Beta-cyfluthrin impairs implantation process by inducing mitochondrial defects and changes in reactive oxygen species-mediated signaling pathways in porcine trophectoderm and uterine luminal epithelial cells.

Pyrethroid insecticides, such as beta-cyfluthrin, are used extensively globally, including in households and agriculture, and have been detected in the milk and urine of humans and cattle. Beta-cyfluthrin exhibits toxic effects, including neurotoxicity and male reproductive toxicity; however, few studies have investigated female reproductive toxicity despite its wide environmental distribution. The present study investigates effects of beta-cyfluthrin on implantation in porcine cells (pTr from the trophectoderm and pLE from the endometrial luminal epithelium). To identify the various physiological changes induced by beta-cyfluthrin, such as apoptosis and lipid peroxidation, flow cytometry analysis and immunofluorescence were performed with various reagents. In addition, the expression of genes and proteins associated with intracellular changes was confirmed using qRT-PCR and western blotting. Beta-cyfluthrin induced cell-cycle arrest and altered intracellular calcium flux. It also disrupted the mitochondrial function and promoted reactive oxygen species (ROS) production, leading to lipid peroxidation. Moreover, ROS induced by beta-cyfluthrin altered mitogen-activated protein kinase (MAPK) pathways and decreased cell migration capability. The expression levels of genes that are significant during early pregnancy were altered by beta-cyfluthrin in both cell lines. The changes resulted in apoptosis and diminished cell proliferation of pTr and pLE. Collectively, the results imply that beta-cyfluthrin disrupts the implantation process by affecting the physiology of the trophectoderm and endometrial luminal epithelial cells. The present study is the first to reveal the cellular mechanisms of beta-cyfluthrin on the female reproductive system and highlights the need for further in-depth research into its hazards.

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.

Norflurazon causes cell death and inhibits implantation-related genes in porcine trophectoderm and uterine luminal epithelial cells.

Norflurazon, an inhibitor of carotenoid synthesis, is a pre-emergent herbicide that prevents growth of weeds. The norflurazon is known to hamper embryo development in non-mammals. However, specific toxic effects of norflurazon on mammalian maternal and fetal cells have not been elucidated. Thus, the hypothesis of this study is that norflurazon may influence the toxic effects between maternal and fetal cells during early pregnancy in pigs. We aimed to examine the toxic effects of norflurazon in porcine trophectoderm (Tr) and uterine luminal epithelium (LE) cells. Norflurazon, administered at 0, 20, 50 or 100 µM for 48 h was used to determine its effects on cell proliferation and cell-cycle arrest. For both uterine LE and Tr cell lines, norflurazone caused mitochondrial dysfunction by inhibiting mitochondrial respiration and ATP production, and down-regulated expression of mRNAs of mitochondrial complex genes. Norflurazon increased cell death by increasing intracellular calcium and regulating PI3K and MAPK cell signaling pathways, as well as endoplasmic reticulum (ER) stress, ER-mitochondrial contact, and autophagy-related target proteins. Norflurazone also inhibited expression of genes required for implantation of blastocysts, including SMAD2, SMAD4, and SPP1. These findings indicate that norflurazon may induce implantation failure in pigs and other mammals through adverse effects on both Tr and uterine LE cells.

Hexaconazole induces developmental toxicities via apoptosis, inflammation, and alterations of Akt and MAPK signaling cascades.

Hexaconazole is a highly effective triazole fungicide that is frequently applied in various countries to elevate crop productivity. Given its long half-life and high water solubility, this fungicide is frequently detected in the environment, including water sources. Moreover, hexaconazole exerts hazardous effects on nontarget organisms. However, little is known about the toxic effects of hexaconazole on animal development. Thus, this study aimed to investigate the developmental toxicity of hexaconazole to zebrafish, a valuable animal model for toxicological studies, and elucidate the underlying mechanisms. Results showed that hexaconazole affected the viability and hatching rate of zebrafish at 96 h postfertilization. Hexaconazole-treated zebrafish showed phenotypic defects, such as reduced size of head and eyes and enlarged pericardiac edema. Moreover, hexaconazole induced apoptosis, DNA fragmentation, and inflammation in developing zebrafish. Various organ defects, including neurotoxicity, cardiovascular toxicity, and hepatotoxicity, were observed in transgenic zebrafish models olig2:dsRed, fli1:eGFP, and l-fabp:dsRed. Furthermore, hexaconazole treatment altered the Akt and MAPK signaling pathways, which possibly triggered the organ defects and other toxic mechanisms. This study demonstrated the developmental toxicity of hexaconazole to zebrafish and elucidated the underlying mechanisms.

Relevance of the endoplasmic reticulum-mitochondria axis in cancer diagnosis and therapy.

Dynamic interactions between organelles are responsible for a variety of intercellular functions, and the endoplasmic reticulum (ER)-mitochondrial axis is recognized as a representative interorganelle system. Several studies have confirmed that most proteins in the physically tethered sites between the ER and mitochondria, called mitochondria-associated ER membranes (MAMs), are vital for intracellular physiology. MAM proteins are involved in the regulation of calcium homeostasis, lipid metabolism, and mitochondrial dynamics and are associated with processes related to intracellular stress conditions, such as oxidative stress and unfolded protein responses. Accumulating evidence has shown that, owing to their extensive involvement in cellular homeostasis, alterations in the ER-mitochondrial axis are one of the etiological factors of tumors. An in-depth understanding of MAM proteins and their impact on cell physiology, particularly in cancers, may help elucidate their potential as diagnostic and therapeutic targets for cancers. For example, the modulation of MAM proteins is utilized not only to target diverse intracellular signaling pathways within cancer cells but also to increase the sensitivity of cancer cells to anticancer reagents and regulate immune cell activities. Therefore, the current review summarizes and discusses recent advances in research on the functional roles of MAM proteins and their characteristics in cancers from a diagnostic perspective. Additionally, this review provides insights into diverse therapeutic strategies that target MAM proteins in various cancer types.

Pyridaben impaired cell cycle progression through perturbation of calcium homeostasis and PI3K/Akt pathway in zebrafish hepatocytes.

Environmental pollution caused by pesticides is a growing concern. Pyridaben, a widely used organochlorine insecticide, is a representative water pollutant. Owing to its extensive usage, it has been detected in various aquatic ecosystems, including rivers and oceans. Pyridaben is highly toxic to aquatic organisms; however, the mechanism of its toxicity in the liver, which is important in toxicant metabolism, has not been studied. Therefore, we employed zebrafish and its well-characterized liver cell line, ZFL to assess pyridaben hepatotoxicity and explore its potential mechanisms of action. Pyridaben led to reduction of the liver size and fluorescence intensity of dsRed-labeled Tg (fabp10a:dsRed) zebrafish. It reduced the viability and proliferation of ZFL cells in vitro by inducing apoptosis and cell cycle arrest. These changes might be primarily linked to uncontrolled intracellular calcium flow in ZFL cells exposed to pyridaben. Additionally, it also downregulates the PI3K/Akt signaling cascade, leading to the inactivation of Gsk3ß and nuclear translocation of ß-catenin. Taken together, our findings suggest that pyridaben could have hepatotoxic effects on aquatic organisms. This study is the first to provide insight into the hepatotoxic mechanism of pyridaben using both in vivo and in vitro models.

Bifenox induces programmed cell death in bovine mammary epithelial cells by impairing calcium homeostasis, triggering ER stress, and altering the signaling cascades of PI3K/AKT and MAPK.

Bifenox (methyl 5-(2,4-dichlorophenoxy)-2-nitrobenzoate), a nitrophenyl ether herbicide, was first introduced in the 1980s to control broadleaf weeds. As a result of its wide and frequent application in diverse agricultural settings and reports on residual traces, potential adverse effects of bifenox have been studied extensively in rat hepatocytes, bovine peripheral lymphocytes, and mice. Despite the reported risks of bifenox exposure in dairy cows, the toxicity of bifenox on bovine lactation system has not been extensively investigated. Therefore, we used bovine mammary epithelial (MAC-T) cells to study the toxic effects of bifenox on mammary glands. We found that bifenox inhibited MAC-T cells proliferation and disturbed the cell cycle, especially in the sub-G1 and G1 phases. Bifenox also disrupted the calcium homeostasis within the cell and impaired mitochondrial membrane potential. We also examined phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase (MAPK) signaling cascades. The findings indicated hyperactivation of phosphorylated protein kinase B (AKT), p70 ribosomal S6 kinase (p70S6K), S6, extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and c-Jun, as well as endoplasmic reticulum (ER) stress caused by bifenox treatment. In conclusion, based on our in vitro study employing MAC-T cells, we report that bifenox can induce damage to the bovine mammary glands, potentially impacting milk production.

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.

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.

Toxic effects of benfluralin on zebrafish embryogenesis via the accumulation of reactive oxygen species and apoptosis.

The dinitroaniline herbicide benfluralin is used weed control in conventional systems and poses a high risk of accumulation in aquatic systems. Previous studies have shown the toxic effects of benfluralin on non-target organisms; however, its developmental toxicity in vertebrates has not yet been reported. This study demonstrated the developmental toxicity of benfluralin and its mechanism of action, using zebrafish as an aquatic vertebrate model. Benfluralin induces morphological and physiological alterations in body length, yolk sac, and heart edema. We also demonstrated a reactive oxygen species (ROS) increase of approximately 325.53 % compared with the control group after 20 µM benfluralin-treatment. In addition, the malformation of the heart and vascular structures was identified using transgenic flk1:eGFP zebrafish models at 20 µM concentration benfluralin exposure. Moreover, benfluralin induced small livers, approximately 59.81 % of normal liver size, via abnormal development of the liver as observed in the transgenic L-fabp:dsRed zebrafish. Benfluralin also inhibits normal growth via abnormal expression of cell cycle regulatory genes and increases oxidative stress, inflammation, and apoptosis. Collectively, we elucidated the mechanisms associated with benfluralin toxicity, which lead to various abnormalities and developmental toxicities in zebrafish. Therefore, this study provides information on the parameters used to assess developmental toxicity in other aquatic organisms, such as herbicides, pesticides, and environmental contaminants.

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.

Bensulide-induced oxidative stress causes developmental defects of cardiovascular system and liver in zebrafish (Danio rerio).

Bensulide is an organophosphate herbicide commonly used in agricultural crops; however, no studies have reported on its toxic effects in the embryonic development of vertebrates, particularly gene expression level and cellular response. Therefore, to identify developmental toxicity, zebrafish eggs 8 h post-fertilization (hpf) were exposed to bensulide concentrations of up to 3 mg/L. The results indicated that exposure to 3 mg/L bensulide inhibited the hatching of all eggs and decreased the size of the body, eyes, and inner ear. There were demonstrated effects observed in the cardiovascular system and liver caused by bensulide in fli1:eGFP and L-fabp:dsRed transgenic zebrafish models, respectively. Following exposure to 3 mg/L bensulide, normal heart development, including cardiac looping, was disrupted and the heart rate of 96 hpf zebrafish larvae decreased to 16.37%. Development of the liver, the main detoxification organ, was also inhibited by bensulide, and after exposure to 3 mg/L bensulide its size reduced to 41.98%. Additionally, exposure to bensulide resulted in inhibition of antioxidant enzyme expression and an increase in ROS levels by up to 238.29%. Collectively, we identified various biological responses associated with the toxicity of bensulide, which led to various organ malformations and cytotoxic effects in zebrafish.

Molinate induces organ defects by promoting apoptosis, inflammation, and endoplasmic reticulum stress during the developmental stage of zebrafish.

Molinate is classified as a thiocarbamate herbicide and is mainly used in paddy fields to culture rice. However, the toxic effects of molinate and the associated mechanisms in the process of development have not been completely elucidated. Therefore, in the present study, we demonstrated that molinate reduced the viability of zebrafish larvae and the probability of successful hatching using zebrafish (Danio rerio), one of the remarkable in vivo models for testing the toxicity of chemicals. In addition, molinate treatment triggered the occurrence of apoptosis, inflammation, and endoplasmic reticulum (ER) stress response in zebrafish larvae. Furthermore, we identified that an abnormal cardiovascular phenotype through wild type zebrafish, neuronal defects through transgenic olig2:dsRed zebrafish, and developmental toxicity in the liver through transgenic lfabp:dsRed zebrafish. Collectively, these results provide evidence of the hazardous effects of molinate on the developmental stage of non-target organisms by elucidating the toxic mechanisms of molinate in developing zebrafish.

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.

Oxyfluorfen induces cell cycle arrest by regulating MAPK, PI3K and autophagy in ruminant immortalized mammary epithelial cells.

Oxyfluorfen, a phenoxy phenyl-type herbicide, causes significant damage to ecosystems through chronically effecting invertebrates, fish, and mammals. Considering its adverse effect on ecosystem conservation, it is necessary to investigate its toxic effects on animals. However, the mechanisms of oxyfluorfen toxicity on bovines are not well established. This study investigated the cytotoxic effect of oxyfluorfen on bovine mammary epithelial cells (MAC-T). We conducted several functional experiments to examine the response of MAC-T to oxyfluorfen under various concentrations (0, 1, 2, 5, and 10 ppm). Oxyfluorfen decreased cell viability and increased apoptotic cells by regulating the expression of apoptotic genes and proteins in MAC-T. In addition, oxyfluorfen-treated cells exhibited reduced PCNA expression with a low 3D spheroid formation as compared to that of control cells. Furthermore, oxyfluorfen treatment suppressed cell cycle progression with a decrease in cyclin D1 and cyclin A2 in MAC-T. Next, we performed western blot analysis to verify intercellular signaling changes in oxyfluorfen-treated MAC-T. The phosphor-AKT protein was increased, whereas MAPK signal pathways were decreased. Particularly, the combination of oxyfluorfen with U0126 or SP600125 completely blocked the ERK1/2 and JNK pathways leading to cell viability in MAC-T. Moreover, oxyfluorfen induced inflammatory gene expression and autophagy by increasing phosphorylation of P62 and LC3B in MAC-T. These results demonstrated that oxyfluorfen has cytotoxic effect on MAC-T, implying that the milk production capacity in cows may eventually harm humans.

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.

Folpet promotes apoptosis of bovine mammary epithelial cells via disruption of redox homeostasis and activation of MAPK cascades.

Folpet, a phthalimide fungicide, is an agrochemical used to prevent fungal diseases in several crops. The toxicity of folpet has been demonstrated in Cyprinus carpio, pigs, and the human respiratory system. However, despite the possibilities of ingestion of folpet through feed, detrimental influences of folpet on dairy cattle have not been documented. Thus, this study aimed to record the harmful effects of folpet on the bovine mammary system and milk production using mammary epithelial cells (MAC-T cells), which play an essential role in the maintenance of yield and quality of milk production. In this study, we first confirmed that folpet exhibited cytotoxicity against MAC-T cells in both 2D and 3D cultures. Folpet treatment caused apoptosis, dysregulated intracellular calcium levels, and mitochondrial membrane potential, leading to cell death. We further demonstrated the induction of oxidative stress upon folpet treatment by assessing reactive oxygen species (ROS) content and lipid peroxidation in MAC-T cells. ROS generation following folpet treatment induced activation of MAPK cascades, including ERK1/2, JNK, and p38 signaling. This is the first report highlighting the detrimental impacts of folpet on bovine mammary glands and, consequently, the dairy industry by elucidating intracellular mechanisms using MAC-T cells.

Tetraconazole interrupts mitochondrial function and intracellular calcium levels leading to apoptosis of bovine mammary epithelial cells.

Tetraconazole is a type of fungicide that eliminates pathogens in plants and fruit. To date, studies have focused on the direct exposure of plants and fruits to residual tetraconazole, but no studies have been conducted on the indirect effects of tetraconzaole. Given the importance of cows as milk-producing animals and their potential exposure to pesticides via plant consumption, we analyzed the mechanism by which tetraconazole influences milk production. Here, we confirmed that tetraconazole-induced apoptosis and inhibited cell viability and proliferation by regulating the cell cycle in bovine mammary epithelial cells (MAC-T). In addition, Ca2+ homeostasis in mitochondria was disrupted by tetraconazole, leading to the depolarization of mitochondrial membrane potential. Consistent with the proliferation-related findings, tetraconazole downregulated AKT, ERK1/2, P38, and JNK signaling pathways and proliferation-related proteins such as CCND1 and PCNA in MAC-T cells. Meanwhile, it upregulated cleaved caspase 3, BAX, and Cytochrome c under the same conditions in MAC-T cells. Furthermore, MAC-T exposed to tetraconazole causes a failure of proper autophagy functioning. In summary, the results of this study indicated that tetraconazole exposure may lead to a failure of milk production from bovine mammary epithelial cells by disrupting calcium homeostasis and mitochondrial function.

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.