Inorganic arsenic-mediated upregulation of TUG1 promotes apoptosis in human bronchial epithelial cells by activating the p53 signaling pathway.

Exposure to arsenic, an environmental contaminant, is known to cause arsenicosis and cancer. Although considerable research has been conducted to understand the underlying mechanism responsible for arsenic-induced cancers, the precise molecular mechanisms remain unknown, especially at the epigenetic regulation level. Long non-coding RNAs (LncRNAs) that have been shown to mediate various biological processes, including proliferation, apoptosis, necrosis, and mutagenesis. There are few studies on LncRNAs and biological damage caused by environmental pollutants. The LncRNAs taurine upregulated gene 1 (TUG1) regulates cell growth both in vitro and in vivo, and contributes its oncogenic role. However, the precise roles and related mechanisms of arsenic-induced cell apoptosis are still not fully understood owing to controversial findings in the literature. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed higher expression levels of TUG1 in people occupationally exposed to arsenic than in individuals living away from the source of arsenic exosure (N = 25). In addition, the results suggested that TUG1 was involved in arsenic-induced apoptosis. Furthermore, knockdown experiments showed that silencing of TUG1 markedly inhibited proliferation, whereas depletion of TUG1 led to increased apoptosis. The TUG1-small interfering RNA (siRNA) combination with arsenic (3 µM/L) slightly increased apoptosis compared with the TUG1-siRNA. Additionally, the knockdown experiments showed that the silencing of TUG1 by siRNA inhibited proliferation and promoted apoptosis by inducing p53, p-p53 (ser392), FAS, BCL2, MDM2, cleaved-caspase7 proteins in 16HBE cells. These results indicated that arsenic mediates the upregulation of TUG1 and induces cell apoptosis via activating the p53 signaling pathway.

As3MT is related to relative RNAs and base modifications of p53 in workers exposed to arsenic.

As3MT is the key enzyme involved in the methylation metabolism of arsenic. It is associated with DNA methylation closely also. This study is to explore the relationships between As3MT and epigenetic changes, and how p53 and relative ncRNAs and mRNAs play roles in the process. In this study, workers from four arsenic plants and individuals who resided in villages far away from the four plants were recruited. Arsenic compounds, relative indices, 28 relative RNAs, and base modifications of exons 5-8 of p53 were detected separately. Several methods were used to analyze the associations between them. Results shown that As3MT RNA was closely associated with all selected lncRNAs, miRNAs, and mRNAs related to miRNA production and maturation, tumorigenesis, and base modifications of p53. There probably exists causal relationship. Base modifications of exons 7 and 8 of p53 had significant synergistic effects on the expression of As3MT RNA and a series of genetic indices. But miR-190, miR-548, and base modifications of exon 5 of p53 had substantial inhibitory effects. Arsenic compounds and relative indices of metabolic transformation may have limited roles. The main novel finding in the present study is that As3MT play special and significant roles in the genotoxicity and carcinogenesis which could be coordinated operation with p53, and influenced by epigenetic factors largely, such as lncRNAs and miRNAs. P53 and relative ncRNAs and mRNAs may regulate the process by interacting with As3MT. The changes may initiate by arsenic, but probability through indirect relationship.

Involvement of a AS3MT/c-Fos/p53 signaling axis in arsenic-induced tumor in human lung cells.

Arsenite methyltransferase (AS3MT) is an enzyme that catalyzes the dimethylation of arsenite (+3 oxidation state). At present, the studies on arsenic carcinogenicity mainly focus on studying the polymorphisms of AS3MT and measuring their catalytic activities. We recently showed that AS3MT was overexpressed in lung cancer patients who had not been exposed to arsenic. However, little is known about the molecular mechanisms of AS3MT in arsenite-induced tumorigenesis. In this study, we showed that AS3MT protein expression was higher in the arsenic-exposed population compared to the unexposed population. AS3MT was also overexpressed in human lung adenocarcinoma (A549) and human bronchial epithelial (16HBE) cells exposed to arsenic (A549: 20-60 µmol/L; 16HBE: 2-6 µmol/L) for 48 h. Furthermore, we investigated the effects of AS3MT on cell proliferation and apoptosis using siRNA. The downregulation of AS3MT inhibited the proliferation and promoted the apoptosis of cells. Mechanistically, AS3MT was found to specifically bind to c-Fos, thereby inhibiting the binding of c-Fos to c-Jun. Additionally, the siRNA-mediated knockdown of AS3MT enhanced the phosphorylation of Ser392 in p53 by upregulating p38 MAPK expression. This led to the activation of p53 signaling and the upregulated expression of downstream targets, such as p21, Fas, PUMA, and Bax. Together, these studies revealed that the inorganic arsenic-mediated upregulation of AS3MT expression directly affected the proliferation and apoptosis of cells, leading to arsenic-induced toxicity or carcinogenicity.

Bio-Pulsed Stimulation Effectively Improves the Production of Avian Mesenchymal Stem Cell-Derived Extracellular Vesicles That Enhance the Bioactivity of Skin Fibroblasts and Hair Follicle Cells.

Mesenchymal stem cell (MSC)-derived extracellular vesicles (exosomes) possess regeneration, cell proliferation, wound healing, and anti-senescence capabilities. The functions of exosomes can be modified by preconditioning MSCs through treatment with bio-pulsed reagents (Polygonum multiflorum Thunb extract). However, the beneficial effects of bio-pulsed small extracellular vesicles (sEVs) on the skin or hair remain unknown. This study investigated the in vitro mechanistic basis through which bio-pulsed sEVs enhance the bioactivity of the skin fibroblasts and hair follicle cells. Avian-derived MSCs (AMSCs) were isolated, characterized, and bio-pulsed to produce AMSC-sEVs, which were isolated, lyophilized, characterized, and analyzed. The effects of bio-pulsed AMSC-sEVs on cell proliferation, wound healing, and gene expression associated with skin and hair bioactivity were examined using human skin fibroblasts (HSFs) and follicle dermal papilla cells (HFDPCs). Bio-pulsed treatment significantly enhanced sEVs production by possibly upregulating RAB27A expression in AMSCs. Bio-pulsed AMSC-sEVs contained more exosomal proteins and RNAs than the control. Bio-pulsed AMSC-sEVs significantly augmented cell proliferation, wound healing, and gene expression in HSFs and HFDPCs. The present study investigated the role of bio-pulsed AMSC-sEVs in the bioactivity of the skin fibroblasts and hair follicle cells as mediators to offer potential health benefits for skin and hair.

Relative miRNA and mRNA expression involved in arsenic methylation.

Three arsenic species in urine are measured using an atomic absorption spectrophotometer. RT-PCR is performed to detect the expression levels of AS3MT, 3 miRNAs, and 17 relative mRNAs in 43 workers producing arsenic trioxide, 36 workers who stopped exposure to arsenic for 85 days, and 24 individuals as the control group. The concentrations of urinary arsenic are very high in workers. A negative correlation between AS3MT and MiR-548c-3p is found. There exist significant changes for most selected miRNAs and mRNAs in workers. There are no significant differences between workers who stopped exposure to arsenic and the control group for most miRNAs and mRNAs, but the MiR-548c-3p levels show significant changes. Similar positive correlations between the expression of AS3MT and all selected mRNAs are found. Negative correlations between the expression of MiR-548c-3p and many relative mRNAs are found as well. AS3MT and MiR-548c-3p may regulate arsenic methylation jointly, which when involved in a group of relative mRNAs may play roles in arsenic metabolism and epigenetic changes caused by this metabolism.

[Expression changes of tumor-related gene mRNA in workers who smelt arsenic].

OBJECTIVE: To explore the changes and influencing factors about expressions of tumor-related gene mRNA in workers who smelt arsenic. METHODS: There were 37 workers who smelt arsenic, 43 workers who stopped exposure to arsenic for 85 days, and 51 individuals as control group which selected by random cluster sampling. Arsenic species( iAs, MMA, and DMA) in urine were determined by atomic absorption spectrophotometer with an As speciation pretreatment system. Real time PCR( RT-PCR)was performed to detect the expressions of 4 tumor-related gene mRNAs. RESULTS: The concentrations of iAs, MMA and DMA in urine of workers who smelt arsenic, stopped exposure to arsenic for 85 days, and control group were( 133. 97 ± 109. 53), ( 208. 93 ±171. 43) and( 820. 35 ± 487. 39) µg/( g·creatinine)( workers who smelt arsenic), ( 123. 31 ± 112. 72), ( 176. 21 ± 157. 19) and( 467. 73 ± 392. 17) µg/( g·creatinine)( workers who stopped exposure to arsenic), ( 1. 55 ±1. 49), ( 0. 10 ±0. 09) and( 10. 47 ±7. 85) µg/( g·creatinine)( control group). Compared to control group, the concentrations of 3 arsenic species were all higher in worker came from arsenic smelting. Compared to workers who were smelting arsenic, DMA are lower in workers who stopped exposure to arsenic for 85 days( P < 0. 05). The relative mRNA expressions of Lin28, Bax, Bcl-2 and Fas in 3 groups were( 8. 88 ± 2. 42), ( 6. 87 ± 1. 10), ( 7. 24 ± 2. 31) and( 8. 23 ±2. 90)( workers who smelt arsenic), ( 6. 21 ± 2. 94), ( 5. 81 ± 1. 72), ( 4. 50 ± 1. 59)and( 6. 89 ± 2. 35)( workers who stopped exposure to arsenic), ( 5. 60 ± 1. 43), ( 5. 56 ±0. 98), ( 4. 88 ± 1. 39) and( 6. 92 ± 1. 87)( control group). Compared to control group, relative mRNA expressions of Lin28, Bax, Bcl-2 and Fas were all higher in worker who were smelting arsenic( P < 0. 05). CONCLUSION: The expressions of tumor-related gene mRNAs are high in workers who smelt arsenic, and the methylation metabolism of arsenic play great role in the process of relative mRNAs expresses.

LincRNAs and base modifications of p53 induced by arsenic methylation in workers.

Arsenic (As) metabolites could induce methylation changes of DNA and base modifications of p53, which play role in the toxicity of As. LincRNAs should play a key regulatory role in the p53 transcriptional response. There were 43 workers producing As trioxide, 36 workers who stopped exposure to As trioxide about 85 days ago, and 24 individuals as control group. Three As species in urine were measured, and primary and secondary methylation indexes, iAs%, MMA% and DMA% were calculated. RT-PCR was performed to detect the expression of 7 LincRNAs and the base modifications of exon 5, 6, 7, and 8 of p53. The concentrations of urinary As were high in workers. Compared to control group, significant changes for 5 LincRNAs in workers producing As trioxide were found (P < 0.05), and there were significant base modifications of p53 in workers came from the two plants (P < 0.05). There exist various correlations between different exon base modifications of p53 and expressions of LincRNAs (P < 0.05). The closely positive correlations between MMA/DMA and MEG3/TUG1/HOTAIR/MALAT1 were found, but negative correlation between DMA/MALAT1 and the base modifications of exon 7 and 8 of p53 were found also (P < 0.05). LincRNAs and base modifications of p53 could be induced by As, MALAT1 and the base modifications of exon 7 and 8 of p53 could play unique roles in epigenic changes. These findings suggest potentially widespread roles of p53 and relative RNAs in arsenic workers, which may be caused by As metabolism.

[Changes in mRNA expression of p53 and related downstream genes in peripheral blood lymphocytes in workers occupationally exposed to arsenic].

OBJECTIVE: To investigate the changes in mRNA expression of p53 and related downstream genes in peripheral blood lymphocytes in workers occupationally exposed to arsenic as well as its influencing factors, and to analyze the mechanism of genetic toxicity of arsenic. METHODS: With cluster random sampling, 79 workers from an arsenic smelting plant were selected as exposure group, and another 24 people without occupational exposure to arsenic were selected as control group. The relative mRNA expression of p53 and related downstream genes in the peripheral blood lymphocytes of the two groups was determined by quantitative realtime PCR. The levels of inorganic arsenic (iAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) in urine were determined by hydride generation-atomic absorption spectrometry. RESULTS: The exposure group had significantly higher levels of iAs, MMA, and DMA than the control group (P<0.01); the exposure group had significantly higher relative mRNA expression (2(-ΔΔCt)) of p53 and four related downstream genes in peripheral blood lymphocytes than the control group (P<0.05); the relative mRNA expression of p53 and related downstream genes was positively correlated with each other (P<0.01), with a correlation coefficient greater than 0.4; the levels of arsenic compounds in urine were positively correlated with the relative mRNA expression of p53 and some of its downstream genes (P<0.05). CONCLUSION: The changes in mRNA expression of p53 and related downstream genes are closely related to the metabolic transformation of inorganic arsenic in workers occupationally exposed to arsenic, and it also plays an important role in genetic toxicity and carcinogenic effect in people exposed to arsenic.

Metabolites of arsenic and increased DNA damage of p53 gene in arsenic plant workers.

Recent studies have shown that monomethylarsonous acid is more cytotoxic and genotoxic than arsenate and arsenite, which may attribute to the increased levels of reactive oxygen species. In this study, we used hydride generation-atomic absorption spectrometry to determine three arsenic species in urine of workers who had been working in arsenic plants,and calculated primary and secondary methylation indexes. The damages of exon 5, 6, 8 of p53 gene were determined by the method developed by Sikorsky, et al. Results show that the concentrations of each urinary arsenic species,and damage indexes of exon 5 and 8 of p53 gene in the exposed population were significantly higher, but SMI was significantly lower than in the control group. The closely positive correlation between the damage index of exon 5 and PMI,MMA, DMA were found, but there was closely negative correlation between the damage index of exon 5 and SMI. Those findings suggested that DNA damage of exon 5 and 8 of p53 gene existed in the population occupationally exposed to arsenic. For exon 5, the important factors may include the model of arsenic metabolic transformation, the concentrations of MMA and DMA, and the MMA may be of great importance.

Increased damage of exon 5 of p53 gene in workers from an arsenic plant.

Mutagenesis is a multistage process. Substitution mutations can be induced by base modified through alteration of pairing property. Mutations of exon 5 and 8 of p53 gene have been found in most arsenicosis patients with precarcinomas and carcinomas, but never in arsenicosis individuals without precarcinomas and carcinomas. This study investigates whether base modification exists in exon 5 and 8 of p53 gene, and explores the dose-effect relationship between damage of exon 5 of p53 gene and urinary arsenic. Concentrations of urinary 8-hydroxydeoxyguanine (8-OHdG) are analyzed to identify the occurrence of DNA damage. The real-time PCR developed by Sikorsky et al. is applied to detect base modification in exon 5 and 8 of p53 gene for apparently healthy participants. Our results show that the mean total arsenic concentrations of two exposed groups from an arsenic plant are significantly elevated compared with the control group, and the damage level of exon 5 of the high-exposed group is significantly higher than that of the control group, but which does not happen in exon 8. The closely correlation between the damage index of exon 5 and urinary organic arsenic concentration are found. Concentration of 8-OHdG of the high-exposed group is significantly higher than that of the control group. These results imply that base modification in exon 5 of p53 gene can be induced by arsenic. In addition, our study suggests that the damage level of exon 5 is a useful biomarker to assess adverse health effect levels caused by chronic exposure to arsenic.

[Real-time PCR used to detect p53 gene damage in workers exposed to arsenic].

OBJECTIVE: To evaluate the relationship between the metabolism of arsenic and the damage of exon 5 and 8 of p53 gene from workers in a arsenic mill, and with real-time PCR technique, to establish the method probing the gene-specific DNA damage in people. METHODS: By real-time PCR, the damages of exon 5 and 8 of p53 gene were probed in 37 workers exposed highly to, 16 manager and logistic employees exposed less to an arsenic mill in Yunnan province, and also 25 local people who did not contact with any white arsenic in near past time. At the same time, the urinary total and organic arsenic of workers were detected. The correlation between metabolism of arsenic and damage of p53 gene was evaluated. RESULTS: Total urinary arsenic concentrations were (1.18 +/- 0.76) mg/L and (0.32 +/- 0.28) mg/L for high and low exposed male workers, and 0.23 mg/L, (0.53 +/- 0. 30) mg/L for high and low exposed females. Organic urinary arsenic concentrations were (0.48 +/- 0.37) mg/L and (0.08 +/- 0.05) mg/L for high and low exposed males, and 0.11 mg/L, (0.30 +/- 0.24) mg/L for high and low exposed females. The total and organic urinary arsenic of high exposed group was higher than that of control male (P < 0.05), all in control group were lower than 0.02 mg/L for reference. The Ct relative value of exon 5 of p53 gene in high exposed group was higher than that in control male (P < 0.05), and the increased tendency of Ct relative value of exon 5 of p53 gene was found in workers with organic arsenic concentration going up (r(s) = 0.355, P = 0.011). The Ct relative value of exon 8 of p53 gene in low exposed group was higher than that in control male (P < 0.05), but the difference between high exposed and low exposed or reference's was not obvious (P > 0.05). CONCLUSION: The damages in exon 5 and 8 of p53 gene in workers exposed to arsenic may be induced. The metabolism of arsenic may be very important in the damage of exon 5. It is feasible for real-time PCR technique used to detect gene-specific DNA damage in people.