MCF-7 cell - derived exosomes were involved in protecting source cells from the damage caused by tributyltin chloride via transport function.

Tributyltin chloride (TBTC) is a ubiquitous environmental pollutant with various adverse effects on human health. Exosomes are cell - derived signaling and substance transport vesicles. This investigation aimed to explore whether exosomes could impact the toxic effects caused by TBTC via their transport function. Cytotoxicity, DNA and chromosome damage caused by TBTC on MCF-7 cells were analyzed with CCK-8, flow cytometry, comet assay and micronucleus tests, respectively. Exosomal characterization and quantitative analysis were performed with ultracentrifugation, transmission electron microscope (TEM) and bicinchoninic acid (BCA) methods. TBTC content in exosomes was detected with Liquid Chromatography-Mass Spectrometry (LC-MS). The impacts of exosomal secretion on the toxic effects of TBTC were analyzed. Our data indicated that TBTC caused significant cytotoxicity, DNA and chromosome damage effects on MCF-7 cells, and a significantly increased exosomal secretion. Importantly, TBTC could be transported out of MCF-7 cells by exosomes. Further, when exosomal secretion was blocked with GW4869, the toxic effects of TBTC were significantly exacerbated. We concluded that TBTC promoted exosomal secretion, which in turn transported TBTC out of the source cells to alleviate its toxic effects. This investigation provided a novel insight into the role and mechanism of exosomal release under TBTC stress.

mTORC1 - TFEB pathway was involved in sodium arsenite induced lysosomal alteration, oxidative stress and genetic damage in BEAS-2B cells.

The mechanistic target of rapamycin (RAPA) complex 1 (mTORC1) - transcription factor EB (TFEB) pathway plays a crucial role in response to nutritional status, energy and environmental stress for maintaining cellular homeostasis. But there is few reports on its role in the toxic effects of arsenic exposure and the related mechanisms. Here, we show that the exposure of bronchial epithelial cells (BEAS-2B) to sodium arsenite promoted the activation of mTORC1 (p-mTORC1) and the inactivation of TFEB (p-TFEB), the number and activity of lysosomes decreased, the content of reduced glutathione (GSH) and superoxide dismutase (SOD) decreased, the content of malondialdehyde (MDA) increased, the DNA and chromosome damage elevated. Further, when mTORC1 was inhibited with RAPA, p-mTORC1 and p-TFEB down-regulated, GSH and SOD increased, MDA decreased, the DNA and chromosome damage reduced significantly, as compared with the control group. Our data revealed for the first time that mTORC1 - TFEB pathway was involved in sodium arsenite induced lysosomal alteration, oxidative stress and genetic damage in BEAS-2B cells, and it may be a potential intervention target for the toxic effects of arsenic.

miR-584-5p / Ykt6 - mediated autophagy - lysosome - exosome pathway as a critical route affecting the toxic effects of lead on HK-2 cells.

Lead is a widespread environmental pollutant with serious adverse effects on human health, but the mechanism underlying its toxicity remains elusive. This study aimed to investigate the role of miR-584-5p / Ykt6 axis in the toxic effect of lead on HK-2 cells and the related mechanism. Our data suggested that lead exposure caused significant cytotoxicity, DNA and chromosome damage to HK-2 cells. Mechanistically, lead exposure down-regulated miR-584-5p and up-regulated Ykt6 expression, consequently, autophagosomal number and autophagic flux increased, lysosomal number and activity decreased, exosomal secretion increased. Interestingly, when miR-584-5p level was enhanced with mimic, autophagosomal number and autophagic flux decreased, lysosomal number and activity increased, ultimately, exosomal secretion was down-regulated, which resulted in significant aggravated toxic effects of lead. Further, directly blocking exosomal secretion with inhibitor GW4869 also resulted in exacerbated toxic effects of lead. Herein, we conclude that miR-584-5p / Ykt6 - mediated autophagy - lysosome - exosome pathway may be a critical route affecting the toxic effects of lead on HK-2 cells. We provide a novel insight into the mechanism underlying the toxicity of lead on human cells.

Lysosome-related exosome secretion mediated by miR-26b / Rab31 pathway was associated with the proliferation and migration of MCF-7 cells treated with BPA.

Bisphenol A (BPA), one of the typical environmental endocrine disruptors (EEDs), can promote the proliferation and migration of cancer cells, but the mechanism of which remains largely unclear. Exosome secretion plays an important role in the stress response of cells to environmental stimuli. This study was designed to explore whether exosome secretion was involved in the toxic effect of BPA on the proliferation and migration of MCF-7 cells, and the related mechanism. Our data shows that the IC50 value of MCF-7 exposure to BPA was about 65.82 µM. The exposure of MCF-7 to 10 µM BPA resulted in a decreased miR-26b expression and the activation of miR-26b/Rab-31 pathway, consequently, the number and activity of lysosomes decreased, the secretion of exosomes increased, cell proliferation and migration were enhanced obviously. Interestingly, miR-26b mimic up-regulated the number and activity of lysosomes via miR-26b/miR-31 pathway, exosome secretion was down-regulated, cell proliferation and migration decreased. Further, when GW4869 was used to directly inhibit the exosome secretion of MCF-7 treated with BPA, their proliferation and migration were down-regulated. Herein, we concluded that the stimulating effect of BPA on the proliferation and migration of MCF-7 cells was associated with the lysosome - related exosome secretion via miR-26b / Rab31 pathway.

Circulating exosome level of indigenous fish may be a novel biomarker for the integrated ecotoxicity effect of water environment.

The deficiency of effective biomarker for the toxic effects of water pollutants greatly limits the application of biological monitoring. This study aimed to investigate the possibility of circulating exosomes of indigenous fish acting as biomarker for the ecotoxicity effect of water environment. The Helong Reservoir in Guangzhou, China, was chosen as the investigating field, of which the water quality belongs to Class V (2013) (GB 3838-2002, China). The clean drinking water source of the upper reaches of the Liuxihe Reservoir was selected as the control. Indigenous fishes including Oreochromis niloticus (Nile tilapia), Labeo rohita (Rohu), Carassius auratus (Crucian carp) were sampled during the period from July 2020 to April 2021. Circulating exosomes of fish samples were isolated by using ultracentrifugation, characterized with transmission electron microscopy (TEM) and quantified by using bicinchoninic acid (BCA) assay. Oxidative stress, DNA and chromosome damage in liver, kidney, brain, gill and blood of fish samples were measured. The results showed that there were significant differences in superoxide dismutase (SOD) activity, glutathione (GSH) and malondialdehyde (MDA) contents, DNA and chromosome damage in fish samples between the Helong Reservoir and the control. Interestingly, there were also significant differences in circulating exosome levels of fish samples between them. Our data suggested that circulating exosome level of indigenous fish may be a novel biomarker for the ecotoxicity effects of water environment.

Indigenous Fish-Based Assessment of Genotoxic Potentials of the Helong Reservoir in Guangzhou, China.

The present study was conducted to assess the genotoxic potential of water from the Helong Reservoir, which was designated as a strategic drinking water source by the Guangdong Provincial Government of China in October 2016. Four kinds of common indigenous fish samples (Labeo rohita, Cirrhinus molitorella, red tilapia, and Oreochromis niloticus) were collected at 6 sampling sites during the period from July to November 2020. Fish from the clean drinking water source of the upper reaches of the Liuxihe Reservoir in Guangzhou were collected as the control. Both the alkaline single cell gel electrophoresis assay and the micronucleus test were used to detect DNA damage and the micronucleus rate in erythrocytes of fish samples, respectively. The results indicated that there was a significant increase in comet tail length, Olive tail moment, and micronucleus rates of all fish samples compared with those of the control (p < 0.05). The order of sensitivity to DNA damage and micronucleus formation was Labeo rohita > Cirrhinus molitorella > red tilapia > Oreochromis niloticus. The results of the 2 kinds of experiments were in perfect agreement with each other. We conclude that there are obvious genotoxic effects from the water in the Helong Reservoir. As a strategic drinking water source, the safety of the Reservoir water quality should be considered. The local government should put the restoration of the Helong Reservoir water quality on the agenda as soon as possible. Environ Toxicol Chem 2021;40:1919-1927. © 2021 SETAC.

Exosomal secretion may be a self-protective mechanism of its source cells under environmental stress: A study on human bronchial epithelial cells treated with hydroquinone.

Accumulating evidence reveals that exosome plays an important role in cell-to-cell communication in both physiological and pathological processes by transferring bioactive molecules. However, the role of exosomal secretion in the adaption of its source cells to the stimuli of environmental chemicals remains elusive. In this study, we revealed that the exposure of hydroquinone (HQ; the main bioactive metabolite of benzene) to human bronchial epithelial cells (16HBE) resulted in decreased ability of cell proliferation and migration, and simultaneously DNA damage and micronuclei formation. Interestingly, when exosomal secretion of HQ treated 16HBE cells was inhibited with the inhibitor GW4869, cellular proliferation and migration were further significantly reduced; concurrently, their DNA damage and micronuclei formation were both further significantly aggravated. Herein, we conclude that exosomal secretion of 16HBE cells may be an important self-protective function against the toxic effects induced by HQ.

EDTA-functionalized magnetic chitosan oligosaccharide and carboxymethyl cellulose nanocomposite: Synthesis, characterization, and Pb(II) adsorption performance.

A novel ethylenediaminetetraacetic acid (EDTA)-functionalized magnetic chitosan oligosaccharide and carboxymethyl cellulose (Fe3O4@CMCCOS-EDTA) nanocomposite adsorbent was successfully fabricated for Pb(II) adsorption. The adsorbent was characterized by Fourier transform infrared, and X-ray photoelectron spectroscopy was used to confirm successful EDTA modification and Pb(II) adsorption. Scanning electron microscopy, transmission electron microscopy, vibrating sample magnetometer, and thermogravimetric analysis were used to study the morphology and properties of magnetic particles. EDTA modification considerably improved the capacity of the adsorbent. The batch adsorption experiment results indicated that the pseudo-second-order (PSO) model and the Langmuir isotherm model reliably described the adsorption behavior. The maximum adsorption capacity (qm) for monolayer chemical adsorption was calculated to be 432.34 mg/g at the pH of 5 and temperature of 308 K. Notably, Fe3O4@CMCCOS-EDTA exhibited a high Pb(II) removal rate of ~100% using an initial metal ion solution of 100 mg/L and 200 mg/L.

Exosomal miR-221 derived from hydroquinone-transformed malignant human bronchial epithelial cells is involved in cell viability of recipient cells.

miR-221, an oncogenic microRNA, can promote cell proliferation and is highly expressed in various types of tumors. However, the role of exosomal miR-221 in benzene-caused carcinogenesis remains elusive. Our study was designed to investigate whether exosomes secreted by the hydroquinone (HQ; an active metabolite of benzene)-transformed malignant cells can transmit miR-221 to normal recipient cells and its possible effects on cell viability. Our investigation revealed that expression levels of miR-221 were significantly increased in HQ-transformed malignant cells relative to normal controls. Furthermore, exposure of control cells to exosomes that were derived from HQ-transformed malignant cells increased miR-221 levels and promoted their proliferation. Analyses of the biological potency of exosomes derived from HQ-transformed malignant cells in which miR-221 levels were decreased using an inhibitor, showed that both miR-221 levels and proliferation of recipient cells were decreased, but still were higher than those of normal 16HBE cells. Our study indicates that exosomal miR-221 derived from HQ-transformed malignant human bronchial epithelial cells is involved in the proliferation of recipient cells.

Exosomes derived from normal human bronchial epithelial cells down-regulate proliferation and migration of hydroquinone-transformed malignant recipient cells via up-regulating PTEN expression.

The gene encoding the tumor suppressor, phosphatase and tensin homolog (PTEN), located on chromosome 10, is frequently expressed at low levels in various tumors, resulting in the stimulation of cell proliferation and migration. However, the role of exosomal PTEN in cell-cell communication during the progress of benzene-induced carcinogenesis remains unclear. The goal of this study was to explore whether exosomes derived from normal human bronchial epithelial cells (16HBE) could transmit PTEN to hydroquinone-transformed malignant recipient cells (16HBE-t) and its possible effects on cell proliferation and migration. Consistent with PTEN expression being down-regulated in transformed cells, we found that its expression was significantly decreased in 16HBE-t relative to 16HBE cells and that purified exosomes secreted by 16HBE, up-regulated PTEN levels in recipient 16HBE-t cells. Thus, down-regulating their proliferation and migration. Further, when exosomes derived from 16HBE cells that had been treated with the PTEN inhibitor SF1670, were incubated with recipient 16HBE-t cells, they exhibited decreased PTEN levels, with a corresponding increase in their proliferation and migration. In conclusion, our study demonstrates that exosomes derived from 16HBE cells can down-regulate proliferation and migration of recipient 16HBE-t cells via transferring PTEN.

Adsorption of Pb(II) from aqueous solution by polyacrylic acid grafted magnetic chitosan nanocomposite.

A creative combination of chitosan with polyacrylic acid (PAA) improves the acidity resistance of chitosan and increases its potential in the field of adsorption. In order to facilitate recovery, magnetic nanoparticles were incorporated in CS-PAA to obtain a magnetic-CS-PAA (MCS-PAA) nanocomposite. The physical and chemical characteristics of the composite adsorbent MCS-PAA were determined by SEM, TEM, FTIR, EDX, XRD, and XPS. This environmental-friendly, magnetic, composite adsorbent showed significantly better adsorption performance than those of the individual adsorbents alone. The maximal adsorption capacity was 204.89 mg/g according to the Langmuir isotherm model, when the concentration of Pb(II) was 100 mg/L at the equilibrium time of 70 min. The main adsorption mechanism was the complexation between the carboxyl, amino, and hydroxyl groups in MCS-PAA and Pb(II). Further, introduction of PAA also improved the acid resistance of CS. The new adsorbent MCS-PAA is thus expected to facilitate a wider range of applications for chitosan in the adsorption of Pb(II).

Adsorption of diclofenac sodium on bilayer amino-functionalized cellulose nanocrystals/chitosan composite.

Residual diclofenac sodium (DS) in the environment is harmful to human health. A promising method for DS removal is the use of adsorbents functionalized with amino groups that can form an ionic bond with the carboxyl group of DS at a suitable pH. In this work, a novel composite adsorbent composed of cellulose nanocrystals (CNC) and chitosan (CS) has been synthesized and functionalized by ethylenediamine (ED) in both layers. Characterization methods, including scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectrometry, and X-ray photoelectron spectroscopy, were used to confirm the morphology and synthetic mechanism of the double- amino-functionalized adsorbent. Based on the optimization of adsorption conditions and modeling of the adsorption mechanism, the DS adsorption process on CNC-ED@CS-ED involves chemical adsorption, and the maximum adsorption capacity obtained from the Langmuir model is 444.44 mg/g. CNC-ED@CS-ED exhibits good adsorption capacity and high sustainability; thus, it is a promising composite material for the removal of DS from wastewater.

miR-222/GAS5 is involved in DNA damage and cytotoxic effects induced by temozolomide in T98G cell line.

Temozolomide (TMZ), a therapeutic DNA alkylator that can cause lethal DNA damage in cancer cells, is widely used for the standard chemotherapy against glioblastoma. However, long-term treatment with TMZ often causes drug resistance and poor prognosis, the mechanism of which remains largely unclear. This study aimed to investigate the possible role of miR-222/GAS5 axis on DNA damage and cytotoxic effects induced by TMZ in glioblastoma cells (T98G). Data suggest that the DNA comet tail length of T98G is positively correlated with the levels of miR-222 (R2  = 0.9808, P < 0.05), and negatively correlated with the levels of GAS5 (R2  = 0.8903, P < 0.05). The optical density value of T98G is negatively correlated with the levels of miR-222 (R2  = 0.7848, P < 0.05), and positively correlated with the levels of GAS5 (R2  = 0.6886, P < 0.05). Furthermore, comet tail length and optical density value are negatively and positively correlated with the levels of O-6-methylguanine-DNA methyltransferase, respectively (R2  = 0.8462, P < 0.05; R2  = 0.7018, P < 0.05). In conclusion, miR-222/GAS5 is involved in DNA damage and cytotoxic effects induced by TMZ, which means that miR-222/GAS5 may have great potential of being used as a biomarker for screening of chemotherapeutic alkylators.

Relationship between Methyl Tertiary Butyl Ether Exposure and Non-Alcoholic Fatty Liver Disease: A Cross-Sectional Study among Petrol Station Attendants in Southern China.

Methyl tertiary butyl ether (MTBE)-A well known gasoline additive substituting for lead alkyls-causes lipid disorders and liver dysfunctions in animal models. However, whether MTBE exposure is a risk factor for non-alcoholic fatty liver disease (NAFLD) remains uncertain. We evaluate the possible relationship between MTBE exposure and the prevalence of NAFLD among 71 petrol station attendants in southern China. The personal exposure concentrations of MTBE were analyzed by Head Space Solid Phase Microextraction GC/MS. NAFLD was diagnosed by using abdominal ultrasonography according to the guidelines for the diagnosis and treatment of NAFLD suggested by the Chinese Hepatology Association. Demographic and clinical characteristics potentially associated with NAFLD were investigated. Mutivariate logistic regression analysis was applied to measure odds ratios and 95% confidence intervals (CI). The result showed that the total prevalence of NAFLD was 15.49% (11/71) among the study subjects. The average exposure concentrations of MTBE were 292.98 ± 154.90 µg/m³ and 286.64 ± 122.28 µg/m³ in NAFLD and non-NAFLD groups, respectively, and there was no statistically significant difference between them (p > 0.05). After adjusting for age, gender, physical exercise, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), alanine aminotransferase (ALT), white blood cell (WBC), total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL), the odds ratios were 1.31 (95% CI: 0.85-1.54; p > 0.05), 1.14 (95% CI: 0.81-1.32; p > 0.05), 1.52 (95% CI: 0.93-1.61; p > 0.05) in the groups (including men and women) with exposure concentrations of MTBE of 100-200 µg/m³, 200-300 µg/m³, and ≥300 µg/m³, respectively, as compared to the group (including men and women) ≤100 µg/m³. Our investigation indicates that exposure to MTBE does not seem to be a significant risk factor for the prevalence of NAFLD among petrol station attendants in southern China.

Health Risk Assessment for Inhalation Exposure to Methyl Tertiary Butyl Ether at Petrol Stations in Southern China.

Methyl tertiary butyl ether (MTBE), a well known gasoline additive, is used in China nationwide to enhance the octane number of gasoline and reduce harmful exhaust emissions, yet little is known regarding the potential health risk associated with occupational exposure to MTBE in petrol stations. In this study, 97 petrol station attendants (PSAs) in southern China were recruited for an assessment of the health risk associated with inhalation exposure to MTBE. The personal exposure levels of MTBE were analyzed by Head Space Solid Phase Microextraction GC/MS, and the demographic characteristics of the PSAs were investigated. Cancer and non-cancer risks were calculated with the methods recommended by the United States Environmental Protection Agency. The results showed that the exposure levels of MTBE in operating workers were much higher than among support staff (p < 0.01) and both were lower than 50 ppm (an occupational threshold limit value). The calculated cancer risks (CRs) at the investigated petrol stations was 0.170 to 0.240 per 106 for operating workers, and 0.026 to 0.049 per 106 for support staff, which are below the typical target range for risk management of 1 × 10(-6) to 1 × 10(-4); The hazard quotients (HQs) for all subjects were <1. In conclusion, our study indicates that the MTBE exposure of PSAs in southern China is in a low range which does not seem to be a significant health risk.

Overexpression of miR-221 in peripheral blood lymphocytes in petrol station attendants: A population based cross-sectional study in southern China.

Benzene is a recognized environmental leukemogen, however, the mechanisms for its carcinogenesis have not been fully elucidated. Recently, miR-221, a suggested oncogene involved in a number of malignancies, has been detected with elevated expression levels in blood cells of patients with leukemia. To explore whether benzene exposure has an effect on the expression of miR-221, a population based cross-sectional study was conducted in southern China, with 97 petrol station attendants as the exposure group and 103 general residents as the control group. Plasma benzene was analyzed by using GC∖MS. miR-221 in peripheral blood lymphocytes were measured by qRT-PCR and the ΔCt value for each sample was calculated by normalizing the Ct value for miR-221 with U6 RNA (i.e., ΔCt = CtmiR-221 - CtU6). Potential confounding factors were taken into account. Pearson correlation, univariate and multivariate logistic regression were performed in statistical analysis. The results showed that the air concentrations of benzene were significantly higher in petrol stations than in control sites (P < 0.05); The levels of benzene and miR-221 in exposure group were both significantly higher than in control group (P < 0.05) and there was a significant positive correlation between the two indexes (r = 0.851, P < 0.05); An association between benzene levels and the ΔCt values for miR-221 was identified by univariate and multivariate logistic analysis (OR 0.274; 95%CI 0.117, 0.396). Our investigation indicates that benzene exposure may be related to elevated miR-221 expression in human lymphocytes.

Histone modifications contribute to cellular replicative and hydrogen peroxide-induced premature senescence in human embryonic lung fibroblasts.

Histone modifications are major post-translational mechanisms responsible for regulation of gene transcription involved in cellular senescence. By using immunofluorescence and Western blot, we showed that the global acetylated levels of histone H3 and H4 were significantly reduced in both replicative and premature senescence of human embryonic lung fibroblasts. However the whole trimethylated level of histone H4 lysine 20 was higher in senescent cells. The alterations in the mRNA and protein levels of histone acetyltransferases (HATs), histone methyltransferase (HMT), and histone deacetylases (HDACs) indicate that differential expression exists between replicative and premature senescent cells. Meanwhile, the reduced activity of HDACs was accompanied by cellular senescence. By employing the quantitative chromatin immunoprecipitation assay in detecting specific histone modifications in senescence-related genes including p53 and p16, it was demonstrated that the mRNA expression of p53 was associated with increased H4 acetylation in replicative senescence and increased H4 acetylation and trimethylation of histone H3 at lysine 4 (H3K4me3) in premature senescence. Both acetylation and trimethylation of H3 were involved in replicative senescence, while the acetylation of histone H3 and H4 was predominant in premature senescence, contributing to the mRNA expression of p16. In summary, the global hypoacetylation of histone H3 and H4 and the hypertrimethylation of histone H4 lysine 20 account for epigenetic characteristics in senescence, controlled by HATs, HMT, and HDACs differentially between replicative and premature senescence. Taken together, these findings suggest that the specific histone modifications are involved in regulating the expression of genes related to senescence of human embryonic lung fibroblasts.

[Epidemiological study on hyperuricemia and gout in Foshan areas, Guangdong province].

OBJECTIVE: To determine the prevalence rates and risk factors of hyperuricemia (HUA) and gout among residents aged over 20 years in Foshan areas. METHODS: A randomly stratified cluster sampling was conducted, and 7403 inhabitants were investigated on their prevalence rates of HUA and gout. RESULTS: (1) The prevalence of HUA was 15.09%, and the standardized rate was 15.27%, in which the prevalence in males was 19.90% and females was 10.54%. The prevalence of gout was 1.04% and the standardized rate was 1.08%, in which the prevalence in males was 1.73% and females was 0.39%. The prevalence of gout in patients with HUA was 6.89%. (2) Average serum uric acid was (336.4 ± 81.5) µmol/L, with (347.1 ± 88.6) µmol/L in males and (289.7 ± 78.6) µmol/L in females. The serum uric acid levels in male patients with HUA was higher than those in women. (3) Age, body mass index, systolic blood pressure, diastolic blood pressure, serum uric acid, blood sugar, triglyceride (TG), total cholesterol were significantly higher in patients with HUA and gout than in the normal group (P < 0.05 - 0.01). The incidence rates of patients with hyperuricemia and gout in the following indices as:overweight and obesity, high blood pressure, high blood sugar were significantly higher than those in the normal group (P < 0.05). Patients having gout in the following indices as age, TG, serum uric acid levels were significantly higher than the HUA group (P < 0.05). (4) Data from non-conditional logistic regression analysis showed that age, overweight, hypertension, diabetes, hyperlipidemia, use of diuretics, family history, alcohol uptake, eating seafood and drinking meat broth, post-menopausal women, and other factors were similar to those factors as patients with hyperuricemia. Tea, fresh vegetables, fruits seemed to be the protective factors. CONCLUSION: Both the prevalence rates of HUA and gout had significantly increased in Foshan areas in recent years. Restricting the intake of food with rich purine, alcohol intake as well as controlling obesity and blood pressure, improving the status of lipid metabolic disorder together with programs as hypertension control etc. were important measures in the strategies on prevention and treatment on hyperuricemia and gout.

Epigenetic silencing of O6-methylguanine DNA methyltransferase gene in NiS-transformed cells.

Nickel (Ni) compounds are potent carcinogens and can induce malignant transformation of rodent and human cells. To uncover the molecular mechanisms of nickel sulfide (NiS)-induced cell transformation, we investigated epigenetic alterations in a set of DNA repair genes. The silencing of the O(6)-methylguanine DNA methyltransferase (MGMT) gene locus and upregulation of DNA methyltransferase 1 (DNMT1) expression was specifically detected in NiS-transformed human bronchial epithelial (16HBE) cells. In addition, we noted epigenetic alterations including DNA hypermethylation, reduced histone H4 acetylation and a decrease in the ratio of Lys-9 acetylated/methylated histone H3 at the MGMT CpG island in NiS-transformed 16HBE cells. Meanwhile, we identified concurrent binding of methyl-CpG-binding protein 2, methylated DNA-binding domain protein 2 and DNMT1 to the CpG island of the MGMT promoter, demonstrating that these components collaborate to maintain MGMT methylation in NiS-transformed cells. Moreover, depletion of DNMT1 by introduction of a small hairpin RNA construct into NiS-transformed cells resulted in a 30% inhibition of cell proliferation and led to increased MGMT gene expression by reversion of the epigenetic modifications at the MGMT promoter region. MGMT suppression and hypermethylation at the CpG island of the MGMT promoter occurred 6 days after NiS treatment, indicating that epigenetic modifications of MGMT might be an early event in tumorigenesis. Taken together, these observations demonstrate that epigenetic silencing of MGMT is associated with DNA hypermethylation, histone modifications and DNMT1 upregulation, which contribute to NiS-induced malignant transformation.

Possible role of DNA polymerase beta in protecting human bronchial epithelial cells against cytotoxicity of hydroquinone.

OBJECTIVE: To explore the toxicological mechanism of hydroquinone in human bronchial epithelial cells and to investigate whether DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone. METHODS: DNA polymerase beta knock-down cell line was established via RNA interference as an experimental group. Normal human bronchial epithelial cells and cells transfected with the empty vector of pEGFP-C1 were used as controls. Cells were treated with different concentrations of hydroquinone (ranged from 10 micromol/L to 120 micromol/L) for 4 hours. MTT assay and Comet assay [single-cell gel electrophoresis (SCGE)] were performed respectively to detect the toxicity of hydroquinone. RESULTS: MTT assay showed that DNA polymerase beta knock-down cells treated with different concentrations of hydroquinone had a lower absorbance value at 490 nm than the control cells in a dose-dependant manner. Comet assay revealed that different concentrations of hydroquinone caused more severe DNA damage in DNA polymerase beta knock-down cell line than in control cells and there was no significant difference in the two control groups. CONCLUSIONS: Hydroquinone has significant toxicity to human bronchial epithelial cells and causes DNA damage. DNA polymerase beta knock-down cell line appears more sensitive to hydroquinone than the control cells. The results suggest that DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.