brains, and identified that the P13k-Akt signaling pathway participates in both single and combined exposure of CPF- and GLY-induced toxicity. Taken together, our results demonstrated that the interaction of CPF and GLY might be synergic and provided novel insights into the molecular mechanisms of fish brains coping with CPF and GLY.

Developmental Neurotoxicity of Trichlorfon in Zebrafish Larvae.

Trichlorfon is an organophosphorus pesticide widely used in aquaculture and has potential neurotoxicity, but the underlying mechanism remains unclear. In the present study, zebrafish embryos were exposed to trichlorfon at concentrations (0, 0.1, 2 and 5 mg/L) used in aquaculture from 2 to 144 h post fertilization. Trichlorfon exposure reduced the survival rate, hatching rate, heartbeat and body length and increased the malformation rate of zebrafish larvae. The locomotor activity of larvae was significantly reduced. The results of molecular docking revealed that trichlorfon could bind to acetylcholinesterase (AChE). Furthermore, trichlorfon significantly inhibited AChE activity, accompanied by decreased acetylcholine, dopamine and serotonin content in larvae. The transcription patterns of genes related to acetylcholine (e.g., ache, chrna7, chata, hact and vacht), dopamine (e.g., drd4a and drd4b) and serotonin systems (e.g., tph1, tph2, tphr, serta, sertb, htrlaa and htrlab) were consistent with the changes in acetylcholine, dopamine, serotonin content and AChE activity. The genes related to the central nervous system (CNS) (e.g., a1-tubulin, mbp, syn2a, shha and gap-43) were downregulated. Our results indicate that the developmental neurotoxicity of trichlorfon might be attributed to disorders of cholinergic, dopaminergic and serotonergic signaling and the development of the CNS.

Neurotoxicity of Chronic Co-Exposure of Lead and Ionic Liquid in Common Carp: Synergistic or Antagonistic?

Previous studies have indicated that the harmful heavy metal lead (Pb) contamination in aquatic systems has caused intelligence development disorders and nervous system function abnormalities in juveniles due to the increased permeability of the blood-brain barrier. Ionic liquids (ILs) are considered "green" organic solvents that can replace traditional organic solvents. Studies have found the presence of ILs in soil and water due to chemical applications or unintentional leakage. Therefore, what would happen if Pb interacted with ILs in a body of water? Could ILs enable Pb to more easily cross the blood-brain barrier? Therefore, we examined the combined exposure of Pb and ILs in common carp at low concentration (18.3 mg L-1 of Pb(CH3COO)2•3 H2O and 11 mg L-1 of the IL 1-methyl-3-octylimidazolium chloride, 5% of their LC50) for 28 days in the present study. The result of a neurobehavioral assay showed that chronic exposure of lead at lower concentrations significantly altered fish movement and neurobehaviors, indicating that lead exposure caused neurotoxicity in the carp. Increases in the neurotransmitter dopamine levels and injuries in the fish brain accounted for neurobehavioral abnormalities induced by lead exposure. Moreover, we also found that lead could easily cross the blood-brain barrier and caused significant bioaccumulation in the brain. Particularly, our study indicated that the ionic liquid could not synergistically promote blood-brain barrier permeability and hence failed to increase the absorption of lead in the fish brain, suggesting that the combined exposure of lead and ILs was not a synergistic effect but antagonism to the neurotoxicity. The results of this study suggested that ILs could recede the Pb induced neurotoxicity in fish.

MicroRNA-16 participates in the cell cycle alteration of HepG2 cells induced by MC-LR.

Microcystin-LR (MC-LR) is a cyclic hepatotoxin produced by cyanobacteria in freshwater, and chronic MC-LR exposure could induce human hepatitis if consumed in drinking water. In recent years, many studies have indicated that microRNAs participate in the hepatotoxicity of MC-LR. The purpose of this study was to investigate the potential function of miR-16 in the hepatocellular toxicity and cell cycle alteration induced by MC-LR in human hepatocellular carcinoma (HepG2) cells after treatment with 10 µM MC-LR. The result of flow cytometry detection showed that a low concentration of MC-LR (10 µM) failed to induce apoptosis but promoted cell cycle G1/S transition in HepG2 cells. In addition, the expression of apoptosis-related genes was suppressed after MC-LR exposure. These results confirm that MC-LR exposure at a low dose can promote the proliferation of HepG2 cells. Furthermore, we also found that microRNA-16 (miR-16) expression was suppressed in HepG2 cells following MC-LR exposure. Hence, we overexpressed miR-16 in HepG2 cells and treated them with MC-LR, and the results showed that miR-16 overexpression induced an increase in the G0/G1 phase and a decrease in the S phase cell cycle populations in HepG2 cells, suggesting that miR-16 can inhibit the cell proliferation of HepG2 cells. In conclusion, our results suggest that miR-16 may play a vital role in the cell cycle alteration of HepG2 cells after MC-LR exposure.

Neurotoxicity of an emerging organophosphorus flame retardant, resorcinol bis(diphenyl phosphate), in zebrafish larvae.

Resorcinol bis(diphenyl phosphate) (RDP), an emerging organophosphorus flame retardant and alternative to triphenyl phosphate (TPHP), is a widespread environmental pollutant. The neurotoxicity of RDP has attracted much attention, as RDP exhibits a similar structure to TPHP, a neurotoxin. In this study, the neurotoxicity of RDP was investigated by using a zebrafish (Danio rerio) model. Zebrafish embryos were exposed to RDP (0, 0.3, 3, 90, 300 and 900 nM) from 2 to 144 h postfertilization. After this exposure, the decreased heart rates and body lengths and the increased malformation rates were observed. RDP exposure significantly reduced the locomotor behavior under light-dark transition stimulation and the flash stimulus response of larvae. Molecular docking results showed that RDP could bind to the active site of zebrafish AChE and that RDP and AChE exhibit potent binding affinity. RDP exposure also significantly inhibited AChE activity in larvae. The content of neurotransmitters (γ-aminobutyric, glutamate, acetylcholine, choline and epinephrine) was altered after RDP exposure. Key genes (α1-tubulin, mbp, syn2a, gfap, shhα, manf, neurogenin, gap-43 and ache) as well as proteins (α1-tubulin and syn2a) related to the development of the central nervous system (CNS) were downregulated. Taken together, our results showed that RDP can affect different parameters related to CNS development, eventually leading to neurotoxicity. This study indicated that more attention should be paid to the toxicity and environmental risk of emerging organophosphorus flame retardants.

Environmental impacts of chlorpyrifos: Transgenerational toxic effects on aquatic organisms cannot be ignored.

Chlorpyrifos (CPF) has been extensively used in the world and frequently found in natural environments, might cause a range of environmental issues and pose a health risk to aquatic species. However, investigation of its toxic effects on offspring after parental exposure has been neglected, especially for aquatic organisms such as fish. In the current study, the effects of chronic CPF exposure (3 and 60 µg/L) on adult zebrafish (F0) was investigated to determine its influence on adult reproductive capacity and offspring (F1 and F2). The results showed the existence of CPF both in F0 ovaries and F1 embryos and larvae, indicating that CPF could be transferred directly from the F0 adult fish to F1 offspring. After 90 d exposure, we observed that F0 female fish showed increased proportion of perinucleolar oocyte in the ovaries, decreased proportion of mature oocyte, and decreased egg production, but not in F1 adult. The transcriptomic analysis revealed that the disruption of metabolism during oocyte maturation in the CPF treatment zebrafish might interfere with F0 oocytes development and quality and ultimately influence offspring survival. For the larvae, the parental CPF exposure distinctly inhibited heart rate at 72 and 120 hpf and increased the mortality of F1 but not F2 larvae. The changes of biochemical indicators confirmed a disturbance in the oxidative balance, induced inflammatory reaction and apoptosis in F1 larvae. Furthermore, the changing profiles of mRNA revealed by RNA-seq confirmed an increased susceptibility in F1 larvae and figured out potential disruptions of ROS metabolism, immune system, apoptosis, and metabolism pathways. Taken together, these results show that chronic CPF treatment can induce reproductive toxicity, and parental transfer of CPF occurs in fish, resulting in transgenerational alters in F1 generation survival and transcription that raising concerns on the ecological risk of CPF in the natural environment.

Effect of Acute Exposure to the Ionic Liquid 1-Methyl-3-octylimidazolium Chloride on the Embryonic Development and Larval Thyroid System of Zebrafish.

Previous studies have shown that ILs can induce toxicity in animals, plants, and cells. However, the effect of imidazolium-based ILs on the hypothalamus-pituitary-thyroid (HPT) axis of fish remains unknown. The present study aimed to evaluate the acute effect of [C8mim]Cl on the embryonic development and thyroid-controlled internal secretion system of zebrafish by determining the thyroid hormone level and the expression of HPT-related genes. The results obtained for embryonic developmental toxicity showed the survival rate, heart beats, and body length of fish had decreased 96 h after exposure to [C8mim]Cl, but the hatching rate had increased by the 48 h time point. The transcription levels of HTP-related genes showed that the genes dio3, tg, ttr, tsh, trhrα, trhrß, trhr2, and tpo were up-regulated, while the expression levels of dio1, trh, tshr, and nis were significantly suppressed. Furthermore, we found that exposure to [C8mim]Cl induced an alteration in the levels of thyroid hormones that increased the T3 but decreased the T4 content. In conclusion, our study indicated that acute exposure to [C8mim]Cl altered the expression of HTP-related genes and disturbed the thyroid hormone level, suggesting that the ionic liquid [C8mim]Cl might pose an aquatic environmental threat to fish.

Negative impacts of microcystin-LR and glyphosate on zebrafish intestine: Linked with gut microbiota and microRNAs?

Microcystin-LR (MC-LR) and glyphosate (GLY) have been classified as a Group 2B and Group 2A carcinogens for humans, respectively, and frequently found in aquatic ecosystems. However, data on the potential hazard of MC-LR and GLY exposure to the fish gut are relatively scarce. In the current study, a subacute toxicity test of zebrafish exposed to MC-LR (35 µg L-1) and GLY (3.5 mg L-1), either alone or in combination was performed for 21 d. The results showed that MC-LR or/and GLY treatment reduced the mRNA levels of tight junction genes (claudin-5, occludin, and zonula occludens-1) and altered the levels of diamine oxidase and D-lactic, indicating increased intestinal permeability in zebrafish. Furthermore, MC-LR and/or GLY treatment remarkably increased the levels of intestinal IL-1ß and IL-8 but decreased the levels of IL-10 and TGF-ß, indicating that MC-LR and/or GLY exposure induced an inflammatory response in the fish gut. MC-LR and/or GLY exposure also activated superoxide dismutase and catalase, generally upregulated the levels of p53, bax, bcl-2, caspase-3, and caspase-9, downregulated the levels of caspase-8 and caused notable histological injury in the fish gut. Moreover, MC-LR and/or GLY exposure also significantly altered the microbial community in the zebrafish gut and the expression of miRNAs (miR-146a, miR-155, miR-16, miR-21, and miR-223). Chronic exposure to MC-LR and/or GLY can induce intestinal damage in zebrafish, and this study is the first to demonstrate an altered gut microbiome and miRNAs in the zebrafish gut after MC-LR and GLY exposure.