Lead acetate induces cartilage defects and bone loss in zebrafish embryos by disrupting the GH/IGF-1 axis.

Skeletal system toxicity due to lead exposure has attracted extensive attention in recent years, but few studies focus on the skeletal toxicity of lead in the early life stages of zebrafish. The endocrine system, especially the GH/IGF-1 axis, plays an important role in bone development and bone health of zebrafish in the early life. In the present study, we investigated whether lead acetate (PbAc) affected the GH/IGF-1 axis, thereby causing skeletal toxicity in zebrafish embryos. Zebrafish embryos were exposed to lead PbAc between 2 and 120 h post fertilization (hpf). At 120 hpf, we measured developmental indices, such as survival, deformity, heart rate, and body length, and assessed skeletal development by Alcian Blue and Alizarin Red staining and the expression levels of bone-related genes. The levels of GH and IGF-1 and the expression levels of GH/IGF-1 axis-related genes were also detected. Our data showed that the LC50 of PbAc for 120 h was 41 mg/L. Compared with the control group (0 mg/L PbAc), after PbAc exposure, the deformity rate increased, the heart rate decreased, and the body length was shortened at various time periods, in the 20-mg/L group at 120 hpf, the deformity rate increased by 50 fold, the heart rate decreased by 34%, and the body length shortened by 17%. PbAc altered cartilage structures and exacerbated bone loss in zebrafish embryos; in addition, PbAc exposure down-regulated the expression of chondrocyte (sox9a, sox9b), osteoblast (bmp2, runx2) and bone mineralization-related genes (sparc, bglap), and up-regulated the expression of osteoclast marker genes (rankl, mcsf). The GH level increased and the IGF-1 level declined significantly. The GH/IGF-1 axis related genes (ghra, ghrb, igf1ra, igf1rb, igf2r, igfbp2a, igfbp3, igfbp5b) were all decreased. These results suggested that PbAc inhibited the differentiation and maturation of osteoblasts and cartilage matrix, promoted the formation of osteoclasts, and ultimately induced cartilage defects and bone loss by disrupting the GH/IGF-1 axis.

[Meta-analysis of the relationship between maternal arsenic exposure and preterm birth].

OBJECTIVE: To systematic evaluate the association between maternal arsenic exposure and preterm birth. METHODS: A literature search was conducted through Pubmed, Web of Science, Embase, China Knowledge Network(CNKI), WanFang Data, Vip Chinese Journal Service Platform(VIP) with a time frame of November 2022 from the beginning of database construction. Meta-analysis of dichotomous variables was performed using Stata MP15 software, and a random or fixed effects model was chosen for the meta-analysis based on the heterogeneity result, with the ratio of ratios(OR) as the effect indicator; subgroup analysis was used to find characteristic changes; funnel plots were used to evaluate publication bias. RESULTS: A total of 15 papers with a sample size of(n=9 892 256 279), 10 prospective cohort studies, 3 retrospective cohort studies, and 2 cross-sectional studies, were included in 6 Chinese and 9 English papers. By Meta-analysis, the combined OR of preterm birth outcome was 1.06(95%CI 1.03-1.09); the result of subgroup analysis by exposure factors and region, the combined OR(95%CI) of hair, blood, urine, drinking water, and placenta were 0.97(0.56-1.69), 1.40(1.22-1.60), 1.04(0.93-1.17), 1.14(1.04-1.24) and 0.69(0.07-6.38). The combined OR(95%CI) were 1.17(1.04-1.31), 1.10(1.05-1.14), 0.69(0.07-6.38) and 1.17(1.01-1.36) for Asia, Americas, Europe and Africa, respectively. For subgroup analysis based on study type, the combined OR(95%CI) was 1.16(1.05-1.28), 1.01(1.01-1.02) and 1.65(0.73-3.74) for prospective cohort studies, retrospective cohort studies, and cross-sectional studies, respectively. CONCLUSION: Maternal arsenic exposure may contribute to the occurrence of preterm birth, and drinking water arsenic levels may be an important indicator for assessing human arsenic exposure and risk of causing preterm birth.

MicroRNA-191 blocking the translocation of GLUT4 is involved in arsenite-induced hepatic insulin resistance through inhibiting the IRS1/AKT pathway.

Environmental exposure to arsenic can cause a variety of health problems. Epidemiological and experimental studies have established a diabetogenic role for arsenic, but the mechanisms responsible for arsenic-induced impairment of insulin action are unclear. MicroRNAs (miRNAs) are involved in various metabolic disorders, particularly in the development of insulin resistance. The present study investigated whether arsenite, an active form of arsenic, induces hepatic insulin resistance and the mechanisms underlying it. After male C57BL/6J mice were exposed to arsenite (0 or 20 ppm) in drinking water for 12 months, intraperitoneal glucose tolerance tests (IPGTTs) and insulin tolerance tests (ITTs) revealed an arsenite-induced glucose metabolism disorder. Hepatic glycogen levels were lower in arsenite-exposed mice. Further, for livers of mice exposed to arsenite, miR-191 levels were higher, and protein levels of insulin receptor substrate 1 (IRS1), p-IRS1, and phospho-protein kinase B (p-AKT) were lower. Further, glucose transporter 4 (GLUT4) had lower levels on the plasma membrane. For insulin-treated L-02 cells, arsenite decreased glucose consumption and glycogen levels, increased miR-191 levels, and inhibited the IRS1/AKT pathway and the translocation of GLUT4 from the cytoplasm to the plasma membrane. For insulin-treated L-02 cells, the decreases of glucose consumption, glycogen levels, GLUT4 on the plasma membrane, and p-AKT levels induced by arsenite were reversed by SC79 (agonist of AKT) and an miR-191 inhibitor; these effects caused by miR-191 inhibitor were restored by IRS1 siRNA. In insulin-treated L-02 cells, miR-191, via IRS1, was involved in the arsenite-induced decreases of glucose consumption and glycogen levels and in inhibition of the translocation of GLUT4. Thus, miR-191 blocking the translocation of GLUT4 was involved in arsenite-induced hepatic insulin resistance through inhibiting the IRS1/AKT pathway. Our study reveals a mechanism for arsenite-induced hepatic insulin resistance, which provides clues for discovering biomarkers for the development of type 2 diabetes and for prevention and treatment of arsenic poisoning.

Role of GH/IGF axis in arsenite-induced developmental toxicity in zebrafish embryos.

Growth hormone (GH)/insulin-like growth factor (IGF) axis plays a critical role in fetal development. However, the effect of arsenite exposure on the GH/IGF axis and its toxic mechanism are still unclear. Zebrafish embryos were exposed to a range of NaAsO2 concentrations (0.0-10.0 mM) between 4 and 120 h post-fertilization (hpf). Development indexes of survival, malformation, hatching rate, heart rate, body length and locomotor behavior were measured. Hormone levels, GH/IGF axis-related genes, and nerve-related genes were also tested. The results showed that survival rate, hatching rate, heart rate, body length and locomotor behavior all decreased, while deformity increased. At 120 hpf, the survival rate of zebrafish in 1.5 mM NaAsO2 group was about 70%, the deformity rate exceeded 20%, and the body length shortened to 3.35 mm, the movement distance of zebrafish decreased approximately 63.6% under light condition and about 52.4% under dark condition. The level of GH increased and those of IGF did not change significantly, while the expression of GH/IGF axis related genes (ghra, ghrb, igf2r, igfbp3, igfbp2a, igfbp5b) and nerve related genes (dlx2, shha, ngn1, elavl3, gfap) decreased. In 1.5 mM NaAsO2 group, the decrease of igfbp3 and igfbp5b was almost obvious, about 78.2% and 72.2%. The expression of nerve genes in 1.5 mM NaAsO2 group all have declined by more than 50%. These findings suggested that arsenite exerted disruptive effects on the endocrine system by interfering with the GH/IGF axis, leading to zebrafish embryonic developmental toxicity.

Role of mitochondrial DNA in oxidative damage induced by sodium arsenite in human bronchial epithelial cells.

Long-term exposure to sodium arsenite was found to induce malignant transformation in human bronchial epithelial (HBE) cell line as evidenced by elevated ROS levels. Although chronic sodium arsenite-induced HBE cell line transformation was associated with elevated ROS generation, it was of interest to determine whether acute sodium arsenite exposure also initiated pulmonary damage. Thus, the aim of this study was to investigate oxidative-stress-related pulmonary damage using a human bronchial epithelial (HBE) cell line. Incubation of ρ+-HBE (in the presence of mitochondrial DNA) cells with various concentrations of sodium arsenite, significantly increased ROS and MDA levels accompanied by decreased SOD activity in a concentration-dependent manner. In contrast, treatment of ρ-HBE (without mitochondrial DNA) cells various concentrations of sodium arsenite a reduction in ROS and MDA levels were noted. However, the SOD activity remained decreased in ρ-HBE cells. This was accompanied by a significant rise in HO-1 protein expressions levels in both cell types with greater changes ρ-HBE cells at the lower sodium arsenite concentrations. Data indicate that acute sodium arsenite exposure exerted a greater effect ρ-HBE cells suggesting that absence of mitochondrial DNA appears to enhance sensitivity to the oxidant actions of inorganic As.

MALAT1 via microRNA-17 regulation of insulin transcription is involved in the dysfunction of pancreatic ß-cells induced by cigarette smoke extract.

Cigarettes contain various chemicals with the potential to influence metabolic health. Exposure to cigarette smoke causes a dysfunction in pancreatic ß-cells and impairs insulin production. However, the mechanisms for cigarette smoke-induced reduction of insulin remain largely unclear. Data from 558 patients with diabetes showed that, with smoking pack-years, homeostatic model assessment (HOMA)-ß (a method for assessing ß-cell function) decreased and that HOMA of insulin resistance increased. For ß-cells (MIN6), cigarette smoke extract (CSE) increased the levels of thioredoxin-interacting protein (TXNIP) and the long noncoding (lnc)RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), and downregulated the levels of the transcription factor, mafA, and microRNA (miR)-17. MALAT1, one of four lncRNAs predicted to regulate miR-17, was knocked down by small interfering RNA (siRNA). For these cells, an miR-17 mimic inhibited TXNIP and enhanced the production of insulin. Knockdown of MALAT1 induced an increase in miR-17, which suppressed TXNIP and promoted the production of insulin. In the sera of patients with diabetes who smoked, there were higher MALAT1 levels and lower miR-17 levels than in the sera of nonsmokers. Thus, CSE inhibits insulin production by upregulating TXNIP via MALAT1-mediated downregulation of miR-17, which provides an understanding of the processes involved in the reduced ß-cells function caused by cigarette smoke.

NF-κB-regulated miR-155, via repression of QKI, contributes to the acquisition of CSC-like phenotype during the neoplastic transformation of hepatic cells induced by arsenite.

Chronic exposure to arsenite can cause various human tumors. For the initiation and recurrence of human liver cancer, the acquisition of CSC-like properties is essential. In various cancers, microRNAs (miRNAs) act as regulators in induction of CSC-like properties. Liver cancers over-express miR-155, but the mechanism relating miR-155 and arsenite-induced liver cancer is unknown. Here, we show that long-term exposure of L-02 cells to arsenite increases miR-155 levels by activation of NF-κB and leads to the acquisition of CSC-like properties. In spheroids formed from arsenite-transformed L-02 cells, the levels of miR-155 positively relate to the levels of CD90, EpCAM, and OCT4. Inhibition of miR-155, by reduction of SOX2 and OCT4, results in suppression of spheroid formation. Luciferase reporter assays indicate that QKI is a target of miR-155. Inhibition of QKI expression by miR-155 promotes arsenite-induced acquisition of CSC-like properties, whereas QKI over-expression has the opposite effect. Collectively, the findings demonstrate that miR-155, driven by NF-κB, reduces QKI expression and is involved in acquisition of the CSC-like phenotype during neoplastic transformation of hepatic cells induced by arsenite.

Circ100284, via miR-217 regulation of EZH2, is involved in the arsenite-accelerated cell cycle of human keratinocytes in carcinogenesis.

Circular RNAs (circRNAs), a class of noncoding RNAs generated from pre-mRNAs, participate in regulation of genes. The mechanism for regulation, however, is unknown. Here, to determine if, in human keratinocyte (HaCaT) cells, circular RNAs are involved in arsenite-induced acceleration of the cell cycle, a circRNA microarray was performed to analyze the variability of circRNAs in arsenite-treated HaCaT (As-HaCaT) cells and in arsenite-transformed (T-HaCaT) cells in comparison to control HaCaT cells. Among the circRNAs up-regulated in both As-HaCaT cells and T-HaCaT cells, hsa:circRNA_100284 (circ100284) had the greatest increase and was chosen for further research. The presence of circ100284 was confirmed in HaCaT cells. In these cells, arsenite induced increases of EZH2 and cyclin D1 and accelerated the cell cycle. MicroRNA (miR)-217 suppressed the expression of EZH2 was involved in regulation of the cell cycle. Further, in HaCaT cells exposed to arsenite, EZH2 regulated the cell cycle by binding to the promoter of CCND1, which codes for cyclin D1. Moreover, knockdown of circ100284 with siRNA inhibited the cell cycle acceleration induced by arsenite, but this inhibition was reversed by co-transfection with circ100284 siRNA and by a miR-217 inhibitor. Knockdown of circ100284 with siRNA or transfected with miR-217 mimic inhibited the capacity of T-HaCaT cells for colony formation, invasion, and migration, effects that were reversed by co-transfection with a miR-217 inhibitor or by epigenetic expression of EZH2. These results suggest that, in HaCaT cells, arsenite increases circ100284 levels, which act as a sponge for miR-217 and up-regulate the miR-217 target, EZH2, which, in turn, up-regulates cyclin D1and CDK4, and thus accelerates the cell cycle and leads to malignant transformation. Thus, circ100284, via miR-217 regulation of EZH2, is involved in the arsenite-accelerated cell cycle of human keratinocytes in carcinogenesis. This establishes a previously unknown mechanism between arsenite-induced acceleration of the cell cycle and carcinogenesis.

Impaired autophagic flux and p62-mediated EMT are involved in arsenite-induced transformation of L-02 cells.

Autophagy is a catabolic process essential for preserving cellular homeostasis, and the epithelial-to-mesenchymal transition (EMT) is involved during tissue development and cancer progression. In arsenite-treated human hepatic epithelial (L-02) cells, arsenite reduced the autophagic flux, which caused accumulation of p62, an adaptor and receptor of autophagy. Further, in arsenite-transformed L-02 cells, the levels of E-cadherin were attenuated, but the levels of vimentin, which is expressed in mesenchymal cells, and Snail, a transcription regulator of the EMT, were up-regulated. Thus, after chronic exposure of L-02 cells to arsenite, the impaired autophagic flux induced the accumulation of p62, which up-regulated the expression of Snail, a protein involved in arsenite-induced EMT of these cells. Knockdown of p62 by siRNA reversed the arsenite-induced EMT and decreased the capacities of arsenite-transformed L-02 cells for colony formation and invasion and migration. Therefore, in arsenite-induced transformation of L-02 cells, the accumulation of p62, by impairing autophagic flux, mediates the EMT via Snail. These results provide a previously unknown mechanism underlying arsenic toxicity and carcinogenicity.

Epigenetic silencing of miR-218 by the lncRNA CCAT1, acting via BMI1, promotes an altered cell cycle transition in the malignant transformation of HBE cells induced by cigarette smoke extract.

Cigarette smoking is the strongest risk factor for the development of lung cancer, the leading cause of cancer-related deaths. However, the molecular mechanisms leading to lung cancer are largely unknown. A long-noncoding RNA (lncRNA), CCAT1, regarded as cancer-associated, has been investigated extensively. Moreover, the molecular mechanisms of lncRNAs in regulation of microRNAs (miRNAs) induced by cigarette smoke remain unclear. In the present investigation, cigarette smoke extract (CSE) caused an altered cell cycle and increased CCAT1 levels and decreased miR-218 levels in human bronchial epithelial (HBE) cells. Depletion of CCAT1 attenuated the CSE-induced decreases of miR-218 levels, suggesting that miR-218 is negatively regulated by CCAT1 in HBE cells exposed to CSE. The CSE-induced increases of BMI1 levels and blocked by CCAT1 siRNA were attenuated by an miR-218 inhibitor. Moreover, in CSE-transformed HBE cells, the CSE-induced cell cycle changes and elevated neoplastic capacity were reversed by CCAT1 siRNA or BMI1 siRNA. This epigenetic silencing of miR-218 by CCAT1 induces an altered cell cycle transition through BMI1 and provides a new mechanism for CSE-induced lung carcinogenesis.

'Environmental standard limit concentration' arsenic exposure is associated with anxiety, depression, and autism-like changes in early-life stage zebrafish.

Arsenic is a worldwide environmental pollutant that can impair human health. Previous studies have identified mental disorders induced by arsenic, but the environmental exposure concentrations in the early life stages associated with these disorders are poorly understood. In the present study, early-life stage zebrafish were used to explore the effects on mental disorders under 'environmental standard limit concentrations' arsenic exposures of 5, 10, 50, 150, and 500 µg/L. The results showed that arsenic exposure at these concentrations changed the locomotor behavior in larval zebrafish and was further associated with anxiety, depression, and autism-like behavior in both larval and juvenile zebrafish. Changes were noted at benchmark dose limit (BMDL) concentrations as low as 0.81 µg/L. Transcriptomics showed that immediate early genes (IEGs) fosab, egr1, egr2a, ier2b, egr3, and jund were decreased after arsenic exposure in larval and juvenile zebrafish. Nervous system impairment and anxiety, depression, and autism-like behaviors in early-life stage zebrafish at 'environmental standard limit concentrations' may be attributed to the downregulation of IEGs. These findings in zebrafish provided new experimental support for an arsenic toxicity threshold for mental disorders, and they suggest that low levels of environmental chemicals may be causative developmental factors for mental disorders.

Sustained high expression of NRF2 inhibits cell apoptosis in arsenite-transformed human keratinocytes.

Our previous study identified that nuclear factor-erythroid-2 p45-related factor 2 (NRF2) was activated in arsenite-induced tumorigenesis. However, the underlying mechanisms of NRF2 mediating apoptosis in arsenic-induced skin carcinogenesis remain unknown. This study explored the dynamic changes in apoptosis rate and the expression of apoptosis proteins in immortalized human keratinocytes (HaCaT) malignant transformation caused by 1.0 µM NaAsO2 at passages 0, 1, 7, 14, 21, 28, and 35. The result showed that the apoptosis rate decreased. The apoptosis-related proteins cleaved-caspase-3/caspase-3 ratio decreased in the later stages (passages 21, 28, and 35). Moreover, the expression of intrinsic ER stress pathway-related CHOP, ATF4, ATF6, and the intrinsic mitochondrial pathway-related Bax protein decreased in the later stages, while Bcl-2 and Mcl-1 increased, and NRF2 protein levels also increased. The apoptosis rate increased by silencing NRF2 expression in arsenite-transformed HaCaT (T-HaCaT) cells. Meanwhile, the expression of pro-apoptotic proteins (cleaved-caspase-3/caspase-3, CHOP, Bax) and ATF4, ATF6 increased. On the contrary, antiapoptotic protein levels (Bcl-2 and Mcl-1) decreased. The ability of colony formation and migration of T-HaCaT cells decreased. In conclusion, arsenite activated NRF2 in the later stages, decreasing apoptosis characterized by inhibiting endoplasmic reticulum stress-depended and mitochondria-depended apoptosis pathway, and further promoting NaAsO2-induced HaCaT cellular malignant transformation.

Reductive stress induced by NRF2/G6PD through glucose metabolic reprogramming promotes malignant transformation in Arsenite-exposed human keratinocytes.

Our previous research found that the nuclear factor-E2-related factor 2 (NRF2) protein was sustained activated in malignant transformation of human keratinocyte (HaCaT cells) caused by NaAsO2, but the role of NRF2 in it remains unknown. In this study, malignant transformation of HaCaT cells and labeled HaCaT cells used to detect mitochondrial glutathione levels (Mito-Grx1-roGFP2 HaCaT cells) were induced by 1.0 µM NaAsO2. Redox levels were measured at passages 0, early stage (passages 1, 7, 14), later stage (passages 21, 28 and 35) of arsenite-treated HaCaT cells. Oxidative stress levels increased at early stage. The NRF2 pathway was sustained activated. Cells and mitochondrial reductive stress levels (GSH/GSSG and NADPH/NADP+) increased. The mitochondrial GSH/GSSG levels of Mito-Grx1-roGFP2 HaCaT cells also increased. The indicators of glucose metabolism glucose-6-phosphate, lactate and the glucose-6-phosphate dehydrogenase (G6PD) levels increased, however Acetyl-CoA level decreased. Expression levels of glucose metabolic enzymes increased. After transfection with NRF2 siRNA, the indicators of glucose metabolism were reversed. After transfection with NRF2 or G6PD siRNA, cells and mitochondrial reductive stress levels decreased and the malignant phenotype was reversed. In conclusion, oxidative stress occurred in the early stage and the NRF2 was sustained high expression. In the later stage, increased NRF2/G6PD through glucose metabolic reprogramming induced reductive stress, thereby leading to malignant transformation.

Melatonin Prevents NaAsO2-Induced Developmental Cardiotoxicity in Zebrafish through Regulating Oxidative Stress and Apoptosis.

Melatonin is an indoleamine hormone secreted by the pineal gland. It has antioxidation and anti-apoptosis effects and a clear protective effect against cardiovascular diseases. Our previous studies demonstrated that embryonic exposure to sodium arsenite (NaAsO2) can lead to an abnormal cardiac development. The aim of this study was to determine whether melatonin could protect against NaAsO2-induced generation of reactive oxygen species (ROS), oxidative stress, apoptosis, and abnormal cardiac development in a zebrafish (Danio rerio) model. We found that melatonin decreased NaAsO2-induced zebrafish embryonic heart malformations and abnormal heart rates at a melatonin concentration as low as 10-9 mol/L. The NaAsO2-induced oxidative stress was counteracted by melatonin supplementation. Melatonin blunted the NaAsO2-induced overproduction of ROS, the upregulation of oxidative stress-related genes (sod2, cat, gpx, nrf2, ho-1), and the production of antioxidant enzymes (Total SOD, SOD1, SOD2, CAT). Melatonin attenuated the NaAsO2-induced oxidative damage, DNA damage, and apoptosis, based on malonaldehyde and 8-OHdG levels and apoptosis-related gene expression (caspase-3, bax, bcl-2), respectively. Melatonin also maintained the control levels of heart development-related genes (nkx2.5, sox9b) affected by NaAsO2. In conclusion, melatonin protected against NaAsO2-induced heart malformations by inhibiting the oxidative stress and apoptosis in zebrafish.

The Potential Key Role of the NRF2/NQO1 Pathway in the Health Effects of Arsenic Pollution on SCC.

Arsenic is widely present in nature and is a common environmental poison that seriously damages human health. Chronic exposure to arsenic is a major environmental poisoning factor that promotes cell proliferation and leads to malignant transformation. However, its molecular mechanism remains unclear. In this study, we found that arsenite can promote the transformation of immortalized human keratinocyte cells (HaCaT) from the G0/G1 phase to S phase and demonstrated malignant phenotypes. This phenomenon is accompanied by obviously elevated levels of NRF2, NQO1, Cyclin E, and Cyclin-dependent kinase 2 (CDK2). Silencing the NRF2 expression with small interfering RNA (siRNA) in arsenite-transformed (T-HaCaT) cells was shown to reverse the malignant phenotype. Furthermore, the siRNA silencing of NQO1 significantly decreased the levels of the cyclin E-CDK2 complex, inhibiting the G0/G1 to S phase cell cycle progression and transformation to the T-HaCaT phenotypes. Thus, we hypothesized that the NRF2/NQO1 pathway played a key role in the arsenite-induced malignancy of HaCaT cells. By increasing the expression of Cyclin E-CDK2, the NRF2/NQO1 pathway can affect cell cycle progression and cell proliferation. A new common health effect mechanism of arsenic carcinogenesis has been identified; thus, it would contribute to the development of novel treatments to prevent and treat skin cancer caused by arsenic.

Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae.

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the specific effects and mechanisms in zebrafish remain unclear. Alizarin red has a high affinity for calcium ions and can help visualize the bone and illustrate skeletal mineral mass. In this study, we aimed to detect lead acetate (PbAc)-induced bone loss in zebrafish larvae by using alizarin red staining. Zebrafish embryos were treated with a series of PbAc concentrations (0, 5, 10, 20 mg/L) between 2 and 120 h post fertilization. Whole-mount skeletal staining was conducted on larvae at 9 days post fertilization, and the total stained area was quantified using ImageJ software. The results indicated that the mineralized tissues were stained in red, and the stained area decreased significantly in the PbAc-exposure group, with a dose-dependent change in bone mineralization. This paper presents a staining protocol for investigating skeletal changes in PbAc-induced bone defects. The method can also be used in zebrafish larvae for the detection of bone loss induced by other chemicals.

Possible differences in the mechanism of malignant transformation of HaCaT cells by arsenite and its dimethyl metabolites, particularly dimethylthioarsenics.

BACKGROUND: As a confirmed human carcinogen, arsenic can cause skin cancer, lung cancer, etc. However, its carcinogenic mechanism is still unclear. In recent years, the oxidative stress hypothesis has become widely accepted. In mammals it has been found that arsenic can be converted to dimethylarsinous acid (DMAIII) and dimethylmonothioarsinic acid (DMMTAV) through a series of methylation and redox reactions. DMAIII and DMMTAV are highly toxic. METHODS: Human keratinocytes (HaCaT) were exposed to different concentrations of NaAsO2 (IAsIII), DMMTAV and DMAIII for 24 h. Reactive oxygen species (hydrogen peroxide and superoxide), oxidative damage markers (8-hydroxydeoxyguanosine and malondialdehyde), and antioxidant markers (glutathione and superoxide dismutase) were measured. In addition, sulfane sulfurs were measured in HaCaT cells and a cell-free system. RESULTS: In the DMMTAV and DMAIII treatment groups, the levels of hydrogen peroxide and superoxide in HaCaT cells were higher than in the IAsIII treatment groups at the same dose. Levels of 8-OHdG and MDA in the DMMTAV and DMAIII treatment groups were also higher than those in the IAsIII treatment groups at the same dose. However, in the DMMTAV and DMAIII treatment groups, the levels of GSH and SOD activity were lower than that in the IAsIII treatment groups. In DMMTAV-treated HaCaT cells, sulfane sulfurs were produced. Further, it was found that DMMTAV could react with DMDTAV to form persulfide in the cell-free system, which may explain the mechanism of the formation of sulfane sulfurs in DMMTAV-treated HaCaT cells. CONCLUSIONS: DMMTAV and DMAIII more readily induce reactive oxygen species (ROS) and cause oxidative damage in HaCaT cells than inorganic arsenic. Further, the persulfide formed by the reaction of DMMTAV and DMDTAV produced from the metabolism of DMMTAV may induce a stronger reductive defense mechanism than GSH against the intracellular oxidative stress of DMMTAV. However, the cells exposed to arsenite are transformed by the continuous nuclear translocation of Nrf2 due to oxidative stress, and the persulfide from dimethylthioarsenics may promote Nrf2 by the combination with thiol groups, especially redox control key protein, Keap1, eventually cause nuclear translocation of sustained Nrf2.

High NF-E2-related factor 2 expression predicts poor prognosis in patients with lung cancer: a meta-analysis of cohort studies.

NF-E2-related factor 2 (Nrf2) plays an important role in tumour proliferation and chemoresistance. Nrf2 expression levels may be associated with prognosis of lung cancer, but previous results have been inconsistent. Pooled hazard ratios (HRs) and odds ratios were calculated to assess the prognostic value of the Nrf2 expression in this meta-analysis. Nine studies with 940 patients were included. A high Nrf2 expression level was significantly related to decreased overall survival (OS) (HR = 1.948, 95% CI = 1.564-2.427), lower response rate (HR = 2.675, 95% CI = 1.553-4.610), and poor progression-free survival (HR = 3.078, 95% CI = 1.791-5.293). Subgroup analysis demonstrated that high-Nrf2-expression was significantly correlated with worse OS of patients possessing epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) or undergoing chemotherapeutic treatments (HR = 2.500, 95% CI = 1.556-4.018). Conversely, this high expression was not significantly related to the OS of patients with surgical resection (HR = 1.750, 95% CI = 0.995-3.080, and p=.052). High Nrf2 expression was significantly correlated with worse OS of patients in advanced stage (HR = 2.500, 95% CI = 1.556-4.018), compared with early cancer stage (HR = 1.609, 95% CI = 0.675-3.835, and p=.283). This meta-analysis suggests that high Nrf2 expression may be a predictive factor of poor outcomes in lung cancer. Therefore, Nrf2 likely plays an important role in prognostic evaluation and may be a therapeutic target for EGFR-TKIs therapy and chemotherapy.

Long-term arsenite exposure decreases autophagy by increased release of Nrf2 in transformed human keratinocytes.

Autophagy dysfunction in arsenite toxicity plays critical roles in cancer development and progression. However, the precise mechanisms of arsenite-induced skin cancer by blocking autophagy remain uncertain. Herein, this study investigated molecular mechanisms of arsenite-induced autophagy dysfunction mediated by nuclear factor erythroid-2 related factor 2 (Nrf2) in human keratinocyte (HaCaT) cells. The effects of long-term arsenite exposure on Nrf2 activation and autophagy were established using a siRNA interference assay and western blots. A specific siRNA of Nrf2 was used to verify that autophagy induced by arsenite can be influenced by Nrf2. Specific inhibitors of PI3K (LY294002) and mTOR (Rapamycin) and siRNA of Nrf2 were employed to verify that upregulation of Nrf2 correlated with activating the PI3K/Akt pathway. Downstream mTOR and Bcl2 were upregulated by Nrf2 signaling, inhibiting autophagy initiation in arsenite-exposed HaCaT cells. In conclusion, our data suggest that long-term exposure to arsenite promotes Nrf2 upregulation via the PI3K/Akt pathway and, along with upregulation of downstream mTOR and Bcl2, contributes to autophagy dysfunction in transformed HaCaT cells. This work provides new insights into the mechanisms underlying arsenite-induced autophagy dysfunction in cancer promotion and malignancy progression.

miR-149 Negative Regulation of mafA Is Involved in the Arsenite-Induced Dysfunction of Insulin Synthesis and Secretion in Pancreatic Beta Cells.

Chronic exposure to arsenic, a potent environmental oxidative stressor, is associated with the incidence of diabetes. However, the mechanisms for arsenite-induced reduction of insulin remain largely unclear. After CD1 mice were treated with 20 or 40 ppm arsenite in the drinking water for 12 months, the mice showed reduced fasting insulin levels, a depression in glucose clearance, and lower insulin content in the pancreas. The levels of glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cells isolated from arsenite-exposed mice were low compared with those for control mice. Immunohistochemistry studies showed that arsenite exposure resulted a reduction of insulin content in the pancreas of mice. Exposure of Min6 cells, a pancreatic beta cell line, to low levels of arsenite led to lower GSIS in a dose- and time-dependent fashion. Since microRNAs (miRNAs) are involved in pancreatic ß-cell function and the pathogenesis of diabetes, we hypothesized that arsenite exposure activates miR-149, decreases insulin transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (mafA), and induces an insulin synthesis and secretion disorder. In arsenite-exposed Min6 cells, mafA activity was lowered by the increase of its target miRNA, miR-149. Luciferase assays illustrated an interaction between miR-149 and the mafA 3' untranslated region. In Min6 cells transfected with an miR-149 inhibitor, arsenite did not regulate GSIS and mafA expression. In control cells, however, arsenite decreased GSIS or mafA expression. Our results suggest that low levels of arsenite affect ß-cell function and regulate insulin synthesis and secretion by modulating mafA expression through miR-149.