Search

Excessive use of hard-to-degrade pesticides threatens the ecological health of aquatic systems. This study aimed to investigate difenoconazole (DFZ) residues in the environment induced neurotoxicity in carp and the underlying mechanisms. A total of thirty-six carps were divided into three groups and exposed to 0, 0.5, and 2.0 mg/L DFZ for 96 h, respectively. The alterations in behavior and blood-brain barrier (BBB) were examined, and potential mechanisms were explored using immunological assays and biochemical methods. The results showed that DFZ exposure caused behavioral freezing, reduced feeding, and neuronal necrosis in carp. Mechanistically, DFZ triggered ROS accumulation and destroyed the balance between oxidation and antioxidation with increased lipid peroxidation product MDA contents and reduced antioxidant enzymes SOD and CAT activities in the carp brain by inhibiting the NF-E2-related factor 2 (Nrf2) pathway. The activation of oxidative stress further reduced tight junction proteins and MMP levels, thereby destroying BBB and leading to DFZ leakage into the brain. Increased BBB permeability additionally led to DFZ activation of nuclear factor kappa-B signaling-mediated inflammatory cytokine storm, exacerbating neuroinflammation. Meanwhile, DFZ exposure activated mitochondria-associated apoptosis in the carp's brain by up-regulating Bcl-2 associated X protein, cleaved-caspase3, and cytochrome C and decreasing B-cell lymphoma-2 levels. Interestingly, the carp's brain initiated a protective autophagic response via the PI3K/AKT/TOR pathway intending to counteract the neurotoxicity of DFZ. Overall, we concluded that accumulation of DFZ at high concentrations in the aquatic systems disrupted the BBB and resulted in neurotoxicity in carp through inhibition of Nrf2 pathway-mediated ROS accumulation. This study provides a reference for monitoring DFZ residues in the environment and a new target for the treatment of DFZ-induced neurotoxicity in carp. [Display omitted] • Difenoconazole (DFZ) resulted in altered behavior and brain tissue damage in carp. • Acute DFZ exposure inhibited Nrf2/Keap1 pathway and caused oxidative stress. • DFZ exposure caused ROS accumulation, BBB disruption, and DFZ leakage into the brain. • Disruption of BBB led to DFZ activation of inflammation via the NF-κB pathway. • Disruption of BBB led to DFZ activation of apoptosis via the mitochondrial pathway. [ABSTRACT FROM AUTHOR]

As the use of pesticides increases year after year, so does the level of residual pesticides in the aquatic environment, posing a serious threat to non-target organisms. Difenoconazole (DFZ), a class of long-lasting fungicides and residues in the marine environment, has been shown to cause damaging effects on different organs of aquatic organisms. However, there is no research on the damage of DFZ to carp spleen tissue. This study aimed to investigate the acute toxic effects of DFZ on the spleen tissue of carp (Cyprinus carpio) by exposing juvenile carp to environmentally relevant concentrations of DFZ. We randomly selected 30 carp, divided them into the Control, Low, and High groups, and then exposed the three groups to 0, 0.488 mg/L DFZ, and 1.953 mg/L DFZ for 96 h respectively. We then investigated the toxic effects caused by DFZ on carp and spleen tissues by detecting changes in spleen histopathologic damage, apoptosis, oxidative stress, inflammation, and blood biochemical parameters. We found that DFZ causes severe histopathology in spleen tissue, including ballooning, structural relaxation, and giant mitochondria. In addition, we found that DFZ caused excessive apoptosis in spleen tissue by TUNEL staining and expression levels of apoptosis-related genes (caspase3, caspase8, caspase9, fas, bax, bcl-2, and p53). The activities and transcript levels of the antioxidant enzymes SOD, CAT, and GSH-Px were significantly down-regulated. In addition, DFZ led to a significant increase in activation of the NF-κB signaling pathway and mRNA levels of pro-inflammatory cytokines il-6, il-1β , and tnf-α , and a substantial decrease in mRNA levels of anti-inflammatory cytokines il-10 and tgf-β1 in spleen tissue. Blood biochemical parameters showed that DFZ exposure significantly reduced erythrocyte, leukocyte, hemoglobin, C3, and IgM levels. Collectively, DFZ exposure induced apoptosis, immunosuppression, oxidative stress, and inflammatory responses in the spleen tissue of carp, resulting in spleen tissue damage. [Display omitted] • Difenoconazole (DFZ) triggers apoptosis of the mitochondrial pathway in carp spleen tissue. • DFZ causes disruption of the antioxidant system in the spleen tissue of carp. • DFZ mediates inflammatory responses in carp spleen tissue through activation of the NF-κB pathway. • DFZ exposure causes immunosuppression in carp. [ABSTRACT FROM AUTHOR]

Vibrio alginolyticus is an important zoonotic marine pathogenic bacterium. Previous studies on the mechanism of innate immune against V. alginolyticus infection have been limited to aquatic animals, however, how V. alginolyticus activates mammalian immune cells has not been fully clarified. Here, ELISA combined RT-qPCR assays were used to detect the secretion and transcription level of pro-inflammatory cytokines and TLRs during V. alginolyticus infection of mice peritoneal macrophages (PMϕs). Western blotting was used to explore the phosphorylation levels of p38, JNK, ERK, AKT and NF-κB protein. Immunofluorescence assay was used to determine the location of NF-κB protein. Inhibition assay was used to study the role of up-regulated TLR in activated signaling pathways and the role of these pathways in the release of pro-inflammatory cytokines. Our data showed that V. alginolyticus can up-regulate the expression levels of IL-1β, IL-6, IL-12 and TNF-α in PMϕs. In addition, V. alginolyticus stimulation activated the phosphorylation of p38, JNK and ERK were TLR2 heterodimers-dependent, whereas inhibitors of SB203580 (p38), SCH772984 (ERK) and SP600125 (JNK) significantly reduced IL-1β, IL-6, IL-12 and TNF-α production. We further revealed that V. alginolyticus activated the signaling pathways of AKT via TLR2 heterodimers. The inhibitor of MK-2206 2HCl (AKT) negatively regulated the IL-1β, IL-6 and TNF-α release levels. Moreover, V. alginolyticus infection of PMϕs resulted in TLR2 heterodimers-mediated activation of NF-κB and induced translocation of phosphorylated NF-κB protein from the cytoplasm into the nucleus via IκBα degradation. V. alginolyticus induced IL-1β, IL-6, IL-12 and TNF-α release were blocked by the specific NF-κB inhibitor, BAY 11–7082. Taken together, our results suggested that activation of the TLR2 heterodimers-mediated downstream signaling pathways NF-κB, MAPK and AKT is responsible for inflammatory response during Vibrio alginolyticus infection in vitro. • V. alginolyticus activate TLR2-mediated MAPK, AKT and NF-κB pathways in mouse PMϕs. • V. alginolyticus activate MAPK and NF-κB pathways to enhance inflammatory response. • Cytokines levels are negatively regulated by AKT pathway in infected PMϕs. [ABSTRACT FROM AUTHOR]