Bacterial Contamination in the Different Parts of Household Washing Machine: New Insights from Chengdu, Western China.

The moist and warm environment in the household washing machine provides ideal living conditions for the growth and survival of various microorganisms. However, the biodiversity of bacterial community in the different parts of washing machine from Chinese households has not been clarified. In this study, we assessed the bacterial communities in sealing strip, detergent drawer, inner drum, water filter and greywater of ten domestic washing machines quantitatively and qualitatively in Chengdu, southwestern China. The microbial cultivation results indicated that the washing machines from Chengdu had a severe microbial contamination reflected by large counts on bacteria, fungi and coliform. Furthermore, the sequencing data showed that the different parts displayed distinctive bacterial compositions. At the level of genus, the anaerobic bacteria of Caproiciproducens and Acidipropionibacterium were predominant in sealing strip. Barnesiella, Shinella and Sellimonas were detected as the characteristic bacteria in detergent drawer. The pathogens of Luteibacter and Corynebacterium at the genus level were the dominant bacteria in inner drum and water filter, respectively. The genera of Azospira, Roseococcus, Elstera and Aquicella, which belonged to the pathogenic phylum of Proteobacteria, were identified as bioindicators for the greywater. Gene function analysis on the sequencing data illustrated that the bacteria from washing machines were potentially associated with bacterial infectious diseases and antimicrobial resistance. This study shows the bacterial diversity in the different parts of washing machines, providing new clues for bacterial contamination in washing machines from Chinese households.

Spatial-temporal distribution and source analysis of atmospheric particulate-bound cadmium from 1998 to 2021 in China.

Cadmium (Cd) is one of the most serious atmospheric heavy metal pollutants in China. PM2.5, PM10, and total suspended particle (TSP) are all important media for population Cd exposure. However, no studies so far have systematically explored the spatial and temporal distribution of atmospheric Cd bound to all these media in China, and the specific industrial sectors that contribute to the airborne Cd level are still unclear at present. In this study, we constructed the spatial and temporal distribution of PM (PM2.5, PM10, and TSP) binding Cd concentrations in China. Quantitative source apportionment of atmospheric Cd was carried out by analyzing the association of 23 industrial or energy-consuming sectors with Cd concentrations. Our results showed PM2.5, PM10, and TSP binding Cd concentrations decreased by 5.8%, 5.9%, and 6.1% per year at the national level, respectively. High PM-Cd concentrations were concentrated and distributed mainly in central and northwestern China. In addition, the medians of atmospheric PM2.5, PM10, and TSP binding Cd concentrations at the national level were 0.0026 µg/m3, 0.0036 µg/m3, and 0.0042 µg/m3, respectively. The main sources of PM-Cd include nonferrous metal smelting (Zn, Pb, Al) (47%), glass production (13%), pesticide production (12%), cement production (10%), and coal consumption (9%). This study analyzes comprehensively the atmospheric PM-bound Cd pollution, identifies the major industrial sectors that affect atmospheric Cd concentrations at the macroscale for the first time, and provides a basis for further reduction in the atmospheric Cd pollution.

Long-term exposure to cadmium disrupts neurodevelopment in mature cerebral organoids.

Cadmium (Cd) is a pervasive environmental pollutant. Increasing evidence suggests that Cd exposure during pregnancy can induce adverse neurodevelopmental outcomes. However, due to the limitations of neural cell and animal models, it is challenging to study the developmental neurotoxicity and underlying toxicity mechanism of long-term exposure to environmental pollutants during human brain development. In this study, chronic Cd exposure was performed in human mature cerebral organoids for 49 or 77 days. Our study found that prolonged exposure to Cd resulted in the inhibition of cerebral organoid growth and the disruption of neural differentiation and cortical layer organization. These potential consequences of chronic Cd exposure may include impaired GFAP expression, a reduction in SOX2+ neuronal progenitor cells, an increase in TUJ1+ immature neurons, as well as an initial increase and a subsequent decrease in both TBR2+ intermediate progenitors and CTIP2+ deep layer cortical neurons. Transcriptomic analyses revealed that long-term exposure to Cd disrupted zinc and copper ion homeostasis through excessive synthesis of metallothionein and disturbed synaptogenesis, as evidenced by inhibited postsynaptic protein. Our study employed mature cerebral organoids to evaluate the developmental neurotoxicity induced by long-term Cd exposure.

Oxidative Stress Induced by Arsenite is Involved in YTHDF2 Phase Separation.

YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) undergoes phase separation in response to the stimulation of high concentration of arsenite, suggesting that oxidative stress, the major mechanism of arsenite toxicity, may play a role in YTHDF2 phase separation. However, whether arsenite-induced oxidative stress is involved in phase separation of YTHDF2 has yet to be established. To explore the effect of arsenite-induced oxidative stress on YTHDF2 phase separation, the levels of oxidative stress, YTHDF2 phase separation, and N6-methyladenosine (m6A) in human keratinocytes were detected after exposure to various concentrations of sodium arsenite (0-500 µM; 1 h) and antioxidant N-acetylcysteine (0-10 mM; 2 h). We found that arsenite promoted oxidative stress and YTHDF2 phase separation in a concentration-dependent manner. In contrast, pretreatment with N-acetylcysteine significantly relieved arsenate-induced oxidative stress and inhibited YTHDF2 phase separation. As one of the key factors to YTHDF2 phase separation, N6-methyladenosine (m6A) levels in human keratinocytes were significantly increased after arsenite exposure, accompanied by upregulation of m6A methylesterase levels and downregulation of m6A demethylases levels. On the contrary, N-acetylcysteine mitigated the arsenite-induced increase of m6A and m6A methylesterase and the arsenite-induced decrease in m6A demethylase. Collectively, our study firstly revealed that oxidative stress induced by arsenite plays an important role in YTHDF2 phase separation driven by m6A modification, which provides new insights into the arsenite toxicity from the phase-separation perspective.

N6-methyladenosine promotes aberrant redox homeostasis required for arsenic carcinogenesis by controlling the adaptation of key antioxidant enzymes.

N6-methyladenosine (m6A), a high-profile RNA epigenetic modification, responds to oxidative stress and temporal-specifically mediates arsenic carcinogenesis. However, how m6A affects aberrant redox homeostasis required for arsenic carcinogenesis is poorly understood. Here, we established arsenic-carcinogenic models of different stages, including As-treated, As-transformed, and As-tumorigenic cell models. We found that arsenic-induced reactive oxygen species (ROS) elevated m6A levels, thus triggering m6A-dependent antioxidant defenses. During arsenic-induced cell transformation, METTL3-upregulated m6A on the mRNAs of SOD1, SOD2, CAT, TXN, and GPX1 promoted the mRNA translation and protein expressions of these antioxidant enzymes by increasing YTHDF1-mediated mRNA stability. Meanwhile, FTO-downregulated m6A on PRDX5 mRNA increased PRDX5 translation and expression by reducing YTHDF2-mediated mRNA decay. After upregulated antioxidant defenses balanced with high levels of ROS induced by arsenic, the m6A balance formed in mRNAs of six key antioxidant enzymes (SOD1, SOD2, CAT, TXN, GPX1, and PRDX5) and promoted high expressions of these antioxidant enzymes to maintain aberrant redox homeostasis. METTL3 inhibitor STM2457, FTO inhibitor FB23-2, or YTHDF1 knockdown disturbed the aberrant redox homeostasis by breaking the m6A balance, causing cell death in arsenic-induced tumors. Our results demonstrated that m6A promotes the formation and maintenance of aberrant redox homeostasis required for arsenic carcinogenesis by time-dependently orchestrating the adaptive expressions of six key m6A-targeted antioxidant enzymes. This study advances our understanding of arsenic carcinogenicity from the novel aspect of m6A-dependent adaptation to arsenic-induced oxidative stress.

N6-methyladenosine upregulates ribosome biogenesis in environmental carcinogenesis.

Many trace metal pollutants in surface water, the atmosphere, and soil are carcinogenic, and ribosome biogenesis plays an important role in the carcinogenicity of heavy metals. However, the contradiction between upregulated ribosome biogenesis and decreased ribosomal DNA copy number in environmental carcinogenesis is not fully understood. Here, from a perspective of the most predominant and abundant RNA epigenetic modification, N6-methyladenosine (m6A), we explored the reason behind this contradiction at the post-transcriptional level using arsenite-induced skin carcinogenesis models both in vitro and in vivo. Based on the m6A microarray assay and a series of experiments, we found for the first time that the elevated m6A in arsenite-induced transformation is mainly enriched in the genes regulating ribosome biogenesis. m6A upregulates ribosome biogenesis post-transcriptionally by stabilizing ribosomal proteins and modulating non-coding RNAs targeting ribosomal RNAs and proteins, leading to arsenite-induced skin carcinogenesis. Using multi-omics analysis of human subjects and experimental validation, we identified an unconventional role of a well-known key proliferative signaling node AKT1 as a vital mediator between m6A and ribosome biogenesis in arsenic carcinogenesis. m6A activates AKT1 and transmits proliferative signals to ribosome biogenesis, exacerbating the upregulation of ribosome biogenesis in arsenite-transformed keratinocytes. Similarly, m6A promotes cell proliferation by upregulating ribosome biogenesis in cell transformation induced by carcinogenic heavy metals (chromium and nickel). Importantly, inhibiting m6A reduces ribosome biogenesis. Targeted inhibition of m6A-upregulated ribosome biogenesis effectively prevents cell transformation induced by trace metals (arsenic, chromium, and nickel). Our results reveal the mechanism of ribosome biogenesis upregulated by m6A in the carcinogenesis of trace metal pollutants. From the perspective of RNA epigenetics, our study improves our understanding of the contradiction between upregulated ribosome biogenesis and decreased ribosomal DNA copy number in the carcinogenesis of environmental carcinogens.

N6-methyladenosine plays a dual role in arsenic carcinogenesis by temporal-specific control of core target AKT1.

High-profile RNA epigenetic modification N6-methyladenosine (m6A), as a double-edged sword for cancer, can either promote or inhibit arsenic-induced skin carcinogenesis. However, the core m6A-target gene determining the duality of m6A and the regulatory mechanism of m6A on the core gene are still poorly understood. Based on m6A microarray detection, integrated multi-omics analysis, and further experiments in vitro and in vivo, we explored the molecular basis for the dual role of m6A in cancer induced by environmental pollutants using models in different stages of arsenic carcinogenesis, including As-treated, As-transformed, and As-tumorigenic cell models. We found that the key proliferative signaling node AKT1 is in the center of the m6A-regulatory network in arsenic carcinogenicity. The m6A level on AKT1 mRNA (3'UTR, CDS, and 5'UTR) dynamically changed in different stages of arsenic carcinogenesis. The m6A writer METTL3-catalyzed upregulation of m6A promotes AKT1 expression by elevating m6A reader YTHDF1-mediated AKT1 mRNA stability in As-treated and As-transformed cells, while the m6A eraser FTO-catalyzed downregulation of m6A promotes AKT1 expression mainly by inhibiting m6A reader YTHDF2-mediated AKT1 mRNA degradation in As-tumorigenic cells. Furthermore, upregulation of m6A inhibits the expression of AKT1 negative regulator PHLPP2 and promotes the expression of AKT1 positive regulator PDK1. These changes in AKT1 regulators result in AKT1 activation by upregulating AKT1 phosphorylation at S473 and T308. Interestingly, the FTO-catalyzed decrease in m6A prevents AKT upregulation in As-treated cells but promotes AKT upregulation in As-tumorigenic cells. Both inhibitors targeting the m6A writer and eraser can inhibit the AKT1-mediated proliferation of As-tumorigenic cells by breaking the balance of m6A regulators. Our results demonstrated that AKT1 is the core hub determining m6A as a double-edged sword. Changed m6A dynamically upregulates the expression and activity of AKT1 in different stages of arsenic carcinogenesis. This study can advance our understanding of the dual role and precise time-specific mechanism of RNA epigenetics involved in the carcinogenesis of hazardous materials.

[Mechanism of Triclosan in the Treatment of Nonalcoholic Fatty Liver Disease Based on Network Pharmacology].

Objective To explore the potential targets of triclosan in the treatment of nonalcoholic fatty liver disease(NAFLD) and to provide new clues for the future research on the application of triclosan. Methods The targets of triclosan and NAFLD were obtained via network pharmacology.The protein-protein interaction network was constructed with the common targets shared by triclosan and NAFLD.The affinity of triclosan to targets was verified through molecular docking.Gene ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment were carried out to analyze the key targets and the potential mechanism of action.NAFLD model was established by feeding male C57BL/6J mice with high-fat diet for 12 weeks.The mice were randomly assigned into a model group and a triclosan group [400 mg/(kg·d),gavage once a day for 8 weeks].The hematoxylin-eosin(HE) staining was used for observation of the pathological changes and oil red O staining for observation of fat deposition in mouse liver.Western blotting was employed to detect the protein level of peroxisome proliferator-activated receptor alpha(PPARα) in the liver tissue. Results Triclosan and NAFLD had 34 common targets,19 of which may be the potential targets for the treatment,including albumin(ALB),PPARα,mitogen-activated protein kinase 8(MAPK8),and fatty acid synthase.Molecular docking predicted that ALB,PPARα,and MAPK8 had good binding ability to triclosan.KEGG pathway enrichment showcased that the targets were mainly enriched in peroxisome proliferator-activated receptor signaling pathway,in which ALB and MAPK8 were not involved.Triclosan alleviated the balloon-like change and lipid droplet vacuole,decreased the lipid droplet area,and up-regulated the expression level of PPARα in mouse liver tissue. Conclusion PPARα is a key target of triclosan in the treatment of NAFLD,which may be involved in fatty acid oxidation through the peroxisome proliferator activated receptor signaling pathway.

Triclosan targeting of gut microbiome ameliorates hepatic steatosis in high fat diet-fed mice.

Antibiotic use provides a promising strategy for the treatment of non-alcoholic fatty liver disease (NAFLD) by regulating the gut microbiota composition. Triclosan, a widely used antibiotic, may improve gut microbiome dysbiosis associated with NAFLD through the suppression of pathogenic gram-negative bacteria. However, the effects of triclosan on gut microbiota and hepatic steatosis and have not been explored in NAFLD mouse model. In this study, C57BL/6J mice were fed with high fat diet (HFD) for continuous 20 weeks and treated with triclosan at 400 mg/kg/d for 8 weeks from week 13. We explored the effects of triclosan on hepatic lipid accumulation and gut microbiome in HFD-fed mice by histological examination and 16 S ribosomal RNA sequencing, respectively. Analysis on the composition of gut microbiota indicated that triclosan suppressed pathogenic gram-negative bacteria, including Helicobacter, Erysipelatoclostridium and Citrobacter, and increased the ratio of Bacteroidetes/Firmicutes in HFD-fed mice. Meanwhile, triclosan increased the relative abundance of beneficial gut microbiomes including Lactobacillus, Bifidobacterium and Lachnospiraceae, which protected against metabolic abnormality. The results of alpha-diversity and beta-diversity also showed the improvement of triclosan on bacterial diversity and richness in HFD-fed mice. Pathway analysis further confirmed that triclosan can regulate nutrient and energy metabolism through the elimination of deleterious bacteria. As a result, triclosan intervention significantly reduced lipid accumulation and alleviated hepatic steatosis in HFD-fed mice. In conclusion, our results suggest that triclosan can alleviate liver steatosis in HFD-fed mice by targeting the gut microbiome.

Alveolar macrophage-derived progranulin mediated pro-inflammatory Il-6 expression via regulating Creb1 in silicosis model.

Progranulin (PGRN) is a secreted factor involved in inflammatory diseases. However, the function of PGRN in silica-induced lung inflammation has not been elucidated. In this study, we demonstrated that PGRN in serum and lung tissues was markedly increased in silicosis mouse model. And immunohistochemistry results showed that PGRN was mainly expressed in alveolar macrophages, which was further confirmed in silica-treated alvelar macrophages cell line (MH-S) in vitro. PGRN promoted pro-inflammatory cytokines transcription such as interleukin (Il)-6, tumor necrosis factor-α (Tnf-α) and Il-1ß in MH-S cells, and the increasing of Il-6 was most obvious. Knockdown of PGRN blocked the silica-induced elevation of intracellular Il-6 in MH-S cells. Furthermore, we also found that PGRN could increase the phosphorylation of Cyclic AMP-responsive element-binding protein 1 (Creb1), a transcriptional regulator of Il-6. Inhibition of p-Creb1 by the phosphorylation inhibitor of Creb1 (666-15) decreased PGRN-induced intracellular Il-6 production in MH-S cells. In conclusion, PGRN was highly increased in silicosis mouse model and upregulated inflammatory cytokines expression. These findings suggested that PGRN might be a key mediator in silica-induced inflammation and provided a new clue for the diagnosis and drug therapy of silicosis.

[Comparative Analysis of Direct Drinking Water and the Relevant Sanitation Standards in China].

Along with the economic and technological development and growing demand for high-quality drinking water, direct drinking water has gained general popularity in China. However, no authoritative policy has been issued, giving a clear definition of direct drinking water and existing standards and regulations concerning direct drinking water are not definitive in nature. Existing water quality parameters are not well supported and sometimes even contradict each other. We elaborated, in this paper, the history of direct drinking water in China and systematically reviewed the existing regulations and standards related to direct drinking water. We also compared and analyzed the important microbiology, toxicology, sensory perception and general chemistry parameters in the standards. This paper is the first ever attempt at an in-depth analysis of the chaotic state of the direct drinking water industry. We have also highlighted the problems in the current standards and regulations for direct drinking water. Our study provides a basis for market regulation and the supervision and management of direct drinking water. In addition, the paper provides helpful information for laying down a definition of direct drinking water, calling for and approving of project proposals concerning the establishment of national standards for direct drinking water, and actually formulating the standards. We have made a number of suggestions: A. defining direct drinking water clearly and formulating the national standards for direct drinking water as soon as possible; B. conducting research on water quality benchmarks to provide scientific support for the formulation of the national standards for direct drinking water; C. giving more attention to the formulation of standards concerning microbiology parameters and their limits and giving consideration to the inclusion of parameters concerning viruses.

Triclosan down-regulates fatty acid synthase through microRNAs in HepG2 cells.

Triclosan is a promising candidate of fatty acid synthase (FASN) inhibitor by blocking FASN activity, but its effect on FASN expression and the underling epigenetic mechanism remain elusive. In this study, the effect of triclosan on FASN mRNA and protein expressions in human HepG2 cells and the regulatory role of microRNAs (miRNAs) in the downregulation of FASN induced by triclosan were explored through experiments and bioinformatics analysis. The results showed that triclosan not only directly inhibited FASN activity, but also significantly decreased FASN mRNA and protein levels in human liver HepG2 cells. Nine miRNAs targeting FASN mRNA degradation were identified by miRNA prediction tools, and the expression levels of these nine miRNAs were then detected by real-time quantitative PCR. Triclosan significantly increased the expressions of the six miRNAs, namely miR-15a, miR-107, miR-195, miR-424, miR-497 and miR-503, leading to the downregulation of FASN. Further investigation revealed that the six triclosan-upregulated miRNAs played an important regulatory role in lipid metabolism and cell cycle by gene ontology annotations and pathway analysis. Consistent with the results of bioinformatics analyses, triclosan significantly reduced the intracellular lipid content by triglyceride assay, oil red O, BODIPY 493/503 and Nile Red staining, thereby inhibiting the growth of HepG2 cells through apoptosis. Taken together, our study reveals that triclosan downregulates FASN expression through a variety of miRNAs, providing new insight for triclosan as a FASN inhibitor candidate to regulate lipid metabolism in human hepatoma cells.

China's drinking water sanitation from 2007 to 2018: A systematic review.

Regular monitoring of drinking water in China is carried out by the Centers for Disease Control and Prevention at all levels and some articles were published for the public to understand the status of drinking water. However, these published articles were limited to small areas and reported almost exclusively in Chinese. In order to give the public a comprehensive understanding of the situation of drinking water sanitation in China and provide specific directions for ensuring drinking water safety in the future, we review the sanitation status of drinking water in different regions of China from 2007 to 2018, the changes over time and the factors affecting drinking water quality by means of systematic review for the first time. Our results show that the quality of drinking water in China has shown an upward trend from 2007 to 2018. The qualification rate of urban drinking water (85.51%) was much higher than that of rural drinking water (51.12%), and the dry season (56.93%) was higher than the wet season (50.54%), and the terminal tap water (59.88%) was higher than the outlet water of waterworks (55.87%). In addition, the regions with low qualification rate of water quality in China were mainly distributed in several southern provinces, such as Yunnan, Guizhou, Hainan, etc. What's more, the qualified rate of the three microbiological indicators was the lowest, all below 85%. All the results indicate that the sanitation status of drinking water in China is unsatisfactory, and the biggest risk affecting water safety is microbial pollution. The central and local governments should work hard to improve people's drinking water quality and continue to strengthen the treatment and supervision of drinking water, especially in rural areas and undeveloped southern areas.

Fat mass and obesity-associated protein regulates lipogenesis via m6 A modification in fatty acid synthase mRNA.

As the first identified N6 -methyladenosine (m6 A) demethylase, fat mass and obesity-associated (FTO) protein is associated with fatty acid synthase (FASN) and lipid accumulation. However, little is known about the regulatory role of FTO in the expression of FASN and de novo lipogenesis through m6 A modification. In this study, we used FTO small interfering RNA to explore the effects of FTO knockdown on hepatic lipogenesis and its underlying epigenetic mechanism in HepG2 cells. We found that knockdown of FTO increased m6 A levels in total RNA and enhanced the expression of YTH domain family member 2 which serves as the m6 A-binding protein. The de novo lipogenic enzymes and intracellular lipid content were significantly decreased under FTO knockdown. Mechanistically, knockdown of FTO dramatically enhanced m6 A levels in FASN messenger RNA (mRNA), leading to the reduced expression of FASN mRNA through m6 A-mediated mRNA decay. The protein expressions of FASN along with acetyl CoA carboxylase and ATP-citrate lyase were further decreased, which inhibited de novo lipogenesis, thereby resulting in the deficiency of lipid accumulation in HepG2 cells and the induction of cellular apoptosis. The results reveal that FTO regulates hepatic lipogenesis via FTO-dependent m6 A demethylation in FASN mRNA and indicate the critical role of FTO-mediated lipid metabolism in the survival of HepG2 cells. This study provides novel insights into a unique RNA epigenetic mechanism by which FTO mediates hepatic lipid accumulation through m6 A modification and indicates that FTO could be a potential target for obesity-related diseases and cancer.

Linc-ROR promotes arsenite-transformed keratinocyte proliferation by inhibiting P53 activity.

Linc-ROR is an oncogenic long non-coding RNA over-expressed in many kinds of cancer that promotes cancer cell proliferation. Arsenite is a determined carcinogen that increases the risk of skin cancer, but the carcinogenic mechanism of arsenite remains unclear. To explore whether and how linc-ROR plays a role in arsenite-induced carcinogenesis of skin cancer, we established arsenite-transformed keratinocyte HaCaT cells by exposing them to 1 µM arsenite for 50 passages. Then we examined the linc-ROR expression during the transformation and explored the effect of linc-ROR on the cell proliferation of arsenite-transformed HaCaT cells. We found that the linc-ROR level in HaCaT cells was gradually increased during arsenite-induced malignant transformation, and the activity of P53 was decreased, but the P53 expression was not significantly altered, indicating that linc-ROR may play a role in arsenite-induced HaCaT cell transformation that is associated with P53 activity but not P53 expression. We further demonstrated that linc-ROR down-regulation by siRNA significantly inhibited the cellular proliferation and restored P53 activity in arsenite-transformed HaCaT cells, suggesting that linc-ROR promotes proliferation of arsenite-transformed HaCaT cells by inhibiting P53 activity. Moreover, linc-ROR siRNA also down-regulated the PI3K/AKT pathway in arsenite-transformed HaCaT cells, and treatment with AKT inhibitor wortmannin restored P53 activity, implying that linc-ROR inhibits P53 activity by activating the PI3K/AKT pathway. Taken together, the present study shows that linc-ROR promotes arsenite-transformed keratinocyte proliferation by inhibiting P53 activity through activating PI3K/AKT, providing a novel carcinogenic mechanism of arsenite-induced skin cancer.

Arsenite Increases Linc-ROR in Human Bronchial Epithelial Cells that Can Be Inhibited by Antioxidant Factors.

Oxidative stress is the main mechanism of arsenite toxicity. Long intergenic non-coding RNA regulator of reprogramming is a newly found stress-response long non-coding RNA that is activated in various stress conditions. However, whether long intergenic non-coding RNA, regulator of reprogramming (linc-ROR) is involved in arsenite-induced oxidative stress has not been explored. In this study, we found that arsenite dose responsively increased the expression of linc-ROR in human bronchial epithelial (HBE) cells, along with elevated oxidative stress demonstrated by increased intracellular reactive oxygen species (ROS) and DNA damage, as well as decreased antioxidant glutathione and superoxide dismutase. We further found that the pre-treatment with N-acetylcysteine, a widely used ROS scavenger, and the over-expression of antioxidant NRF2 protein, both significantly reduced arsenite-induced oxidative stress in arsenite-treated HBE cells, and the linc-ROR over-expression was also inhibited, suggesting that oxidative stress is a key factor for the increase of linc-ROR in arsenite-treated HBE cells. Moreover, our results of bio-informatic analysis showed that arsenite-induced oxidative stress might modulate linc-ROR expression via 3 genes and the up-regulated linc-ROR in arsenite-induced oxidative stress may get involved in cellular processes such as cellular stress response, RNA metabolism, and DNA repair. Collectively, our study demonstrates that oxidative stress plays the key role in arsenite-induced over-expression of linc-ROR, and linc-ROR may be a new clue for exploring the mechanism of arsenite toxicity.

Long non-coding RNA ROR confers arsenic trioxide resistance to HepG2 cells by inhibiting p53 expression.

Arsenic trioxide is an effective drug in the treatment of hematologic malignancies, but it has no obvious therapeutic effect on liver cancer. Long non-coding RNA ROR is a newly found long-noncoding RNA that has been reported to get involved in the regulation of chemo-resistance in multiple cancers. However, whether and how long non-coding RNA ROR gets involved in the resistance to arsenic trioxide in liver cancer has not been explored. In this study, We found that cellular apoptosis was increased by arsenic trioxide in liver cancer HepG2 cells; P53 expression was also increased by arsenic trioxide at both mRNA level and protein level, indicating that P53-dependent apoptosis is the main mechanism for arsenic trioxide to induce cytotoxicity in liver cancer HepG2 cells. Meanwhile, we found an obvious increase in the level of long non-coding RNA ROR in arsenic trioxide-treated HepG2 cells. By measuring the level of reactive oxygen species, glutathione, superoxide dismutase, and malondialdehyde, the product of lipid peroxidation, we further demonstrated that oxidative stress was a potential factor for both the activation of P53 expression and the increase in long non-coding RNA ROR expression. Through the knock-down of long non-coding RNA ROR by siRNA, we revealed that the activated long non-coding RNA ROR ameliorated arsenic trioxide-induced apoptosis by inhibiting P53 expression. Together, our study reported that long non-coding RNA ROR conferred arsenic trioxide resistance to liver cancer cells through inhibiting P53 expression, and long non-coding RNA ROR might be a novel sensitizing target for liver cancer treatment.

N6-methyladenosine mediates arsenite-induced human keratinocyte transformation by suppressing p53 activation.

N6-methyladenosine (m6A), the most abundant and reversible RNA modification, plays critical a role in tumorigenesis. However, whether m6A can regulate p53, a leading antitumor protein remains poorly understood. In this study, we explored the regulatory role of m6A on p53 activation using an arsenite-transformed keratinocyte model, the HaCaT-T cell line. We created the cell line by exposing human keratinocyte HaCaT cells to 1 µM arsenite for 5 months. We found that the cells exhibited an increased m6A level along with an aberrant expression of the methyltransferases, demethylase, and readers of m6A. Moreover, the cells exhibited decreased p53 activity and reduced p53 phosphorylation, acetylation, and transactivation with a high nucleus export rate of p53. Knockdown of the m6A methyltransferase, METTL3 significantly decreased m6A level, restoring p53 activation and inhibiting cellular transformation phenotypes in the arsenite-transformed cells. Further, using both a bioinformatics analysis and experimental approaches, we demonstrated that m6A downregulated the expression of the positive p53 regulator, PRDM2, through the YTHDF2-promoted decay of PRDM2 mRNAs. We showed that m6A upregulated the expression of the negative p53 regulator, YY1 and MDM2 through YTHDF1-stimulated translation of YY1 and MDM2 mRNA. Taken together, our study revealed the novel role of m6A in mediating arsenite-induced human keratinocyte transformation by suppressing p53 activation. This study further sheds light on the mechanisms of arsenic carcinogenesis via RNA epigenetics.

Genotoxicity assessment of triclocarban by comet and micronucleus assays and Ames test.

The widespread usage and ubiquitous distribution of triclocarban (3,4,4'-trichlorocarbanilide, TCC) have raised public concerns about its health effects. At present, there is little information about the genotoxicity of TCC. In this study, we used a battery of genotoxicity testing methods including salmonella reverse mutation test (Ames test), comet assay and micronucleus assay to detect the effects of TCC on gene mutation, DNA breakage, and chromosome damage. The results of Ames test showed that TCC at 0.1-1000 µg/plate did not significantly increase the number of revertant colonies in the four standard Salmonella typhimurium strains, i.e., TA97, TA98, TA100, and TA102, when compared to the vehicle control. The results from comet assay demonstrated that exposure to 5, 10, or 15 µM TCC for 24 h did not significantly increase the percentage of comet cells, tail length (TL), DNA in tail (T DNA%), or olive tail moment (OTM) in keratinocyte HaCaT and hepatic L02 cells. Moreover, TCC did not markedly enhance the frequency of micronucleated cells or micronuclei in HaCaT and L02 cells in the micronucleus assay. Taken together, the results indicated that TCC did not exhibit any genotoxic effects. Our study provides additional information for the safety profile of TCC.

Inhibition of base excision repair by natamycin suppresses prostate cancer cell proliferation.

Prostate cancer (PCa) progression is characterized by increased expression and transcriptional activity of the androgen receptor (AR). In the advanced stages of prostate cancer, AR significantly upregulates the expression of genes involved in DNA repair. Upregulation of expression for base excision repair (BER) related genes is associated with poor patient survival. Thus, inhibition of the BER pathway may prove to be an effective therapy for prostate cancer. Using a high throughput BER capacity screening assay, we sought to identify BER inhibitors that can synergize with castration therapy. An FDA-approved drug library was screened to identify inhibitors of BER using a fluorescence-based assay suitable for HTS. A gel-based secondary assay confirmed the reduction of BER capacity by compounds identified in the primary screen. Five compounds were then selected for further testing in the independently derived, androgen-dependent prostate cancer cell lines, LNCaP and LAPC4, and in the nonmalignant prostate derived cell lines PNT1A and RWPE1. Further analysis led to the identification of a lead compound, natamycin, as an effective inhibitor of key BER enzymes DNA polymerase ß (pol ß) and DNA Ligase I (LIG I). Natamycin significantly inhibited proliferation of PCa cells in an androgen depleted environment at 1 µM concentration, however, growth inhibition did not occur with nonmalignant prostate cell lines, suggesting that BER inhibition may improve efficacy of the castration therapies.