Exposure to acifluorfen induces developmental toxicity in the early life stage of zebrafish.

Acifluorfen, a selective herbicide from the diphenyl ether family, targets broad leaf weeds. Diphenyl ether inhibits chlorophyll production in green plants by inhibiting protoporphyrinogen oxidase (PPO), causing cellular damage. Despite its known impacts on plants, the influence of acifluorfen on zebrafish embryo development remains unclear. In this study, we explored the LC50 of acifluorfen in early-stage wild-type zebrafish, determining it to be 54.99 mg/L. Subsequent examinations revealed morphological changes in zebrafish, including reduced body length. Using the cmlc2:dsRED transgenic model, we observed heart dysfunction in acifluorfen-exposed zebrafish, marked by an enlarged heart area, edema, and decreased heart rate. In response to dose-dependent acifluorfen exposure, the inhibition of angiogenesis in the brain was observed in transgenic zebrafish models (fli1a:eGFP). Organ malformations, specifically in the liver and pancreas, were noted, in lfabp:dsRED;elastase:eGFP transgenic models, indicating reduced organ size in acifluorfen-exposed zebrafish. Furthermore, acifluorfen heightened the expression of apoptosis-related genes (casp8, casp9, and tp53) in zebrafish embryos. We then determined whether acifluorfen affected the viability of zebrafish liver (ZFL) cells based on its effects on liver development in vivo. The results indicated that the proliferation of ZFL cells decreased significantly in a dose-dependent manner. Additionally, acifluorfen-treated ZFL cells exhibited a slight increase in apoptotic cells stained with annexin V and propidium iodide. In summary, these findings establish a baseline concentration for acifluorfen's effects on aquatic ecosystems and non-target organisms.

Bifenox induces hepatotoxicity and vascular toxicity in zebrafish embryos via ROS production and alterations in signaling pathways.

Existing evidence shows that currently used pesticides pose toxicological risks to exposed wildlife. Chemically, bifenox belongs to diphenyl ethers, a well-known group of herbicides. Its mechanism of action primarily involves inducing lipid peroxidation and blocking protoporphyrinogen oxidases. Toxicity of diphenyl ether herbicides has been elucidated in animal cells; however, in vivo toxicological evaluations of bifenox are required to determine its unexpected effects. This study aimed to determine the negative effects of bifenox, and its effects on higher eukaryotes. We found that early stages of zebrafish embryo exposed to bifenox demonstrated increased mortality and physiological defects, based on the LC50 value. Bifenox severely inhibited blood vessel growth by reducing key elements of complex connectivity; fluorescently tagged transgenic lines (fli1a:EGFP) showed morphological changes. Additionally, transgenic lines that selectively identified hepatocytes (fabp10a:DsRed) showed reduced fluorescence, indicating that bifenox may inhibit liver development. To evaluate the level of oxidative stress, we used 2',7'-dichlorofluorescein diacetate (DCFH-DA) probes in zebrafish embryos to identify the underlying mechanisms causing developmental damage. Our findings demonstrate that exposure to bifenox causes abnormalities in the hepatic and cardiovascular systems during zebrafish embryogenesis. Therefore, this study provides new information for the evaluation of toxicological risks of bifenox in vertebrates.

Bifenox compromises porcine trophectoderm and luminal epithelial cells in early pregnancy by arresting cell cycle progression and impairing mitochondrial and calcium homeostasis.

Bifenox is a widely used herbicide that contains a diphenyl ether group. However its global usage, the cell physiological effects that induce toxicity have not been elucidated. In this study, the effect of bifenox was examined in porcine trophectoderm and uterine epithelial cells to investigate the potential toxicity of the implantation process. To uncover the toxic effects of bifenox, cell viability and apoptosis following treatment with bifenox were evaluated. To investigate the underlying cellular mechanisms, mitochondrial and calcium homeostasis were investigated in both cell lines. In addition, the dysregulation of cell signal transduction and transcriptional alterations were also demonstrated. Bifenox reduced cell viability and significantly increased the number of cells arrested at the sub-G1 stage. Moreover, bifenox depolarized the mitochondrial membrane and upregulated the calcium flux into the mitochondria in both cell lines. Cytosolic calcium flux increased in porcine trophectoderm (pTr) cells and decreased in porcine luminal epithelium (pLE) cells. In addition, bifenox activated the mitogen-activated protein kinase and phosphoinositide 3-kinase signaling pathways. Furthermore, bifenox inhibited the expression of retinoid receptor genes, such as RXRA, RXRB, and RXRG. Chemokine CCL8 was also downregulated at the mRNA level, whereas CCL5 expression remained unchanged. Overall, the results of this study suggest that bifenox deteriorates cell viability by arresting cell cycle progression, damaging mitochondria, and controlling calcium levels in pTr and pLE cells. The present study indicates the toxic potential of bifenox in the trophectoderm and luminal epithelial cells, which can lead to implantation disorders in early pregnancy.

Developmental toxicity of flufenacet including vascular, liver, and pancreas defects is mediated by apoptosis and alters the Mapk and PI3K/Akt signal transduction in zebrafish.

Release of agrochemicals from agricultural fields could unintentionally harm organisms that not targeted by pesticides. Flufenacet is one of the oxyacetamide herbicide applied in cultivation fields of crops and this has a possibility of unintentional exposure to diverse ecosystems including streams and surface water. Despite these environmental risks, limited information regarding toxicity of flufenacet on vertebrates is available. This study is aimed to assess environmental hazards and underlying toxic mechanisms of flufenacet by using a zebrafish model. Mortality measurements and morphological observations after the treatment of flufenacet suggested developmental toxicity of flufenacet in zebrafish. In addition, its toxicity on specific organs was evaluated using transgenic fluorescent zebrafish embryo. Adverse effects of flufenacet on vascular and hepatopancreatic development were demonstrated using Tg(flk1:EGFP) and Tg(fabp10a:DsRed; ela3l:EGFP) respectively. To address intracellular actions of flufenacet in zebrafish, cellular responses including apoptosis, cell cycle modulation, and Mapk and Akt signaling pathway were verified in transcriptional and protein levels. These results demonstrated developmental toxicity of flufenacet using the zebrafish model, providing essential information for assessing its potential hazards on vertebrates that are not directly targeted by the pesticide and for elucidating molecular mechanisms.

Dimethenamid promotes oxidative stress and apoptosis leading to cardiovascular, hepatic, and pancreatic toxicities in zebrafish embryo.

Dimethenamid, one of the acetamide herbicides, is widely used on soybeans and corns to inhibit weed growth. Although other acetamide herbicides have been reported to have several toxicities in non-target organisms including developmental toxicity, the toxicity of dimethenamid has not yet been studied. In this research, we utilized the zebrafish animal model to verify the developmental toxicity of dimethenamid. It not only led to morphological abnormalities in zebrafish larvae but also reduced their viability. ROS production and inflammation responses were promoted in zebrafish larvae. Also, uncontrolled apoptosis occurred when the gene expression level related to the cell cycle and apoptosis was altered by dimethenamid. These changes resulted in toxicities in the cardiovascular system, liver, and pancreas are observed in transgenic zebrafish models including fli1a:EGFP and L-fabp:dsRed;elastase:GFP. Dimethenamid triggered morphological defects in the heart and vasculature by altering the mRNA levels related to cardiovascular development. The liver and pancreas were also damaged through not only the changes of their morphology but also through the dysregulation in their function related to metabolic activity. This study shows the developmental defects induced by dimethenamid in zebrafish larvae and the possibility of toxicity in other non-target organisms.

Fluchloralin induces developmental toxicity in heart, liver, and nervous system during early zebrafish embryogenesis.

The zebrafish is a prominent vertebrate model popularly used for toxicity testing because of its rapid development and transparent embryos. Fluchloralin, a dinitroaniline herbicide used to control weeds, inhibits microtubule formation and cell division. The structurally homologous substances ethalfluralin and pendimethalin, which belong to the dinitroaniline family, were found to be genotoxic and to exert developmental toxicity via mitochondrial dysfunction in a zebrafish model. To date, developmental toxicity of fluchloralin in zebrafish has not been reported. In the present study, we identified morphological changes in developing zebrafish, including decreased survival rate and body length, and increased yolk sac edema. In dose-dependent response to fluchloralin exposure, inhibition of neurogenesis in the spinal cord and motor neuron defects were observed in transgenic zebrafish models (olig2:dsRed). Zebrafish exposed to fluchloralin also displayed organ dysfunction in the heart, liver, and pancreas in cmlc2:dsRed and lfabp:dsRed;elastase:GFP transgenic models. Fluchloralin increased cell death in the brain by promoting apoptosis, visualized via acridine orange staining, and by activating apoptosis signaling proteins, including cytochrome c1, zBax, and Bcl-XL. This study provides novel evidence supporting the necessity of controlling pollutants in aquatic environments.

Terbutryn causes developmental toxicity in zebrafish (Danio rerio) via apoptosis and major organ malformation in the early stages of embryogenesis.

Terbutryn (2-(ethylamino)-4-(tert-butylamino)-6-(methylthio)-1,3,5-triazine) is a substituted symmetrical triazine herbicide used in agricultural fields to prevent undesired vegetation growth by inhibiting photosynthesis in target weeds. Although terbutryn has various benefits, long-term exposure, misuse, or abuse of terbutryn may cause non-target toxicity and severe ecosystem pollution. To provide a detailed description of the embryonic developmental toxicity of terbutryn, zebrafish (Danio rerio) were exposed to 2, 4, and 6 mg/L of terbutryn and the morphological changes, pathological abnormalities, and developmental endpoints were assessed relative to that of a solvent control. The results showed that terbutryn induces a loss of survivability, reduction in body and eye size, and edema in the yolk sac. Through fluorescence microscopy, blood vessels, motor neurons, and liver development were investigated using transgenic zebrafish models based on fluorescently tagged genes (fllk1:eGFP, olig2:dsRed, and L-fabp:dsRed). Furthermore, cell death by apoptosis in zebrafish caused by terbutryn exposure was evaluated via acridine orange staining, which is a selective fluorescent staining agent. To support the preceding results, gene expression alterations caused by terbutryn exposure in zebrafish larvae were assessed. The overall results indicate that exposure to terbutryn induces apoptosis and disrupts organ development. These embryonic developmental toxicity results suggest that terbutryn should be applied in the right areas at the appropriate rates, concentrations, and quantities.

Developmental defects induced by thiabendazole are mediated via apoptosis, oxidative stress and alteration in PI3K/Akt and MAPK pathways in zebrafish.

Thiabendazole, a benzimidazole fungicide, is widely used to prevent yield loss in agricultural land by inhibiting plant diseases derived from fungi. As thiabendazole has a stable benzimidazole ring structure, it remains in the environment for an extended period, and its toxic effects on non-target organisms have been reported, indicating the possibility that it could threaten public health. However, little research has been conducted to elucidate the comprehensive mechanisms of its developmental toxicity. Therefore, we used zebrafish, a representative toxicological model that can predict toxicity in aquatic organisms and mammals, to demonstrate the developmental toxicity of thiabendazole. Various morphological malformations were observed, including decreased body length, eye size, and increased heart and yolk sac edema. Apoptosis, reactive oxygen species (ROS) production, and inflammatory response were also triggered by thiabendazole exposure in zebrafish larvae. Furthermore, PI3K/Akt and MAPK signaling pathways important for appropriate organogenesis were significantly changed by thiabendazole. These results led to toxicity in various organs and a reduction in the expression of related genes, including cardiovascular toxicity, neurotoxicity, and hepatic and pancreatic toxicity, which were detected in flk1:eGFP, olig2:dsRED, and L-fabp:dsRed;elastase:GFP transgenic zebrafish models, respectively. Overall, this study partly determined the developmental toxicity of thiabendazole in zebrafish and provided evidence of the environmental hazards of this fungicide.

Establishment of Immortalized Human Endometriotic Stromal Cell Line from Ectopic Lesion of a Patient with Endometriosis.

Endometriosis is an estrogen-dependent inflammatory disease characterized by the growth of endometrial-like tissues containing endometrial stromal cells and glandular epithelium outside the uterine cavity. An insufficient response to progesterone contributes to disease progression and systemic inflammation during the pathogenesis of endometriosis. Patients with endometriosis usually experience painful symptoms, dysmenorrhea, and infertility, which contribute to a significant reduction in their quality of life. To determine the possible molecular mechanisms of endometriosis and explore novel therapeutic targets, we derived primary human ovarian endometriotic stromal cells (hOESCs) from a patient of reproductive age with ovarian endometriosis. In this study, we successfully established immortalized human ovarian endometriotic stromal cell lines (ihOESCs) using primary stromal cells obtained from endometriotic lesions to overcome short lifespan and growth inhibition. Immortalization of hOESCs with human telomerase reverse transcriptase (hTERT) transfection led to cells that maintained a proliferative state under passage culture conditions without mutagenesis during cellular senescence. The morphology and karyotype of ihOESCs were unchanged compared with those of hOESCs. Moreover, ihOESCs were continuously positive for vimentin and negative for E-cadherin expression. Following decidual stimuli and inflammatory responses, both hOESCs and ihOESCs sensitively express decidualization markers and proinflammatory cytokines. Collectively, we characterized ihOESCs to maintain their phenotypic and functional properties with a longer lifespan and normal physiological responses than those of hOESCs. These immortalized cells could aid in a detailed understanding of the pathological mechanisms of endometriosis.

Embryonic exposure to chloroxylenol induces developmental defects and cardiovascular toxicity via oxidative stress, inflammation, and apoptosis in zebrafish.

Chloroxylenol is an extensively consumed anti-microbial compound. Since its usage is on the rise due to the coronavirus pandemic and ban on other antimicrobial ingredients, recent studies have suggested the necessity of estimating its potential for ecotoxicity. The detrimental effect of chloroxylenol on zebrafish (Danio rerio) viability has been reported; however, research on the mechanisms underlying its toxicity is quite limited. Therefore, we applied the zebrafish model for elucidating responses against chloroxylenol to predict its toxicity toward human health and ecology. Zebrafish exposed to chloroxylenol (0, 0.5, 1, 2.5, 5, and 10 mg/L) at the embryonic stage (from 6 h post-fertilization (hpf) to 96 hpf) showed impaired viability and hatchability, and pathological phenotypes. To address these abnormalities, cellular responses such as oxidative stress, inflammation, and apoptosis were confirmed via in vivo imaging of a fluorescent dye or measurement of the transcriptional changes related to each response. In particular, developmental defects in the cardiovascular system of zebrafish exposed to 0, 0.5, 1, and 2.5 mg/L of chloroxylenol from 6 to 96 hpf were identified by structural analyses of the system in the flk1:eGFP transgenic line. Additional experiments were conducted using human umbilical vein endothelial cells (HUVECs) to predict the adverse impacts of chloroxylenol on the human vascular system. Chloroxylenol impairs the viability and tube formation ability of HUVECs by modulating ERK signaling. The findings obtained using the zebrafish model provide evidence of the possible risks of chloroxylenol exposure and suggest the importance of more in-depth ecotoxicological studies.

Exposure to the herbicide fluridone induces cardiovascular toxicity in early developmental stages of zebrafish.

Fluridone is a systemic herbicide used to control a range of invasive aquatic plants in irrigation systems, lake, and reservoirs. Since aquatic herbicides are more likely to have a hazardous impact on ecosystems than terrestrially applied herbicides, a risk assessment is needed to determine whether to expand or limit their use. The aim of this study was to investigate the developmental toxicity of fluridone using zebrafish. Diverse toxicological results were observed for the sub-lethal endpoints, including lack of hatching, reduced heartbeat and disturbed blood circulation through dysmorphic heart, and edema formation. Abnormal apoptosis was observed in the brain and yolk sac of fluridone-exposed larvae. A computational analysis was used to predict chemical properties in non-target organisms and revealed that fluridone was highly relevant in the cardiovascular system. Double transgenic zebrafish (fli1a:EGFP;cmlc2:dsRed) were used to evaluate the effects of fluridone on the cardiovascular system during embryonic development. Ectopic growth of sub-intestinal vessels and sprouting angiogenesis in the hindbrain region were highly inhibited. Additionally, essential genes involved in the VEGF signaling and heart development were differentially expressed in dose-dependent manner. Collectively, our toxicological findings in fluridone exposure highlight defects in the cardiovascular development causing embryonic lethality that could damage aquatic communities and natural ecosystems.

Melatonin inhibits endometriosis development by disrupting mitochondrial function and regulating tiRNAs.

Endometriosis is a benign gynecological disease characterized by abnormal growth of endometrial-like cells outside the uterus. Melatonin, a hormone secreted by the pineal gland, has been shown to have therapeutic effects in various diseases, including endometriosis. However, the underlying molecular mechanisms are yet to be elucidated. The results of this study demonstrated that melatonin and dienogest administration effectively reduced surgically induced endometriotic lesions in a mouse model. Melatonin suppressed proliferation, induced apoptosis, and dysregulated calcium homeostasis in endometriotic cells and primary endometriotic stromal cells. Melatonin also caused mitochondrial dysfunction by permeating through the mitochondrial membrane to disrupt redox homeostasis in the endometriotic epithelial and stromal cells. Furthermore, melatonin affected oxidative phosphorylation systems to decrease ATP production in End1/E6E7 and VK2/E6E7 cells. This was achieved through messenger RNA-mediated downregulation of respiratory complex subunits. Melatonin inhibited the PI3K/AKT and ERK1/2 pathways and the mitochondria-associated membrane axis and further suppressed the migration of endometriotic epithelial and stromal cells. Furthermore, we demonstrated that tiRNAGluCTC and tiRNAAspGTC were associated with the proliferation of endometriosis and that melatonin suppressed the expression of these tiRNAs in primary endometriotic stromal cells and lesions in a mouse model. Thus, melatonin can be used as a novel therapeutic agent to manage endometriosis.

Triadimenol promotes the production of reactive oxygen species and apoptosis with cardiotoxicity and developmental abnormalities in zebrafish.

Various types of fungicides, especially triazole fungicides, are used to prevent fungal diseases on farmlands. However, the developmental toxicity of one of the triazole fungicides, triadimenol, remains unclear. Therefore, we used the zebrafish animal model, a representative toxicological model, to investigate it. Triadimenol induced morphological alterations in the eyes and body length along with yolk sac and heart edema. It also stimulated the production of reactive oxygen species and expression of inflammation-related genes and caused apoptosis in the anterior regions of zebrafish, especially in the heart. The phosphorylation levels of Akt, ERK, JNK, and p38 proteins involved in the PI3K and MAPK pathways, which are important for the development process, were also reduced by triadimenol. These changes led to malformation of the heart and vascular structures, as observed in the flk1:eGFP transgenic zebrafish models and a reduction in the heart rate. In addition, the expression of genes associated with cardiac and vascular development was also reduced. Therefore, we elucidated the mechanisms associated with triadimenol toxicity that leads to various abnormalities and developmental toxicity in zebrafish.

Oxamyl exerts developmental toxic effects in zebrafish by disrupting the mitochondrial electron transport chain and modulating PI3K/Akt and p38 Mapk signaling.

Oxamyl, a carbamate insecticide, is mainly used to control nematodes in the agricultural field. Although oxamyl is a widely used insecticide that is associated with ecological concerns, limited studies have examined the toxic effects of oxamyl on the developmental stage and the underlying mechanisms. In this study, the developmental toxicity of oxamyl was demonstrated using zebrafish, which is a representative model as it is associated with rapid embryogenesis and a toxic response similar to that of other vertebrates. The morphological alteration of zebrafish larvae was analyzed to confirm the sub-lethal toxicity of oxamyl. Analysis of transgenic zebrafish (olig2:dsRED and flk1:eGFP line) and mRNA levels of genes associated with individual organ development revealed that oxamyl exerted toxic effects on the development of neuron, notochord, and vascular system. Next, the adverse effect of oxamyl on the mitochondrial electron transport chain was examined. Treatment with oxamyl altered the PI3K/Akt signaling and p38 Mapk signaling pathways in zebrafish. Thus, this study elucidated the mechanisms underlying the developmental toxicity of oxamyl and provided information on the parameters to assess the developmental toxicity of other environmental contaminants.

Developmental toxicity of prometryn induces mitochondrial dysfunction, oxidative stress, and failure of organogenesis in zebrafish (Danio rerio).

Prometryn, 2-methylthio-4,6-bis(isopropylamino)-1,3,5-triazine, is a selective thiomethyl triazine herbicide widely used to control unwanted weeds and harmful insects by inhibiting electron transport in target organisms. Despite having various advantages, herbicides pose as a major threat to the environment and human health due to persistent contamination, bioaccumulation, and damage to non-target organisms. In this study, the developmental toxicity of 5, 10, and 20 mg/L prometryn in zebrafish (Danio rerio) embryos was evaluated and compared to that of the solvent control for 96 h. Several transgenic zebrafish models (fli1a:eGFP, flk1:eGFP, olig2:dsRed and L-fabp:dsRed) were visually assessed to detect fluorescently tagged genes. Results showed that prometryn shortened body length, and induced yolk sac, heart edema, abnormal heart rate, and loss of viability. Fluorescence microscopy revealed that prometryn exposure caused defects in organ development, reactive oxygen species accumulation, and apoptotic cell death. Mitochondrial bioenergetics were also evaluated to determine the effect of prometryn on the electron transport chain activity and metabolic alterations. Prometryn was found to interfere with mitochondrial function, ultimately inhibiting energy metabolism and embryonic development. Collectively, our findings suggest that prometryn is a potential contaminate for non-target sites and organisms, especially aquatic, and emphasize the need to consider the toxic effects of prometryn.

Ethalfluralin induces developmental toxicity in zebrafish via oxidative stress and inflammation.

Ethalfluralin, of dinitroaniline herbicide family, is an effective weed controller. Following residue detection in herbicide-treated fields, ethalfluralin was reported to interfere with early stages of implantation in some vertebrate species. However, the role of ethalfluralin in the development of zebrafish embryos has not been elucidated yet. Therefore, in the present study, we investigated the morphological and physiological changes that occur in the embryonic development of zebrafish due to ethalfluralin exposure. Results indicated that ethalfluralin decreased survival rate along with reduction in the hatching ratio and heartbeat. It was observed to cause edema in the heart and yolk sac, and apoptosis in the anterior region of the developing zebrafish larvae; as visualized through acridine orange and TUNEL staining. In addition, ethalfluralin increased the expression of the apoptosis-associated genes including tp53, cyc1, casp8, casp9, and casp3. The Seahorse Mito Stress analysis revealed that ethalfluralin slightly reduced mitochondrial respiration in live zebrafish embryos. Reactive oxygen species (ROS) production was also observed to be elevated in zebrafish larvae in response to ethalfluralin. Treatment with ethalfluralin decreased blood vessel formation in brain and intestine in flk1 transgenic zebrafish embryos. The decrease in angiogenesis related gene expression was specifically observed in vegfc, flt1, and kdrl, and in the intestinal vasculature related genes apoa4a, aqp3, fabp2, and vil1. Moreover, an increase in inflammatory genes such as cox2a, cox2b, cxcl-c1c, il8, mcl1a, mcl1b, and nf-κb was observed using real-time PCR analysis. Collectively, these results indicate that oxidative stress generated by exposure to ethalfluralin induced ROS generation, apoptosis, inflammation and anti-angiogenic effects, and therefore, ethalfluralin may be toxic to the development of zebrafish embryos.

Targeting Thymidylate Synthase and tRNA-Derived Non-Coding RNAs Improves Therapeutic Sensitivity in Colorectal Cancer.

Some colorectal cancer (CRC) patients are resistant to 5-fluorouracil (5-FU), and high expression levels of thymidylate synthase (TS) contribute to this resistance. This study investigated whether quercetin, a representative polyphenol compound, could enhance the effect of 5-FU in CRC cells. Quercetin suppressed TS levels that were increased by 5-FU in CRC cells and promoted the expression of p53. Quercetin also induced intracellular and mitochondrial reactive oxygen species (ROS) production and Ca2+ dysregulation in a 5-FU-independent pathway in CRC cells. Furthermore, quercetin decreased mitochondrial membrane potential in CRC cells and inhibited mitochondrial respiration. Moreover, quercetin regulated the expression of specific tiRNAs, including tiRNAHisGTG, and transfection of a tiRNAHisGTG mimic further enhanced the apoptotic effect of quercetin in CRC cells. An enhanced sensitivity to 5-FU was also confirmed in colitis-associated CRC mice treated with quercetin. The treatment of quercetin decreased survival rates of the CRC mouse model, with reductions in the number of tumors and in the disease activity index. Also, quercetin suppressed TS and PCNA protein expression in the distal colon tissue of CRC mice. These results suggest that quercetin has the potential to be used as an adjuvant with 5-FU for the treatment of CRC.

Dinitramine induces cardiotoxicity and morphological alterations on zebrafish embryo development.

Dinitramine (DN), an herbicide in the dinitroaniline family, is used in agricultural areas to prevent unwanted plant growth. Dinitroaniline herbicides inhibit cell division by preventing microtubulin synthesis. They are strongly absorbed by the soil and can contaminate groundwater; however, the mode of action of these herbicides in non-target organisms remains unclear. In this study, we examined the developmental toxicity of DN in zebrafish embryos exposed to 1.6, 3.2, and 6.4 mg/L DN, compared to embryos exposed to DMSO (control) for 96 h. Visual assessments using transgenic zebrafish (fli1:eGFP) indicated abnormal cardiac development with enlarged ventricles and atria, decreased heartbeats, and impaired cardiac function. Along with cardiac development, vessel formation and angiogenesis were suppressed through activation of the inflammatory response. In addition, exposure to 6.4 mg/L DN for 96 h induced cell death, with upregulation of genes related to apoptosis. Our results showed that DN induced morphological changes and triggered an inflammatory response and apoptotic cell death that can impair embryonic growth and survival, providing an important mechanism of DN in aquatic organisms.

Diflubenzuron leads to apoptotic cell death through ROS generation and mitochondrial dysfunction in bovine mammary epithelial cells.

Pesticides, which are used in agriculture and forestry to eliminate insects, are a major cause of environmental pollution. Among them, diflubenzuron (DFB), 1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl) urea, is a common benzoylurea insecticide that hinders larval development, primarily in Aedes aegypti larvae. Many experts have announced the biological toxicity of DFB in various species. However, the toxicity of benzoylurea pesticides, including DFB, to bovine mammary epithelial cells (MAC-T) is unclear. Therefore, in this study, we confirmed the cytotoxic effects of DFB on the viability and proliferation of MAC-T cells. Additionally, we observed that DFB induced lipid peroxidation through reactive oxygen species (ROS) production, resulting in an increase in transcriptional gene expression related to inflammatory response. Moreover, we demonstrated mitochondrial dysfunction including depolarization of the mitochondrial membrane, perturbation of calcium homeostasis, and, eventually, apoptosis. Furthermore, we identified DFB-triggered signaling pathways related to ROS generation and cell proliferation, as well as their interactions, by treating the cells with pharmacological inhibitors in combination with DFB. DFB attenuated the phosphorylation of AKT, P70S6K, S6, and ERK1/2 and facilitated the phosphorylation of JNK and c-Jun. These results show that DFB can induce apoptotic cell death via ROS generation and mitochondrial dysfunction in MAC-T cells.

Fluroxypyr-1-methylheptyl ester interferes with the normal embryogenesis of zebrafish by inducing apoptosis, inflammation, and neurovascular toxicity.

Fluroxypyr-1-methylheptyl ester (FPMH) is a synthetic auxin herbicide used to regulate the growth of post-emergence broad-leaved weeds. Although acute exposure to FPMH increases the mortality of several fish species in the juvenile stage, the developmental toxicity of FPMH in aquatic vertebrates has not yet been investigated. In the present study, we assessed the developmental toxicity of FPMH using zebrafish models that offer many advantages for studying toxicology. During embryogenesis, survival rates gradually decreased with increasing FPMH concentrations and exposure times. At 120 h post-fertilization, FPMH-exposed zebrafish larvae showed various abnormalities such as small eye size, heart defects, enlarged yolk sac, and shortened body length. The study results confirmed the induction of apoptosis in the anterior body of zebrafish and upregulation of inflammatory gene expression. Further, defects in vascular networks, especially the loss of central arteries and abnormal aortic arch structures, were seen in the fli1:eGFP transgenic zebrafish model. Neurotoxicity of FPMH was examined using mbp:eGFP zebrafish and which displayed compromised myelination following FPMH administration. Our study has demonstrated the mechanisms underlying FPMH toxicity in developing zebrafish that is a representative model of vertebrates.