Repeated radon exposure induced ATM kinase-mediated DNA damage response and protective autophagy in mice and human bronchial epithelial cells.

OBJECTIVE: Radon ( 222 Rn) is a naturally occurring radioactive gas that has been closely linked with the development of lung cancer. In this study, we investigated the radon-induced DNA strand breaks, a critical event in lung carcinogenesis, and the corresponding DNA damage response (DDR) in mice and human bronchial epithelial (BEAS-2B) cells. METHODS: Biomarkers of DNA double-strand breaks (DSBs), DNA repair response to DSBs, ataxia-telangiectasia mutated (ATM) kinase, autophagy, and a cell apoptosis signaling pathway as well as cell-cycle arrest and the rate of apoptosis were determined in mouse lung and BEAS-2B cells after radon exposure. RESULTS: Repeated radon exposure induced DSBs indicated by the increasing expressions of γ-Histone 2AX (H2AX) protein and H2AX gene in a time and dose-dependent manner. Additionally, a panel of ATM-dependent repair cascades [i.e. non-homologous DNA end joining (NHEJ), cell-cycle arrest and the p38 mitogen activated protein kinase (p38MAPK)/Bax apoptosis signaling pathway] as well as the autophagy process were activated. Inhibition of autophagy by 3-methyladenine pre-treatment partially reversed the expression of NHEJ-related genes induced by radon exposure in BEAS-2B cells. CONCLUSIONS: The findings demonstrated that long-term exposure to radon gas induced DNA lesions in the form of DSBs and a series of ATM-dependent DDR pathways. Activation of the ATM-mediated autophagy may provide a protective and pro-survival effect on radon-induced DSBs.

Hepatotoxicity induced in rats by chronic exposure to F-53B, an emerging replacement of perfluorooctane sulfonate (PFOS).

A plethora of studies have shown the prominent hepatotoxicity caused by perfluorooctane sulfonate (PFOS), yet the research on the causality of F-53 B (an alternative for PFOS) exposure and liver toxicity, especially in mammals, is largely limited. To investigate the effects that chronic exposure to F-53 B exert on livers, in the present study, male SD rats were administrated with F-53 B in a certain dose range (0, 1, 10, 100, 1000 µg/L, eight rats per group) for 6 months via drinking water and the hepatotoxicity resulted in was explored. We reported that chronic exposure to 100 and 1000 µg/L F-53 B induced remarkable histopathological changes in liver tissues such as distinct swollen cells and portal vein congestion. In addition, the increase of cytokines IL-6, IL-2, and IL-8 upon long-term administration of F-53 B demonstrated the high level of inflammation. Moreover, F-53 B exposure was revealed to disrupt the lipid metabolism in the rat livers, mainly manifesting as the upregulation of some proteins involved in lipid synthesis and degradation, including ACC, FASN, SREBP-1c as well as ACOX1. These findings provided new evidence for the adverse effects caused by chronic exposure to F-53 B in rodents. It is crucial for industries, regulatory agencies as well as the public to remain vigilant about the adverse health effects associated with the emerging PFOS substitutes such as F-53 B. Implementation of regular monitoring and risk assessments is of great importance to alleviate environmental concerns towards PFOS alternatives exposure, and furthermore, to minimize the latent health risks to the public health.

Repeated radon exposure induced epithelial-mesenchymal transition-like transformation via disruption of p53-dependent mitochondrial function.

Backgrouds: As a human carcinogen, radon and its progeny are the second most important risk factor for lung cancer after smoking. The tumor suppressor gene, p53, is reported to play an important role in the maintenance of mitochondrial function. In this work, we investigated the association between p53 and p53-responsive signaling pathways and radon-induced carcinogenesis. Methods: After repeated radon exposure, the malignant characteristics, cell cycle arrest, cell apoptotic rate, adenosine triphosphate (ATP) content, reactive oxygen species (ROS) level, mitochondrial DNA (mtDNA) copy number as well as indicative biomarkers involved in mitochondrial energy metabolism were evaluated in BEAS-2B cells or BALB-c mouse lung tissue. Results: Radon exposure induced epithelial-mesenchymal transition (EMT)-like transformation in BEAS-2B cells, as indicated by increased cell proliferation and migration. Additional mitochondrial alterations, including decreased ATP content, increased ROS levels, mtDNA copy numbers, cell apoptosis, and G2/M cell cycle arrest were observed. Radon exposure caused an energy generation shift from aerobic respiration to glycolysis as reflected by increased expression of TIGAR and p53R2 proteins and decreased expression of SCO2 protein in BEAS-2B cells, and increased expression of p53, SCO2 and TIGAR proteins in mouse lung tissue, respectively. The effects of p53 deficiency on the prevention of mitochondrial dysfunction suggested a protective role of p53 in radon-induced malignant-like features in BEAS-2B cells. Conclusions: Repeated radon exposure induced EMT-like transformation in BEAS-2B cells via disruption of mitochondrial function. Activation of p53 and p53-responsive signaling pathways in BEAS-2B cells and BALB-c mice may confer a protective mechanism for radon-induced lung injury.

Protection of melatonin against long-term radon exposure-caused lung injury.

Radon is one of the major pathogenic factors worldwide. Recently, epidemiological studies have suggested that radon exposure plays an important role in lung injury, which could further cause cancer. However, the toxic effects and underlying mechanism on lung injury are still not clear. Here, we identified the detailed toxic effects of long-term radon exposure. Specifically, the manifestations were inflammatory response and cell apoptosis in dose- and time-dependent manners. In detail, it caused the mitochondrial dysfunction and oxidative stress as determined by the abnormal levels of mitochondrial DNA copy number, adenosine triphosphate, mitochondrial membrane potential, superoxide dismutase, and cycloxygenase-2. Furthermore, we found that melatonin treatment ameliorated mitochondrial dysfunction and attenuated the levels of oxidative stress caused by long-term radon exposure, which could further inhibit the lung tissue apoptosis as determined by the decreased levels of cleaved caspase 3. Our study would provide potential therapeutic application of melatonin on lung tissue injury caused by long-term radon exposure.

SQR mediates therapeutic effects of H2S by targeting mitochondrial electron transport to induce mitochondrial uncoupling.

Hydrogen sulfide (H2S) is a gasotransmitter and a potential therapeutic agent. However, molecular targets relevant to its therapeutic actions remain enigmatic. Sulfide-quinone oxidoreductase (SQR) irreversibly oxidizes H2S. Therefore, SQR is assumed to inhibit H2S signaling. We now report that SQR-mediated oxidation of H2S drives reverse electron transport (RET) at mitochondrial complex I, which, in turn, repurposes mitochondrial function to superoxide production. Unexpectedly, complex I RET, a process dependent on high mitochondrial membrane potential, induces superoxide-dependent mitochondrial uncoupling and downstream activation of adenosine monophosphate-activated protein kinase (AMPK). SQR-induced mitochondrial uncoupling is separated from the inhibition of mitochondrial complex IV by H2S. Moreover, deletion of SQR, complex I, or AMPK abolishes therapeutic effects of H2S following intracerebral hemorrhage. To conclude, SQR mediates H2S signaling and therapeutic effects by targeting mitochondrial electron transport to induce mitochondrial uncoupling. Moreover, SQR is a previously unrecognized target for developing non-protonophore uncouplers with broad clinical implications.

Nonylphenol induces pancreatic damage in rats through mitochondrial dysfunction and oxidative stress.

The organic alkylphenol 4-nonylphenol (NP) is regarded to be an endocrine disrupting chemical (EDC), one of the widely diffused and stable environmental contaminants. Due to its hydrophobicity and long half-life, NP can easily accumulate in living organisms, including humans, where it displays a series of toxic effects. It has been widely reported that NP affects male reproduction. In addition, there is increasing evidence suggesting that NP is detrimental to various organs, including the pancreas. This study investigated the adverse effects of NP exposure on the pancreas. Sprague-Dawley rats were treated with different doses of NP for 90 consecutive days. The data suggested that the body weights of the rats treated with NP decreased, and the highest dose of NP treatment (180 mg kg-1) dramatically increased water consumption by rats. Meanwhile, H&E staining and immunohistochemistry indicated that islets in the pancreases shrunk when the rats were treated with the indicated doses of NP. TUNEL staining demonstrated that NP exposure up-regulated the level of apoptosis in the pancreases in a dose-dependent manner. Besides this, NP exposure inhibited the secretion of insulin and disrupted glucose tolerance. The levels of reactive oxygen species (ROS) and intracellular calcium ([Ca2+]i) in the islets were up-regulated in the groups of rats treated with NP, but the levels of Mitochondrial Membrane Potential (MMP) were down-regulated. These results suggest that NP-induced pancreatic damage in rats occurs through mitochondrial dysfunction and oxidative stress, which causes disruption of glucose tolerance and decrease in insulin secretion.

Cytogenetic evaluation for the genotoxicity of bisphenol-A in Chinese hamster ovary cells.

Bisphenol A (BPA), identified as an endocrine disruptor, is an important man-made compound used in a wide range of consumer products. The MTT assay, comet assay, micronucleus test, chromosome aberration test, and Ames assay were conducted to assess the cytotoxic, genotoxic, cytogenetic effects, and mutagenic activity of BPA. After BPA exposure, we showed significant increases in cytotoxicity and level of DNA damage indicated by Olive tail moment, tail length, and % tail DNA in a similar dose- and time-dependent manner. Significant increases in micronucleus frequency and conventional chromosome aberrations were also observed after BPA treatment. The major types of structural aberrations were breaks, gaps, and fragments. However, no positive mutagenic activity of BPA was observed in any of the tester strains. Taken together, the data obtained in this study clearly demonstrated that BPA is not mutagenic but could exhibit significant genotoxic and cytogenetic effects in Chinese hamster ovary cells.

Pharmacokinetics, tissue distribution, and metabolites of a polyvinylpyrrolidone-coated norcantharidin chitosan nanoparticle formulation in rats and mice, using LC-MS/MS.

A novel formulation containing polyvinylpyrrolidone (PVP) K(30)-coated norcantharidin (NCTD) chitosan nanoparticles (PVP-NCTD-NPs) was prepared by ionic gelation between chitosan and sodium tripolyphosphate. The average particle size of the PVP-NCTD-NPs produced was 140.03 ± 6.23 nm; entrapment efficiency was 56.33% ± 1.41%; and drug-loading efficiency was 8.38% ± 0.56%. The surface morphology of NCTD nanoparticles (NPs) coated with PVP K(30) was characterized using various analytical techniques, including X-ray diffraction and atomic force microscopy. NCTD and its metabolites were analyzed using a sensitive and specific liquid chromatography-tandem mass spectrometry method with samples from mice and rats. The results indicated the importance of the PVP coating in controlling the shape and improving the entrapment efficiency of the NPs. Pharmacokinetic profiles of the NCTD group and PVP-NCTD-NP group, after oral and intravenous administration in rats, revealed that relative bioavailabilities were 173.3% and 325.5%, respectively. The elimination half-life increased, and there was an obvious decrease in clearance. The tissue distribution of NCTD in mice after the intravenous administration of both formulations was investigated. The drug was not quantifiable at 6 hours in all tissues except for the liver and kidneys. The distribution of the drug in the liver and bile was notably improved in the PVP-NCTD-NP group. The metabolites and excretion properties of NCTD were investigated by analyzing rat feces and urine samples, collected after oral administration. A prototype drug and two metabolites were found in the feces, and seven metabolites in the urine. The primary elimination route of NCTD was via the urine. The quantity of the parent drug eliminated in the feces of the PVP-NCTD-NP group, was 32 times greater than that of the NCTD group, indicating that the NPs dramatically increased the reduction quantity from liver to bile. We conclude that PVP-NCTD-NPs are an adequate formulation for enhancing the absorption of NCTD, and significantly improving therapeutic effects targeting the hepatic system. Decarboxylation and hydroxylation were the dominant metabolic pathways for NCTD. Metabolites were mainly excreted into rat kidney and finally into urine.

[Molecular mechanism of radiosensitizing effect of paclitaxel].

BACKGROUND & OBJECTIVE: Paclitaxel is a radiosensitizer which may stabilize microtubules, block the G2/M phase of the cell cycle and thus modulate the radioresponsiveness of tumor cells. However, its potential molecular mechanisms of radiosensitization have not been well understood yet. This study was to investigate the radiosensitizing effect of paclitaxel on human oral epithelium carcinoma (KB) cell line and to explore the molecular mechanism of radiosensitization. METHODS: The survival of KB cells following the treatment with paclitaxel and/or radiation was determined by colony-forming assay. The radiosensitizing effect was evaluated by calculating the sensitizing enhancement ratio (SER) with multi-target single hit model. The cell cycle distribution was analyzed by flow cytometry. Differentially expressed genes related to paclitaxel radiosensitization were screened using human Oligo microarray. Expressions of protein regulating cytokinesis 1 (PRC1) and cyclin B2 genes were confirmed by real-time quantitative PCR. RESULTS: The proliferation of KB cells was significantly inhibited by paclitaxel combined with ionizing radiation. The SERD0 and SERDq were (2.40 +/- 1.87) and (12.23 +/- 2.81) respectively, when the concentration of paclitaxel was 20 nmol/l. After the treatment with paclitaxel in combination with irradiation, the percentage of G1 phase cells decreased from (48.32 +/- 2.40)% to (15.73 +/- 7.00)% (P<0.01), and the percentage of G2/M phase cells increased from (13.66 +/- 2.16)% to (52.51 +/- 5.02)% (P<0.01). In total 176 differentially expressed genes were identified to be related to paclitaxel radiosensitization. Ten genes were found to regulate cell division, two of which were up-regulated and eight were down-regulated after the treatment. Moreover, the expression of PRC1 and cyclin B2 was decreased. CONCLUSION: The radiosensitizing effect of paclitaxel on KB cells may be due to the down-regulated expression of PRC1 and cyclin B2, resulting in inhibition of mitotic spindle formation and cell necrosis.

Effect of Nd3+ ion on carboxylation activity of ribulose-1,5-bisphosphate carboxylase/oxygenase of spinach.

Neodymium (Nd), as a member of rare earth elements, proved to enhance the photosynthesis rate and organic substance accumulation of spinach through the increase in carboxylation activity of Rubisco. Although the oxygenase activity of spinach Rubisco was slightly changed with the Nd(3+) treatment, the specific factor of Rubisco was greatly increased. It was partially due to the promotion of Rubisco activase (R-A) activity but mainly to the formation of Rubisco-Rubisco activase super-complex, a heavier molecular mass protein (about 1200kD) comprising both Rubisco and Rubisco activase. This super-complex was found during the extraction procedure of Rubisco by the gel electrophoresis and Western-blot studies. The formation of Rubisco-R-A super-complex suggested that the secondary structure of the protein purified from the Nd(3+)-treated spinach was different from that of the control. Extended X-ray absorption fine structure study of the 'Rubisco' purified from the Nd(3+)-treated spinach revealed that Nd was bound with four oxygen atoms and two sulfur atoms of amino acid residues at the Nd-O and Nd-S bond lengths of 2.46 and 2.89A, respectively.