Fangjie Cao

Quizalofop-P-ethyl exposure increases estrogen axis activity in male and slightly decreases estrogen axis activity in female zebrafish (Danio rerio)

The herbicide Quizalofop-P-ethyl (QpE) exerts toxic effects in fish, but limited information is currently available on its effects on the endocrine system. In the current study, adult zebrafish (Danio rerio) were exposed to different concentrations (0, 2, 20, 200μg/L) of QpE for 30days. In males, QpE exposure significantly increased plasma estradiol (E2) and vitellogenin (VTG) levels, concomitant with up-regulation of hepatic esr1 and vtg gene expression. In females, plasma sex hormone levels and VTG concentrations were not altered significantly, but an increased expression of hepatic esr1 in addition to decreased expression of hepatic vtg, esr2a and esr2b was observed. Marked histological lesions were also observed in the gonads of both males and females. Moreover, QpE exposure significantly increased transcriptional profiles of some genes in the HPG axis and liver in males, while the majority of these genes were down-regulated in females. Docking studies showed QpE forming stable interactions with the ligand-binding domain (LBD) of zebrafish ESR1 and ESR2a, suggesting QpE may bind to estrogen receptors (ESRs). This study for the first time reveals QpE as an endocrine-disrupting chemical (EDC) disrupting the zebrafish endocrine system in a sex-specific manner, whereby it increases estrogen axis activity in males and slightly decreases estrogen axis activity in females, which may be accounted for by QpE regulating steroidogenesis and/or activating ESR(s).

Developmental toxicity and potential mechanisms of pyraoxystrobin to zebrafish (Danio rerio)

As a newly developed, highly efficient strobilurin fungicide, pyraoxystrobin has been reported to be highly toxic to some aquatic organisms. However, the toxicity of pyraoxystrobin to different life stages of fish and the potential underlying mechanisms are still unknown. Hence, in the present study, the acute toxicity of pyraoxystrobin to different life stages of zebrafish (embryo, larva, and adult) was assessed. The developmental toxicity of pyraoxystrobin to zebrafish embryos and its effects on gene transcription in the embryo were also investigated. The results showed that the 96-h LC50 values of pyraoxystrobin to embryos [2h post-fertilization (hpf)], 12h post-hatching (hph) larvae (84 hpf), 72 hph larvae (144 hpf), and adult zebrafish were 4.099, 1.069, 3.236, and 5.970µg/L, respectively. This suggests that pyraoxystrobin has very high toxicity to different life stages of zebrafish, while the newly hatched larvae constitute the most sensitive period of zebrafish to pyraoxystrobin. Decreased heart rate, hatching inhibition, growth regression, and morphological deformities were observed in zebrafish embryos after acute exposure to different concentrations of pyraoxystrobin. The rate of malformation increased in a time- and concentration-dependent manner in embryos, and the most pronounced abnormality was pericardial edema and yolk sac edema. Pyraoxystrobin (2 and 4μg/L) significantly altered the mRNA levels of genes related to mitochondrial respiratory chain and ATP synthesis (NDI, uqcrc, and ATPo6), oxidative stress (Mn-Sod, Cat, and Gpx), apoptosis (p53, Bcl2, Bax, and Cas3), and immune system (TNFα, IFN, and IL-1b) in zebrafish embryos. This result indicates that the alteration of these genes is a potential mechanism underlying the toxic effects of pyraoxystrobin on zebrafish.

Developmental toxicity, oxidative stress and immunotoxicity induced by three strobilurins (pyraclostrobin, trifloxystrobin and picoxystrobin) in zebrafish embryos

Strobilurins is the most widely used class of fungicides, but is reported highly toxic to some aquatic organisms. In this study, zebrafish embryos were exposed to a range concentrations of three strobilurins (pyraclostrobin, trifloxystrobin and picoxystrobin) for 96 h post-fertilization (hpf) to assess their aquatic toxicity. The 96-h LC50 values of pyraclostrobin, trifloxystrobin and picoxystrobin to embryos were 61, 55, 86 μg/L, respectively. A series of symptoms were observed in developmental embryos during acute exposure, including decreased heartbeat, hatching inhibition, growth regression, and morphological deformities. Moreover, the three fungicides induced oxidative stress in embryos through increasing reactive oxygen species (ROS) and malonaldehyde (MDA) contents, inhibiting superoxide dismutase (SOD) activity and glutathione (GSH) content as well as differently changing catalase (CAT) activity and mRNA levels of genes related to antioxidant system (Mn-sod, Cu/Zn-sod, Cat, Nrf2, Ucp2 and Bcl2). In addition, exposure to the three strobilurins resulted in significant upregulation of IFN and CC-chem as well as differently changed expressions of TNFa, IL-1b, C1C and IL-8, which related to the innate immune system, suggesting that these fungicides caused immunotoxicity during zebrafish embryo development. The different response of enzymes and genes in embryos exposed to the three fungicides might be the cause that leads to the difference of their toxicity. This work made a comparison of the toxicity of three strobilurins to zebrafish embryos on multi-levels and would provide a better understanding of the toxic effects of strobilurins on aquatic organisms.

Short-term developmental effects and potential mechanisms of azoxystrobin in larval and adult zebrafish ( Danio rerio )

Previous study indicated that azoxystrobin had high acute toxicity to zebrafish, and larval zebrafish were more sensitive to azoxystrobin than adult zebrafish. The objective of the present study was to investigate short-term developmental effects and potential mechanisms of azoxystrobin in larval and adult zebrafish. After zebrafish embryos and adults were exposed to 0.01, 0.05 and 0.20 mg/L azoxystrobin (equal to 25, 124 and 496 nM azoxystrobin, respectively) for 8 days, the lethal effect, physiological responses, liver histology, mitochondrial ultrastructure, and expression alteration of genes related to mitochondrial respiration, oxidative stress, cell apoptosis and innate immune response were determined. The results showed that there was no significant effect on larval and adult zebrafish after exposure to 0.01 mg/L azoxystrobin. However, increased ROS, MDA concentration and il1b in larval zebrafish, as well as increased il1b, il8 and cxcl-c1c in adult zebrafish were induced after exposure to 0.05 mg/L azoxystrobin. Reduced mitochondrial complex III activity and ATP concentration, increased SOD activity, ROS and MDA concentration, decreased cytb, as well as increased sod1, sod2, cat, il1b, il8 and cxcl-c1c were observed both in larval and adult zebrafish after exposure to 0.20 mg/L azoxystrobin; meanwhile, increased p53, bax, apaf1 and casp9, alteration of liver histology and mitochondrial ultrastructure in larval zebrafish, and alteration of mitochondrial ultrastructure in adult zebrafish were also induced. The results demonstrated that azoxytrobin induced short-term developmental effects on larval zebrafish and adult zebrafish, including mitochondrial dysfunction, oxidative stress, cell apoptosis and innate immune response. Statistical analysis indicated that azoxystrobin induced more negative effects on larval zebrafish, which might be the reason for the differences of developmental toxicity between larval and adult zebrafish caused by azoxystrobin. These results provided a new insight into potential mechanisms of azoxystrobin in larval zebrafish and adult zebrafish.

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.

Effects of penthiopyrad on the development and behaviour of zebrafish in early-life stages

The agricultural use of succinate dehydrogenase inhibitor (SDHI) fungicides has increased dramatically in the US and Europe. As the SDHI fungicides, boscalid, flutolanil and thifluzamide had been reported to induce a series of toxic effects on zebrafish. However, the toxic effects of penthiopyrad on zebrafish have not been reported yet. This study aimed to assess the acute toxicity of penthiopyrad to zebrafish in early-life stages and investigate behavioural response of larvae and the effects on lipid metabolism and pigmentation under sub-lethal exposure of penthiopyrad. Based on results of the acute toxicity tests of zebrafish embryo and larvae, penthiopyrad had an acute toxicity to early-life stages of zebrafish and induced a series of deformities during development. Based on the results of sub-lethal exposure for 8 days, penthiopyrad resulted in significant decreases in swimming velocity, acceleration speed, distance moved and inactive time of larvae at 0.3, 0.6 and 1.2 mg/L. Penthiopyrad induced the disorders of lipid metabolism via affecting fatty acid synthesis and β-oxidation, in accordance with remarkable changes in the content of triglycerides and cholesterol and the expression of key genes (hmgcrα, pparα1, srebf1, cyp51 and acca1) at 1.2 mg/L. In addition, the disorder of melanin synthesis and distribution was caused by penthiopyrad in larvae in accordance with changes in body colour and related gene expression at 8 dpe.

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.