Combined Exposure to Diazinon and Nicotine Exerts a Synergistic Adverse Effect In Vitro and Disrupts Brain Development and Behaviors In Vivo.

A real-life environment during pregnancy involves multiple and simultaneous exposures to toxic chemicals. Perinatal exposures to toxic chemicals have been reported to exert an inhibitory effect on mouse neural development and behaviors. However, the effect of combined exposures of organophosphate and nicotine has not been previously reported. In this study, we investigated whether a combined exposure of diazinon and nicotine can have a synergistic effect. The effects of the combined chemical exposure on cell viability and neuronal differentiation were examined using mouse Sox1-GFP cells. Additionally, mice were maternally administered 0.18 mg/kg diazinon, a no adverse effect level (NOAEL) dose, combined with 0.4, 1, and 2 mg/kg nicotine. Mice offspring underwent behavior tests to assess locomotor, depressive, cognitive, and social behaviors. Morphological change in the brain was investigated with immunolocalization. We revealed that the combined exposure to diazinon and nicotine can have a synergistic adverse effect in vitro. In addition, the chemical-treated mouse offspring showed abnormalities in motor learning, compulsive-like behaviors, spatial learning, and social interaction patterns. Moreover, 0.18 mg/kg diazinon and 2 mg/kg nicotine co-exposure resulted in an increase in tyrosine hydroxylase (TH)-positive dopaminergic neurons. Thus, the findings suggest that perinatal co-exposure to nicotine and diazinon can result in abnormal neurodevelopment and behavior, even at low-level administration.

Mitochondrial dynamics when mitochondrial toxic chemicals exposed in 3D cultured mouse embryonic stem cell.

Mitochondria need to use considerable energy for the intracellular organelles that produce ATP. They are abundant in the cells of organs, such as muscles, liver, and kidneys. The heart, which requires a lot of energy, is also rich in mitochondria. Mitochondrial damage can induce cell death. Doxorubicin, acetaminophen, valproic acid, amiodarone, and hydroxytamoxifen are representative substances that induce mitochondrial damage. On the other hand, the effects of this substance on the progress of cardiomyocyte-differentiating stem cells have not been investigated. Therefore, a 3D cultured embryonic body toxicity test was performed. The results confirmed that the cytotoxic effects on cardiomyocytes were due to mitochondrial damage in the stage of cardiomyocyte differentiation. After drug treatment, the cells were raised in the embryoid body state for four days to obtain the ID50 values, and the levels of mRNA expression associated with the mitochondrial complex were examined. The mitochondrial DNA copy numbers were also compared to prove that the substance affects the number of mitochondria in EB-state cardiomyocytes. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-022-00161-1.

Effects of Sodium Arsenite on the Myocardial Differentiation in Mouse Embryonic Bodies.

Arsenic in inorganic form is a known human carcinogen; even low levels of arsenic can interfere with the endocrine system. In mammalian development, arsenic exposure can cause a malformation of fetuses and be lethal. This study examined the effects of sodium arsenite (SA) as the inorganic form of arsenic in embryonic bodies (EBs) with three germ layers in the developmental stage. This condition is closer to the physiological condition than a 2D cell culture. The SA treatment inhibited EBs from differentiating into cardiomyocytes. A treatment with 1 µM SA delayed the initiation of beating, presenting successful cardiomyocyte differentiation. In particular, mitochondria function analysis showed that SA downregulated the transcription level of the Complex IV gene. SA increased the fission form of mitochondrion identified by the mitochondria number and length. In addition, a treatment with D-penicillamine, an arsenic chelator, restored the beat of EBs against SA, but not mitochondrial dysfunction. These findings suggest that SA is a toxicant that induces mitochondrial damage and interferes with myocardial differentiation and embryogenesis. This study suggests that more awareness of SA exposure during pregnancy is required because even minuscule amounts have irreversible adverse effects on embryogenesis through mitochondria dysfunction.

Pre-validation of an alternative test method for prediction of developmental neurotoxicity.

Exposure to neurodevelopmental toxicants can cause permanent brain injury. Hance, determining the neurotoxicity of unknown substances is essential for the safety of substance. As an alternative method to animal studies, developmental neurotoxicity test (DNT) and the first discriminant function (DF) were established in previous study. This study aimed to increase the predictability of the DNT method and perform a mobility test. Two endpoints of 29 newly investigated substances were used to establish a second-generation DF (2nd GDF). As two endpoints, the half-inhibitory concentration of the cell viability (IC50) was determined using a cell counting kit-8 assay. The half-inhibitory concentration of differentiation (ID50) was determined by measuring the green fluorescent protein (GFP) intensity in 46C cells. The substances were treated dose-dependently to measure IC50 and ID50. The 2nd GDF classified 29 chemicals accurately as toxic and non-toxic. Four participants of three independent laboratories were enrolled to test the mobility. The results of the test set were highly accurate in reproducibility (100% of accuracy, sensitivity, and specificity) and mobility (accuracy 93.33%, sensitivity 90.91%, and specificity 100%). In conclusion, the protocol is transferable, reproducible, and accurate. Therefore, this could be a standardizing method for determining a neurotoxicant as an alternative for animal experiments.

Melatonin mitigates the adverse effect of hypoxia during myocardial differentiation in mouse embryonic stem cells.

BACKGROUND: Hypoxia causes oxidative stress and affects cardiovascular function and the programming of cardiovascular disease. Melatonin promotes antioxidant enzymes such as superoxide dismutase, glutathione reductase, glutathione peroxidase, and catalase. OBJECTIVES: This study aims to investigate the correlation between melatonin and hypoxia induction in cardiomyocytes differentiation. METHODS: Mouse embryonic stem cells (mESCs) were induced to myocardial differentiation. To demonstrate the influence of melatonin under hypoxia, mESC was pretreated with melatonin and then cultured in hypoxic condition. The cardiac beating ratio of the mESC-derived cardiomyocytes, mRNA and protein expression levels were investigated. RESULTS: Under hypoxic condition, the mRNA expression of cardiac-lineage markers (Brachyury, Tbx20, and cTn1) and melatonin receptor (Mtnr1a) was reduced. The mRNA expression of cTn1 and the beating ratio of mESCs increased when melatonin was treated simultaneously with hypoxia, compared to when only exposed to hypoxia. Hypoxia-inducible factor (HIF)-1α protein decreased with melatonin treatment under hypoxia, and Mtnr1a mRNA expression increased. When the cells were exposed to hypoxia with melatonin treatment, the protein expressions of phospho-extracellular signal-related kinase (p-ERK) and Bcl-2-associated X proteins (Bax) decreased, however, the levels of phospho-protein kinase B (p-Akt), phosphatidylinositol 3-kinase (PI3K), B-cell lymphoma 2 (Bcl-2) proteins, and antioxidant enzymes including Cu/Zn-SOD, Mn-SOD, and catalase were increased. Competitive melatonin receptor antagonist luzindole blocked the melatonin-induced effects. CONCLUSIONS: This study demonstrates that hypoxia inhibits cardiomyocytes differentiation and melatonin partially mitigates the adverse effect of hypoxia in myocardial differentiation by regulating apoptosis and oxidative stress through the p-AKT and PI3K pathway.