Elevated aerobic glycolysis driven by p62-mTOR axis promotes arsenic-induced oncogenic phenotypes in human mammary epithelial cells.

Chronic arsenic exposure is considered to increase the risk of breast cancer. p62 is a multifunctional adaptor protein that controls myriad cellular processes and is overexpressed in breast cancer tissues. Although previous studies have indicated the involvement of p62 accumulation in arsenic tumorigenesis, the underlying mechanism remains obscure. Here, we found that 0.1 µM or 0.5 µM arsenite exposure for 24 weeks induced oncogenic phenotypes in human mammary epithelial cells. Elevated aerobic glycolysis, cell proliferation capacity, and activation of p62-mTOR pathway, as indicated by increased protein levels of p62, phosphorylated-mTOR (p-mTOR) and hypoxia-inducible factor 1α (HIF1α), were observed in chronically arsenite-exposed cells, and of note in advance of the onset of oncogenic phenotypes. Moreover, p62 silencing inhibited acquisition of oncogenic phenotypes in arsenite-exposed cells. The protein levels of p-mTOR and HIF1α, as well as aerobic glycolysis and cell proliferation, were suppressed by p62 knockdown. In addition, re-activation of p­mTOR reversed the inhibitory effects of p62 knockdown. Collectively, our data suggest that p62 exerts an oncogenic role via mTORC1 activation and acts as a key player in glucose metabolism during arsenite-induced malignant transformation, which provides a new mechanistic clue for the arsenite carcinogenesis.

Sleep Deprivation and Central Appetite Regulation.

Research shows that reduced sleep duration is related to an increased risk of obesity. The relationship between sleep deprivation and obesity, type 2 diabetes, and other chronic diseases may be related to the imbalance of appetite regulation. To comprehensively illustrate the specific relationship between sleep deprivation and appetite regulation, this review introduces the pathophysiology of sleep deprivation, the research cutting edge of animal models, and the central regulatory mechanism of appetite under sleep deprivation. This paper summarizes the changes in appetite-related hormones orexin, ghrelin, leptin, and insulin secretion caused by long-term sleep deprivation based on the epidemiology data and animal studies that have established sleep deprivation models. Moreover, this review analyzes the potential mechanism of associations between appetite regulation and sleep deprivation, providing more clues on further studies and new strategies to access obesity and metabolic disease.

Developmental arsenic exposure induces dysbiosis of gut microbiota and disruption of plasma metabolites in mice.

Arsenic is a notorious environmental pollutant. Of note, developmental arsenic exposure has been found to increase the risk of developing a variety of ailments later in life, but the underlying mechanism is not well understood. Many elements of host health have been connected to the gut microbiota. It is still unclear whether and how developmental arsenic exposure affects the gut microbiota. In the present study, we found that developmental arsenic exposure changed intestinal morphology and increased intestinal permeability and inflammation in mouse pups at weaning. These alterations were accompanied by a significant change in gut microbiota, as evidenced by considerably reduced gut microbial richness and diversity. In developmentally arsenic-exposed pups, the relative abundance of Muribaculaceae was significantly decreased, while the relative abundance of Akkermansia and Bacteroides was significantly enhanced at the genus level. Metabolome and pathway enrichment analyses indicated that amino acid and purine metabolism was promoted, while glycerophospholipid metabolism was inhibited. Interestingly, the relative abundance of Muribaculaceae and Akkermansia showed a strong correlation with most plasma metabolites significantly altered by developmental arsenic exposure. These data indicate that gut microbiota dysbiosis may be a critical link between developmental arsenic exposure and metabolic disorders and shed light on the mechanisms underlying increased susceptibility to diseases due to developmental arsenic exposure.

Metabolic reprogramming in the arsenic carcinogenesis.

Chronic exposure to arsenic has been associated with a variety of cancers with the mechanisms undefined. Arsenic exposure causes alterations in metabolites in bio-samples. Recent research progress on cancer biology suggests that metabolic reprogramming contributes to tumorigenesis. Therefore, metabolic reprogramming provides a new clue for the mechanisms of arsenic carcinogenesis. In the present manuscript, we review the latest findings in reprogramming of glucose, lipids, and amino acids in response to arsenic exposure. Most studies focused on glucose reprogramming and found that arsenic exposure enhanced glycolysis. However, in vivo studies observed "reverse Warburg effect" in some cases due to the complexity of the disease evolution and microenvironment. Arsenic exposure has been reported to disturb lipid deposition by inhibiting lipolysis, and induce serine-glycine one-carbon pathway. As a dominant mechanism for arsenic toxicity, oxidative stress is considered to link with metabolism reprogramming. Few studies analyzed the causal relationship between metabolic reprogramming and arsenic-induced cancers. Metabolic alterations may vary with exposure doses and periods. Identifying metabolic alterations common among humans and experiment models with human-relevant exposure characteristics may guide future investigations.

Comparative Study on In Vitro Culture of Mouse Bone Marrow Mesenchymal Stem Cells.

In vitro culture of mesenchymal stem cells (MSCs) from mouse bone marrow (BM) has been hampered because of the low yield of MSCs during isolation and the contamination of hematopoietic cells during expansion. The lack of specific mouse BM-MSC markers increases the difficulty. Several techniques have been reported to improve the purity and in vitro growth of mouse BM-MSCs. However, systematic report on comparison of characteristics in primary BM-MSCs between different culture conditions is rare. Here, we studied the effects of oxygen concentrations and initial medium replacement intervals, along with cell passages, on mouse BM-MSCs isolated with differential adhesion method. BM-MSCs exhibited elevated proliferative and clonogenic abilities in 5% oxygen compared with 10% and 21% oxygen, as well as a better expression of the MSC marker Sca-1. Adipogenic and osteogenetic differentiation of BM-MSCs can be observed in both 21% and 5% oxygen. Adipogenic differentiation appeared stronger under normoxia conditions. BM-MSCs showed increased proliferative capacity and adipogenic/osteogenetic differentiation potential when initial medium replacement interval was 4 days compared with 1 day. As passage number increased, cells were more MSC-like in morphology and in expression of surface markers (positive for CD29, CD44, and Sca-1 and negative for CD11b, CD19, and CD45). These data provide new insight into optimizing the culture method and understanding the biological characteristics of mouse BM-MSCs during in vitro expansion.

Disruption of Intracellular ATP Generation and Tight Junction Protein Expression during the Course of Brain Edema Induced by Subacute Poisoning of 1,2-Dichloroethane.

The aim of this study was to explore changes in intracellular ATP generation and tight junction protein expression during the course of brain edema induced by subacute poisoning of 1,2-dichloroethane (1,2-DCE). Mice were exposed to 1.2 g/m3 1,2-DCE for 3.5 h per day for 1, 2, or 3 days, namely group A, B, and C. Na+-K+-ATPase and Ca2+-ATPase activity, ATP and lactic acid content, intracellular free Ca2+ concentration and ZO-1 and occludin expression in the brain were measured. Results of present study disclosed that Ca2+-ATPase activities in group B and C, and Na+/K+-ATPase activity in group C decreased, whereas intracellular free Ca2+ concentrations in group B and C increased significantly compared with control. Moreover, ATP content decreased, whereas lactic acid content increased significantly in group C compared with control. On the other hand, expressions of ZO-1 and occludin at both the protein and gene levels in group B and C decreased significantly compared with control. In conclusion, findings from this study suggest that calcium overload and depressed expression of tight junction associated proteins, such as ZO-1 and occludin might play an important role in the early phase of brain edema formation induced by subacute poisoning of 1,2-DCE.

Strain differences in arsenic-induced oxidative lesion via arsenic biomethylation between C57BL/6J and 129X1/SvJ mice.

Arsenic is a common environmental and occupational toxicant with dramatic species differences in its susceptibility and metabolism. Mouse strain variability may provide a better understanding of the arsenic pathological profile but is largely unknown. Here we investigated oxidative lesion induced by acute arsenic exposure in the two frequently used mouse strains C57BL/6J and 129X1/SvJ in classical gene targeting technique. A dose of 5 mg/kg body weight arsenic led to a significant alteration of blood glutathione towards oxidized redox potential and increased hepatic malondialdehyde content in C57BL/6J mice, but not in 129X1/SvJ mice. Hepatic antioxidant enzymes were induced by arsenic in transcription in both strains and many were higher in C57BL/6J than 129X1/SvJ mice. Arsenic profiles in the liver, blood and urine and transcription of genes encoding enzymes involved in arsenic biomethylation all indicate a higher arsenic methylation capacity, which contributes to a faster hepatic arsenic excretion, in 129X1/SvJ mice than C57BL/6J mice. Taken together, C57BL/6J mice are more susceptible to oxidative hepatic injury compared with 129X1/SvJ mice after acute arsenic exposure, which is closely associated with arsenic methylation pattern of the two strains.

Roles of CYP2e1 in 1,2-dichloroethane-induced liver damage in mice.

The aim of this study was to explore the roles of cytochrome P450 2E1 (CYP2E1) in 1,2-dichloroethane (1,2-DCE)-induced liver damage. Two parts were included in this study: first, effect of 1,2-DCE on microsomal expression of CYP2E1, and second, potential of an inhibitor of CYP2E1 to reduce 1,2-DCE-induced liver damage. In part one, mice were exposed to 0, 0.225, 0.45, or 0.9 g/m3 1,2-DCE for 10 days, 3.5 h per day through static inhalation. In part two, mice were divided into blank control, solvent control, inhibitor control, 1,2-DCE-poisoned group, and low or high intervention group. In part one, compared to the control, serum alanine aminotransferase (ALT) activities and hepatic malondialdehyde (MDA) levels in 0.9 g/m3 1,2-DCE group, and microsomal CYP2E1 protein expression and activity in both 0.45 and 0.9 g/m3 1,2-DCE groups increased significantly; conversely, hepatic nonprotein sulfhydryl (NPSH) levels in both 0.45 and 0.9 g/m3 1,2-DCE groups and hepatic SOD activities in 0.9 g/m3 1,2-DCE group decreased significantly. In part two, microsomal CYP2E1 protein expression and activity decreased significantly in both low and high intervention groups compared to 1,2-DCE-poisoned group. Along with the changes of CYP2E1, hepatic MDA levels and serum ALT activities decreased; conversely, hepatic NPSH levels and SOD activities increased significantly in high intervention group. Taken together, our results suggested that 1,2-DCE could enhance CYP2E1 protein expression and enzymatic activity, which could cause oxidative damage in liver, serving as an important mechanism underlying 1,2-DCE-induced liver damage. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1430-1438, 2016.

Excitotoxicity Induced by Realgar in the Rat Hippocampus: the Involvement of Learning Memory Injury, Dysfunction of Glutamate Metabolism and NMDA Receptors.

Realgar is a type of mineral drug containing arsenic. The nervous system toxicity of realgar has received extensive attention. However, the underlying mechanisms of realgar-induced neurotoxicity have not been clearly elucidated. To explore the mechanisms that contribute to realgar-induced neurotoxicity, weanling rats were exposed to realgar (0, 0.3, 0.9, 2.7 g/kg) for 6 weeks, and cognitive ability was tested using the Morris water maze (MWM) test and object recognition task (ORT). The levels of arsenic in the blood and hippocampus were monitored. The ultrastructures of hippocampal neurons were observed. The levels of glutamate (Glu) and glutamine (Gln) in the hippocampus and hippocampal CA1 region; the activities of glutamine synthetase (GS) and phosphate-activated glutaminase (PAG); the mRNA and protein expression of glutamate transporter 1 (GLT-1), glutamate/aspartate transporter (GLAST), and N-methyl-D-aspartate (NMDA) receptors; and the level of intracellular Ca(2+) were also investigated. The results indicate that the rats developed deficiencies in cognitive ability after a 6-week exposure to realgar. The arsenic contained in realgar and the arsenic metabolites passed through the blood-brain barrier (BBB) and accumulated in the hippocampus, which resulted in the excessive accumulation of Glu in the extracellular space. The excessive accumulation of Glu in the extracellular space induced excitotoxicity, which was shown by enhanced GS and PAG activities, inhibition of GLT-1 mRNA and protein expression, alterations in NMDA receptor mRNA and protein expression, disturbance of intracellular Ca(2+) homeostasis, and ultrastructural changes in hippocampal neurons. In conclusion, the findings from our study indicate that exposure to realgar induces excitotoxicity and that the mechanism by which this occurs may be associated with disturbances in Glu metabolism and transportation and alterations in NMDA receptor expression.

Effects of lead exposure on D-serine metabolism in the hippocampus of mice at the early developmental stages.

The aim of this study was to explore the mechanisms of lead neurotoxicity by focusing on the alteration of D-serine metabolism in the hippocampus of mice at the early life. Mother mice and their offspring were exposed to 0, 0.5, 1.0 and 2.0 g/L lead in lead acetate via drinking water from the first day of gestation until the postnatal day (PND) 40. Morris water maze was used to measure the spatial learning and memory ability of PND 40 mice. Expressions of serine racemase (SR), D-amino acid oxidase (DAAO), alanine-serine- cysteine transporter-1 (asc-1) and subunits of N-methyl-D-aspartate receptor (NMDAR) in the hippocampus of PND 10, 20 and 40 mice were examined by western blot and real time RT-PCR. Findings from this study disclosed that the spatial learning ability of mice tested by place trial could be significantly impaired by 0.5 g/L lead exposure, and the spatial memory ability tested by probe trail could be impaired by 1.0 g/L lead exposure. Exposure to 2.0 g/L lead in the water could significantly inhibit the protein and mRNA expression of SR; conversely enhance the expression of DAAO protein and mRNA in the hippocampus during the early developmental stages. However, the protein expressions of DAAO and asc-1 in the hippocampus were significantly enhanced by 0.5 g/L lead exposure at different developmental stages. On the other hand, the protein and mRNA expressions of both NR1 and NR2A were inhibited significantly by 1.0 g/L lead exposure since PND 10, and by 0.5 g/L lead exposure since PND 20. Noteworthy, the protein expression of NR2B was inhibited significantly by 0.5 g/L lead exposure in PND 10 mice, and by 1.0 g/L lead exposure in PND 20 mice, but there was no significant group difference in PND 40 mice. Meanwhile, expressions of asc-1 and NR2B mRNA were not affected obviously by lead exposure. In conclusion, chronic lead exposure during brain development might affect D-serine metabolism by enhancing its degradation, which might be related to the inhibited expression of NMDAR subunits, and furthermore contribute to deficits in learning and memory ability in mice.

Roles of aquaporins and matrix metalloproteinases in mouse brain edema formation induced by subacute exposure to 1,2-dichloroethane.

The aim of this study was to explore the effects of 1,2-dichloroethane (1,2-DCE) on expression of aquaporins (AQPs) and matrix metalloproteinases (MMPs) in the process of brain edema formation. Two parts were included in this study, establishment of animal model of brain edema, and mechanism of brain edema induced by subacute exposure to 1,2-DCE. In part one, mice were exposed to 0, 1.1, 1.2 or 1.3g/m(3) 1,2-DCE, 3.5h per day for 3days. Pathological analysis and water content detection in the brain were examined. In part two, mice were exposed to 1.2g/m(3) 1,2-DCE, 3.5h per day for 1, 2 or 3days, named group D, E and F, respectively. Expression of AQP4, MMP2 and MMP9 in the brain was determined by immunochemical staining, western blot and real time PCR. According to the results of part one, the 1.2g/m(3) dose was chosen for part two, a follow-up time-course study. In part two, protein expression of MMP2 and MMP9 in group F, and AQP4 in group E and F significantly increased compared to the control. Similarly, mRNA levels of AQP4 in group F, and MMP9 in group E and F significantly increased. Our results suggested that exposure to 1,2-DCE might up-regulate the expression of AQP4 protein and MMP9 mRNA at the early phase of brain edema, and AQP4 may play an important role in the brain edema formation.

[Influence of 1, 2-dichloroethane on open field behavior and levels of neurotransmitters in brain of mice].

OBJECTIVE: To explore the effects of 1,2-dichloroethane (1,2-DCE) on the behavior and the brain neurotransmitter levels in mice. METHODS: Thirty mice were randomly divided into four groups, which were control group and groups of low, middle and high exposure (225, 450 and 900 mg/m3) to 1,2-DCE for 10 days (3.5 h a day) by inhalation. After the last exposure, the open field test was performed immediately. After exposure all mice were killed and the brain tissues were taken up rapidly. The levels of aspartate (Asp), glutamate (Glu) and gamma-aminobutyric acid (GABA) in the brain were detected by high performance liquid chromatography (HPLC). RESULTS: Levels of Asp and Glu in all exposure groups increased with doses. As compared to the control group, levels of Glu in all exposure groups increased significantly (P < 0.05). Levels of GABA in the low exposure group were significantly lower than those in control group, but those in the high exposure group were significantly higher than those in control group. The results of the open field test showed that effect of low exposure to 1,2-DCE on the behavior was stimulant, but the high exposure to 1,2-DCE inhibited behavior of exploration, excitement and sport. CONCLUSIONS: Subacute exposure to 1,2-DCE could result in the change of amino acid neurotransmitter content and ratio in the brain, thereby change the behavior of mice appeared, which might be the mechanism of neurotoxicity caused by 1,2-DCE in part.