Toxicity of diuron in HepG2 cells and zebrafish embryos.

Diuron is an herbicide, which is used to control a wide variety of annual and perennial broadleaf, grassy weeds, and mosses. However, the toxicity of diuron in HepG2 cells and zebrafish embryos was unclear. In this study, HpeG2 cells and zebrafish embryos were exposed to different concentrations of diuron for 24 h and 48 h, respectively. Results reveal the diuron caused cytotoxicity and the generation of reactive oxygen species (ROS) in the treated HepG2 cells. The effects of diuron on the expression of catalase and superoxide dismutase (SOD1 and SOD2), an antioxidant enzyme, were investigated. Results showed that only SOD1 was significantly induced after treated diuron 48 h, but the expression of catalase and SOD2 was unaffected. Additionally, the cytotoxicity of diuron was not attenuated in cells pretreated with of N-acetyl-cysteine (NAC), a well-known antioxidant, indicating that oxidative stress could not contribute to cellular death in the treated HepG2 cells. In zebrafish embryos, results from proteomic analysis show that 332 differentially upregulated proteins and 199 down-regulated proteins were detected in the treated embryos (P < 0.05). In addition to the up-regulated antioxidant proteins (prdx3, cat, prdx4, txnrd1, prdx1, sod1, prdx2, and sod2), some decreased proteins were related to cytoskeleton formation, tight junction, and gap junction, which could be related to the malformation of the treated zebrafish embryos. In summary, diuron caused cytotoxicity in HepG2 cells, and the mechanisms of toxicity in zebrafish were addressed using the proteomic analysis.

Proteomic analysis of ametryn toxicity in zebrafish embryos.

Ametrym (AMT) is the most widely used herbicide and frequently detected in the aquatic environment. AMT also represent a potential health risk to aquatic organisms and animals, including humans. However, little data are available on their toxicity to zebrafish (Danio rerio). The aim of the present study was to evaluate the toxicological effects of AMT exposure on zebrafish embryos. In the acute toxicity test, 6 hpf embryos were exposed to various concentrations of AMT for 24 or 48 h. The results indicated that AMT induced malformation in larvae. To investigate the toxicological mechanism on the protein expression level. A proteomic approach was employed to investigate the proteome alterations of zebra fish embryos exposed to 20 mg/L AMT for 48 h. Among 2925 unique proteins identified, 298 differential proteins (> or <1.3-fold, P < 0.05) were detected in the treated embryos as compared to the corresponding proteins in the untreated embryos. Gene ontology analysis showed that these up-regulated proteins were most involved in glycolysis, lipid transport, protein polymerization, and nucleotide binding, and the down-regulated proteins were related to microtubule-based process, protein polymerization, oxygen transport. Moreover, KEGG pathway analysis indicated that tight junction, ribosome, and oxidative phosphorylation were inhibited in the treated embryos. These findings provide new insight into the mechanisms of toxicity induced by AMT.

Protective effects of Scoparia dulcis L. extract on high glucose-induced injury in human retinal pigment epithelial cells.

Diabetic retinopathy (DR) is a major cause of vision loss in diabetic patients. Hyperglycemia-induced oxidative stress and the accumulation of inflammatory factors result in blood-retinal barrier dysfunction and the pathogenesis of DR. Scoparia dulcis L. extract (SDE), a traditional Chinese medicine, has been recently recognized for its various pharmacological effects, including anti-diabetic, anti-hyperlipidemia, anti-inflammatory, and anti-oxidative activities. However, there is no relevant research on the protective effect of SDE in DR. In this study, we treated high glucose (50 mM) in human retinal epithelial cells (ARPE-19) with different concentrations of SDE and analyzed cell viability, apoptosis, and ROS production. Moreover, we analyzed the expression of Akt, Nrf2, catalase, and HO-1, which showed that SDE dose-dependently reduced ROS production and attenuated ARPE-19 cell apoptosis in a high-glucose environment. Briefly, we demonstrated that SDE exhibited an anti-oxidative and anti-inflammatory ability in protecting retinal cells from high-glucose (HG) treatment. Moreover, we also investigated the involvement of the Akt/Nrf2/HO-1 pathway in SDE-mediated protective effects. The results suggest SDE as a nutritional supplement that could benefit patients with DR.

Recuperative herbal formula Jing Si maintains vasculature permeability balance, regulates inflammation and assuages concomitants of "Long-Covid".

Coronavirus disease 2019 (COVID-19) is a worldwide health threat that has long-term effects on the patients and there is currently no efficient cure prescribed for the treatment and the prolonging effects. Traditional Chinese medicines (TCMs) have been reported to exert therapeutic effect against COVID-19. In this study, the therapeutic effects of Jing Si herbal tea (JSHT) against COVID-19 infection and associated long-term effects were evaluated in different in vitro and in vivo models. The anti-inflammatory effects of JSHT were studied in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and in Omicron pseudotyped virus-induced acute lung injury model. The effect of JSHT on cellular stress was determined in HK-2 proximal tubular cells and H9c2 cardiomyoblasts. The therapeutic benefits of JSHT on anhedonia and depression symptoms associated with long COVID were evaluated in mice models for unpredictable chronic mild stress (UCMS). JSHT inhibited the NF-ƙB activities, and significantly reduced LPS-induced expression of TNFα, COX-2, NLRP3 inflammasome, and HMGB1. JSHT was also found to significantly suppress the production of NO by reducing iNOS expression in LPS-stimulated RAW 264.7 cells. Further, the protective effects of JSHT on lung tissue were confirmed based on mitigation of lung injury, repression in TMRRSS2 and HMGB-1 expression and reduction of cytokine storm in the Omicron pseudotyped virus-induced acute lung injury model. JSHT treatment in UCMS models also relieved chronic stress and combated depression symptoms. The results therefore show that JSHT attenuates the cytokine storm by repressing NF-κB cascades and provides the protective functions against symptoms associated with long COVID-19 infection.

Low concentrations of 4-ABP promote liver carcinogenesis in human liver cells and a zebrafish model.

4-Aminobiphenyl (4-ABP) is a human bladder cancer carcinogen found in the manufacture of azo dyes and the composition of cigarette smoke in the environment. To determine whether low concentrations of 4-ABP induced or promote liver carcinogenesis and investigate the underlying mechanism, we have established the liver cell carcinogenesis model in human liver cell lines and zebrafish to evaluate liver cancer development associated with long-term exposure to low concentrations of 4-ABP. Results show that repeated 4-ABP exposure promoted cellular proliferation and migration via the involvement of ROS in Ras/MEK/ERK pathway in vitro. Also, 4-ABP (1, 10, and 100 nM) induces hepatocellular carcinoma (HCC) formation in HBx, Src (p53-/-) transgenic zebrafish at four months of age and in wild-type zebrafish at seven months of age. In addition, we observed a correlation between the Ras-ERK pathway and 4-ABP-induced HCC in vitro and in vivo. Our finding suggests low concentrations of 4-ABP repeated exposure is a potential risk factor for liver cancer. To our knowledge, this is the first report on the promotion of liver carcinogenesis in human liver cells and zebrafish following 4-ABP exposure.

The cytotoxicity and genotoxicity of single and combined fenthion and terbufos treatments in human liver cells and zebrafish embryos.

The mechanism of genotoxicity of the individual and combined pesticides of terbufos and fenthion were evaluated using HepG2 cells and zebrafish embryos. We determined genotoxicity by neutral comet assay and phosphorylation of H2AX (γH2AX), which indicated that cells treated with terbufos and/or fenthion caused DNA double-strand breaks (DSBs). The combination of these pesticides at the equimolar concentration (40 µM) exhibited less toxicity, genotoxicity, and did not impact DNA homologous recombination (HR) repair activity compare to terbufos or fenthion alone treatment. In HepG2 cells, terbufos, fenthion and their combination decreased only Xrcc2 expression (one of DNA HR repair genes). Moreover, the combined pesticides decreased Xrcc6 expression (one of DNA non-homologous end joining (NHEJ) repair genes). In addition, only terbufos or fenthion decreased XRCC2 protein expression, while Ku70 was impacted in all of the treated cells irrespective of up or down regulation. In zebrafish embryos, only fenthion impaired HR genes (Rad51 and Rad18) expression at 24 h. After 48 h exposure to pesticides, the combined pesticides elevated HR genes (Rad51 and Xrcc2) expression while terbufos or fenthion inhibited the expression of these four genes (Rad51, Rad18, Xrcc2, Xrcc6). In addition, the hatching rate of zebrafish embryos with fenthion or the combined pesticide at 72 hpf was significantly impaired. Collectively, terbufos and/or fenthion in combining caused DSBs in HepG2 cells and zebrafish embryos. Moreover, the specific mechanism of combined pesticide both HepG2 and zebrafish embryos revealed antagonism interaction.

4-Aminobiphenyl suppresses homologous recombination repair by a reactive oxygen species-dependent p53/miR-513a-5p/p53 loop.

4-Aminobiphenyl (4-ABP), a well-known human carcinogen, can cause oxidative DNA damage and induce miR-513a-5p. However, the interplay between miR-513a-5p and DNA damage remains unclear. In our result of ChIP assay, we speculated that p53 as transcription factor could regulate miR-513a-5p expression. In addition, we found that miR-513a-5p-induced by 4-ABP could suppress p53 expression and HR repair activity. On the other hand, the levels of p53, miR-513a-5p, and γH2AX were attenuated by 5 mM N-acetyl-l-cysteine (NAC) pretreatment, indicating that the reactive oxygen species (ROS)-dependent p53-miR-513a-5p was involved in DSB repair in 4-ABP-treated cells. These findings indicated that the ROS/p53/miR-513a-5p/p53 loop axis plays a relevant role in regulating HR repair which may facilitate our understanding of molecular mechanisms regarding how miR-513a-5p impacts DSB repair in 4-ABP-treated cells.

4-Aminobiphenyl inhibits the DNA homologous recombination repair in human liver cells: The role of miR-630 in downregulating RAD18 and MCM8.

4-Aminobiphenyl (4-ABP), a well-known human carcinogen, has been shown to cause oxidative DNA damage and induce miR-630 expression in HepG2 cells treated with 18.75 µM-300 µM for 24 h. However, the underlying mechanism regarding the epigenetic regulation of miR-630 on DNA damage repair in liver cells is still not understood and needs to be investigated. In present study, our results showed that miR-630 was upregulated, resulting in mediating a decrease of DNA homologous recombination (HR) repair in L-02, HepG2 or Hep3B cells. Results from a luciferase reporting experiment showed that RAD18 and MCM8 were the potential targets of miR-630 during DNA damage induction. The downregulation of RAD18 or MCM8 by miR-630 was accompanied by inhibition of HR repair. Conversely, inhibiting miR-630 enhanced the expression of RAD18 and MCM8, and rescued HR repair. Additionally, we proved that the transcription factor CREB was related to miR-630 biogenesis in liver cells. Moreover, the levels of CREB, miR-630 expression, and double-strand breaks (DSBs) were attenuated by 5 mM N-acetyl-L-cysteine (NAC) pretreatment, indicating that reactive oxygen species (ROS)-dependent CREB-miR-630 was involved in DSB repair. These findings indicated that the ROS/CREB/-miR-630 axis plays a relevant role in the regulation of RAD18 and MCM8 in HR repair, which may facilitate our understanding of molecular mechanisms regarding the role of miR-630 downregulating DNA damage repair in liver cells.

Molecular mechanisms of discrotophos-induced toxicity in HepG2 cells: The role of CSA in oxidative stress.

Dicrotophos (Dic), an insecticide and acaricide, is used against a variety of sucking, boring and chewing pests. It was proven that Dic induced oxidative DNA damage in HepG2 cells. However, the molecular mechanisms of this compound were still unclear. First of all, the cytotoxicity and oxidative DNA damage were confirmed. Next, using RNA-seq for detecting differential expressed genes (DEGs) in cells treated with 50 µM Dic for 24 h, we showed that the dysregulation of these genes, irrespective of up (1298 genes) or down (2125 genes) regulation, could be attributed to some diverse pathways/metabolisms using KEGG analysis, particularly in DNA damage responses (DDRs) such as oxidative phosphorylation, nucleotide excision repair and cell cycle arrest. Validation of some randomly selected DDR genes confirmed RNA-seq results. We further demonstrated that Dic induced ROS overproduction, the loss of mitochondrial depolarization and cell cycle arrest in the G0/G1 phase. In addition, we also definitely clarified the role of CSA, a nucleotide excision repair enzymes in Dic-treated cells. Collectively, our results showed that various mechanisms of Dic-induced toxicity in HepG2 cells including downregulation of some genes related to nucleotide excision repair including CSA and increased oxidative stress.