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.

Cao F ，Souders CL 2nd ，Li P ，Pang S ，Liang X ，Qiu L ，Martyniuk CJ ... -  《-》

Developmental toxicity of the fungicide ziram in zebrafish (Danio rerio).

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 LC50 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.

Cao F ，Souders CL 2nd ，Li P ，Adamovsky O ，Pang S ，Qiu L ，Martyniuk CJ ... -  《-》

Developmental toxicity of the triazole fungicide cyproconazole in embryo-larval stages of zebrafish (Danio rerio).

Cao F ，Souders CL 2nd ，Li P ，Pang S ，Qiu L ，Martyniuk CJ ... -  《-》

Biological impacts of organophosphates chlorpyrifos and diazinon on development, mitochondrial bioenergetics, and locomotor activity in zebrafish (Danio rerio).

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.

Cao F ，Souders CL 2nd ，Li P ，Pang S ，Qiu L ，Martyniuk CJ ... -  《-》

Sub-lethal toxicity assessment of the phenylurea herbicide linuron in developing zebrafish (Danio rerio) embryo/larvae.

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 XFe24 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.

Maharaj S ，El Ahmadie N ，Rheingold S ，El Chehouri J ，Yang L ，Souders CL 2nd ，Martyniuk CJ ... -  《-》