Interleukin-9 promotes EMT-mediated PM2.5-induced pulmonary fibrosis by activating the STAT3 pathway.

This study investigated the impact of PM2.5 on promoting EMT in PM2.5-induced pulmonary fibrosis (PF) development and explored molecular mechanisms of the IL-9/STAT3/Snail/TWIST1 signaling pathway in PF owing to PM2.5. Four groups of male SD rats were formed: control (0 mg/kg.bw), low (1 mg/kg.bw), medium (5 mg/kg.bw), and high-dose (25 mg/kg.bw) PM2.5 groups. Experimental rats were subjected to PM2.5 exposure via intratracheal instillation, given once weekly for 16 weeks. 24 h after the final exposure, blood, BALF, and lung tissues were collected. Pulmonary epithelial cells underwent cultivation and exposure to varying PM2.5 concentrations with/without inhibitors for 24 h, after which total protein was extracted for relevant protein assays. The findings demonstrated that PM2.5 damaged lung tissue to different degrees and led to PF in rats. Rats subjected to PM2.5 exposure exhibited elevated concentrations of IL-9 protein in both serum and BALF, and elevated levels of IL-9 and its receptor, IL-9R, in lung tissues, compared to control counterparts. Furthermore, PM2.5-exposed groups demonstrated significantly augmented protein levels of p-STAT3, Snail, TWIST1, Vimentin, COL-I, and α-SMA, while displaying notably diminished levels of E-Cadherin compared to control group. The same findings were observed in PM2.5-treated cells. In BEAS-2B cells co-treated with Stattic (STAT3 inhibitor) and PM2.5, the opposite results occurred. Similar results were obtained for cells co-treated with IL-9-neutralizing antibody and PM2.5. Our findings suggest PM2.5 mediates PF development by promoting IL-9 expression, leading to STAT3 phosphorylation and upregulation of Snail and TWIST1 expression, triggering EMT occurrence and progression in lung epithelial cells.

TXNIP-NLRP3-GSDMD axis-mediated inflammation and pyroptosis of islet ß-cells is involved in cigarette smoke-induced hyperglycemia, which is alleviated by andrographolide.

Epidemiologic surveys have indicated that cigarette smoking is an important risk factor for diabetes, but its mechanisms remain unclear. Andrographolide, an herb traditionally utilized in medicine, provides anti-inflammatory benefits for various diseases. In the present work, 265 patients with Type 2 diabetes (T2D) were investigated, and male C57BL/6 mice were exposed to cigareete smoke (CS) and/or to intraperitoneally injected andrographolide for 3 months. To elucidate the mechanism of CS-induced hyperglycemia and the protective mechanism of andrographolide, MIN6 cells were exposed to cigarette smoke extract (CSE) and/or to andrographolide. Our data from 265 patients with T2D showed that urinary creatinine and serum inflammatory cytokines (interleukin 6 (IL-6), IL-8, IL-1ß, and tumor necrosis factor α (TNF-α)) increased with smoking pack-years. In a mouse model, CS induced hyperglycemia, decreased insulin secretion, and elevated inflammation and pyroptosis in ß-cells of mice. Treatment of mice with andrographolide preserved pancreatic function by reducing the expression of inflammatory cytokines; the expression of TXNIP, NLRP3, cleaved caspase 1, IL-1ß; and the N-terminal of gasdermin D (GSDMD) protein. For MIN6 cells, CSE caused increasing secretion of the inflammatory cytokines IL-6 and IL-1ß, and the expression of TXNIP and pyroptosis-related proteins; however, andrographolide alleviated these changes. Furthermore, silencing of TXNIP showed that the blocking effect of andrographolide may be mediated by TXNIP. In sum, our results indicate that CS induces hyperglycemia through TXNIP-NLRP3-GSDMD axis-mediated inflammation and pyroptosis of islet ß-cells and that andrographolide is a potential therapeutic agent for CS-induced hyperglycemia.

A novel mitochondria-targeting compound exerts therapeutic effects against melanoma by inducing mitochondria-mediated apoptosis and autophagy in vitro and in vivo.

Melanoma is the most invasive skin cancer, with a high mortality rate. However, existing therapeutic drugs have side effects, low reactivity, and lead to drug resistance. As the power source in cells, mitochondria play an important role in the survival of cancer cells and are an important target for tumor therapy. This study aimed to develop a new anti-melanoma compound that targets mitochondria, evaluate its effect on the proliferation and metastasis of melanoma cells, and explore its mechanism of action. The novel mitochondria-targeting compound, SCZ0148, was synthesized by modifying the structure of cyanine. Then, A375 and B16 cells were incubated with different concentrations of SCZ0148, and different doses of SCZ0148 were administered to A375 and B16 xenograft zebrafish. The results showed that SCZ0148 targeted mitochondria, had dose- and time-dependent effects on the proliferation of melanoma cell lines, and had no obvious side effects on normal cells. In addition, SCZ0148 induced melanoma cell apoptosis through the reactive oxygen species-mediated mitochondrial pathway of apoptosis and promoted autophagy. SCZ0148 significantly inhibited the migration of melanoma cells via a matrix metalloprotein 9-mediated pathway. Similarly, SCZ0148 inhibited melanoma cell proliferation in a concentration-dependent manner in vivo. In summary, SCZ0148 may be a novel anti-melanoma compound that targets mitochondria.

Vitamin B complex blocks the dust fall PM2 .5 -induced acute lung injury through DNA methylation in rats.

This study aimed to explore whether vitamin B complex (folic acid, B6 , and B12 ) could avert DNA methylation changes associated with inflammation induced by acute PM2.5 exposure. Sprague-Dawley rats were administered by gavage with different concentrations of vitamin B complex once a day for 28 days, and then by intratracheal instillation with saline or PM2.5 once every 2 days for three times. Vitamin B continued to be taken during the PM2.5 exposure. Rats were sacrificed 24 h after the last exposure. The results showed that vitamin B complex could block the pathological changes and injury in lungs induced by PM2.5 . Meanwhile, vitamin B complex could prevent the abnormal DNA methylation of IL-4 and IFN-γ to antagonize the imbalance of IL-4/IFN-γ associated with inflammation. It was further found that vitamin B complex could regulate DNA methyltransferases (DNMTs) and increase the S-adenosylmethionine (SAM)/S-Adenosyl-L-homocysteine (SAH) ratio to reverse the hypomethylation of genomic DNA and the abnormal DNA methylation of IL-4 and IFN-γ. In conclusion, vitamin B complex has a protective effect on acute lung injury by attenuating abnormal DNA methylation induced by PM2.5 in rats. This study may provide a new insight into the physiological function of vitamin B to prevent the health effects induced by PM2.5 .

α-Lipoic acid protects against microcystin-LR induced hepatotoxicity through regeneration of glutathione via activation of Nrf2.

Microcystins (MCs), as the most dominant bloom-forming strains in eutrophic surface water, can induce hepatotoxicity by oxidative stress. Alpha-lipoic acid (α-LA) is a super antioxidant that can induce the synthesis of antioxidants, such as glutathione (GSH), by nuclear factor erythroid 2-related factor 2 (Nrf2). However, the potential molecular mechanism of α-LA regeneration of GSH remains unclear. The present study aimed to investigate whether α-LA could reduce the toxicity of MCs induced in human hepatoma (HepG2), Bel7420 cells, and BALB/c mice by activating Nrf2 to regenerate GSH. Results showed that exposure to 10 µM microcystin-leucine arginine (MC-LR) reduced viability of HepG2 and Bel7402 cells and promoted the formation of reactive oxygen species (ROS) compared with untreated cells. Moreover, the protection of α-LA included reducing the level of ROS, increasing superoxide dismutase activity, and decreasing malondialdehyde. Levels of reduced glutathione (rGSH) and rGSH/oxidized glutathione were significantly increased in cells cotreated with α-LA and MC-LR compared to those treated with MC-LR alone, indicating an ability of α-LA to attenuate oxidative stress and MC-LR-induced cytotoxicity by increasing the amount of rGSH. α-LA can mediate GSH regeneration through the Nrf2 pathway under the action of glutathione reductase in MC-LR cell lines. Furthermore, the data also showed that α-LA-induced cytoprotection against MC-LR is associated with Nrf2 mediate pathway in vivo. These findings demonstrated the potential of α-LA to resist MC-LR-induced oxidative damage of liver.

NF-κB-regulation of miR-155, via SOCS1/STAT3, is involved in the PM2.5-accelerated cell cycle and proliferation of human bronchial epithelial cells.

Air pollution, especially fine particulate matter (PM2.5, particles <2.5 µm in size), induces adverse health effects on the respiratory system. Uncontrolled proliferation of bronchial epithelial cells, resulting from deregulated cell cycle progression, contributes to pulmonary homeostatic imbalance. Although dysregulation of miRNAs is involved in a variety of pathophysiologic processes, the role of miRNAs in lung injury caused by PM2.5 is unclear. In the present study, we found that different concentrations of PM2.5 caused a biphasic effect on proliferation of human bronchial epithelial (HBE) cells. PM2.5 induced an aberrant cell cycle and proliferation of HBE cells, and up-regulated miR-155 levels with a concentration-dependent manner. High miR-155 expression, mediated by NF-κB activation, produced an accelerated G1/S phase and cell proliferation though the STAT3 pathway, which targeted SOCS1. These findings indicate that NF-κB-mediated miR-155 induces an altered cell cycle through epigenetic modulation of the SOCS1/STAT3 signaling pathway and provide a mechanism for the biphasic effect of different concentrations of PM2.5 in inducing respiratory injury.

Awareness, attitude and behavior regarding proton pump inhibitor among medical staff in the Southwest of China.

BACKGROUND: Proton pump inhibitors (PPIs) are one of the most frequently prescribed classes of drug in the world and there is a growing number of publications on correct versus incorrect use of PPIs worldwide. The knowledge of PPIs among the medical staff is essential for improving the rationality of PPI application. The present study aimed to investigate awareness, attitude and behavior toward PPI use among medical staff in the Southwest of China. METHODS: The present descriptive-analytical study was conducted on 900 medical staff from three professional groups (300 doctors, 300 nurses and 300 pharmacists) in China. The study data were collected through a self-designed questionnaire which included demographics, awareness, attitude and behavior toward PPI use. The study was carried out in 22 hospitals in Luzhou between February and June 2018. RESULTS: Of 900 surveys issued, 851valid questionnaires (295doctors, 268 nurses and 288 pharmacists) were returned. Of all respondents, 33.25% were men and 66.75% were women. The score related to PPI awareness score of medical staff was low (59.47 ± 15.75). The level of awareness of pharmacist was significantly higher than that of doctors and nurses (P < 0.01), which was related to gender, age, occupation, educational level, professional title, hospital nature and hospital grade. Similarly, on the attitude towards PPI use, the pharmacists scored also significantly higher than doctors and the nurses (P < 0.01). Three hundred eighty-one of 851 medical staff had used PPI in the past 1 year, of which omeprazole was the most widely used. Among doctors, nurses and pharmacists, the usage rate of PPI was 50.85, 42.16, 40.97%, respectively. The use frequency was related to occupation and professional title. The score about the behavior toward PPIs of the nurses was also significantly lower than that of doctors and pharmacists (P < 0.01). CONCLUSIONS: The study indicated that the medical staff lack of awareness concerning rational use of PPI in China, especially nurse. Thus, it is necessary to call for action on the improvement of PPI awareness and medication-taking behaviors to reduce PPI overuse and to promote the rationality of PPI application.

Man-made mineral fiber effects on the expression of anti-oncogenes P53 and P16 and oncogenes C-JUN and C-FOS in the lung tissue of Wistar rats.

Man-made mineral fibers (MMMFs) are substitutes for asbestos. MMMFs are widely used as insulation, but their molecular mechanisms underlying the tumorigenic effects in vivo have been poorly studied. For this reason, this work aimed to explore the properties and carcinogenic molecular mechanisms of MMMFs. The three MMMFs, rock wool (RW), glass fibers (GFs), and ceramic fibers (CFs), were prepared into respirable dust. Particle size, morphology, and chemical composition were analyzed by laser particle analyzer, scanning electron microscope, and X-ray fluorescence spectrometer, respectively. The Wistar rats were administered multiple intratracheal instillations of three MMMFs once a month. Then, several parameters (e.g. body mass, lung mass, and lung histology) were measured at 1, 3, and 6 months. After that, levels of P53, P16, C-JUN, and C-FOS mRNA and protein were measured by quantitative real-time reverse transcription polymerase chain reaction and Western blotting. This work found that exposure to MMMFs could influence the growth of body mass and increase lung mass. General conditions showed white nodules and irregular atrophy. In addition, MMMFs could lead to inactivation of anti-oncogene P16 and activation of proto-oncogenes (C-JUN and C-FOS) in the mRNA and protein levels, in which GF and CF were more obvious compared with RW. The effect of MMMFs was different, which may be related to the physical and chemical characteristics of different MMMFs. In conclusion, MMMFs (GF and CF) could induce an unbalanced expression of cancer-related genes in the lung tissues of rats. The understanding of the determinants of toxicity and carcinogenicity provides a scientific basis for developing and introducing new safer MMMF products, and the present study provides some useful insights into the carcinogenic mechanism of MMMFs.

The mechanism of interaction between tri-para-cresyl phosphate and human serum protein: A multispectroscopic and in-silico study.

Organophosphate flame retardants (OPFRs) pose the significant risks to the environment and human health and have become a serious public health issue. Tricresyl phosphates (TCPs), a group of aryl OPFRs, exhibit neurotoxicity and endocrine disrupting toxicity. However, the binding mechanisms between TCPs and human serum albumin (HSA) remain unknown. In this study, through fluorescence and ultraviolet-visible (UV-vis) absorption spectroscopy, molecular docking and molecular dynamics (MD), tri-para-cresyl phosphate (TpCP) was selected to explore potential interactions between HSA and TCPs. The results of the fluorescence spectroscopy demonstrated that a decrease in the fluorescence intensity of HSA and a blue shift were observed with the increasing concentrations of TpCP. The binding constant (Ka) was 2.575 × 104 L/mol, 4.701 × 104 L/mol, 5.684 × 104 L/mol and 9.482 × 104 L/mol at 293 K, 298 K, 303 K, and 310 K, respectively. The fluorescence process between HSA and TpCP involved a mix of static and dynamic quenching mechanism. The gibbs free energy (ΔG0) of HSA-TpCP system was -24.452 kJ/mol, -25.907 kJ/mol, -27.363 kJ/mol, and - 29.401 kJ/mol at 293 K, 298 K, 303 K, and 310 K, respectively, suggesting that the HSA-TpCP reaction was spontaneous. The enthalpy change (ΔH0) and thermodynamic entropy change (ΔS0) of the HSA-TpCP system were 60.83 kJ/mol and 291.08 J/(mol·>k), respectively, indicating that hydrophobic force was the major driving force in the HSA-TpCP complex. Furthermore, multispectral analysis also revealed that TpCP could alter the microenvironment of tryptophan residue and the secondary structure of HSA and bind with the active site I of HSA. Molecular docking and MD simulations confirmed that TpCP could spontaneously form a stable complex with HSA, which was consistent with the fluorescence experimental results. This study provides novel insights into the mechanisms of underlying the transportation and distribution of OPFRs in humans.

A proteomic study on gastric impairment in rats caused by microcystin-LR.

Microcystins (MCs) are the most common cyanobacterial toxins. Epidemiological investigation showed that exposure to MCs can cause gastro-intestinal symptoms, gastroenteritis and gastric cancer. MCs can also accumulate in and cause histopathological damage to stomach. However, the exact mechanisms by which MCs cause gastric injury were unclear. In this study, Wistar rats were administrated 50, 75 or 100 µg microcystin-LR (MC-LR)/kg, body mass (bm) via tail vein, and histopathology, response of anti-oxidant system and the proteome of gastric tissues at 24 h after exposure were studied. Bleeding of fore-stomach and gastric corpus, inflammation and necrosis in gastric corpus and exfoliation of mucosal epithelial cells in gastric antrum were observed following acute MC-LR exposure. Compared with controls, activities of superoxide dismutase (SOD) were significantly greater in gastric tissues of exposed rats, while activities of catalase (CAT) were less in rats administrated 50 µg MC-LR/kg, bm, and concentrations of glutathione (GSH) and malondialdehyde (MDA) were greater in rats administrated 75 or 100 µg MC-LR/kg, bm. These results indicated that MC-LR could disrupt the anti-oxidant system and cause oxidative stress. The proteomic results revealed that MC-LR could affect expressions of proteins related to cytoskeleton, immune system, gastric functions, and some signaling pathways, including platelet activation, complement and coagulation cascades, and ferroptosis. Quantitative real-time PCR (qRT-PCR) analysis showed that transcriptions of genes for ferroptosis and gastric function were altered, which confirmed results of proteomics. Overall, this study illustrated that MC-LR could induce gastric dysfunction, and ferroptosis might be involved in MC-LR-induced gastric injury. This study provided novel insights into mechanisms of digestive diseases induced by MCs.

α-LA attenuates microcystin-LR-induced hepatocellular oxidative stress in mice through Nrf2-mediated antioxidant and detoxifying enzymes.

Microcystins constitute a class of toxins synthesized by cyanobacteria and are known to inflict significant damage on the antioxidant defense system of living organisms, primarily targeting the liver. α-Lipoic acid (α-LA) is universally recognized as a potent antioxidant in biological systems. It exerts its beneficial effects through multiple mechanisms-directly neutralizing reactive oxygen species (ROS) and free radicals, and indirectly enhancing antioxidant defenses by facilitating the regeneration of glutathione (GSH). However, the precise modus operandi of α-LA's protective effect against Microcystin-LR-induced hepatotoxicity remains incompletely elucidated. The present study, therefore, employed α-LA to explore its protective role against Microcystin-LR exposure in mice. A model of Microcystin-LR-induced hepatic injury was established by administering Microcystin-LR into the peritoneal cavity of BALB/c mice daily over a two-week period. Thereafter, BALB/c mice were pre-treated with varying concentrations of α-LA via oral gavage for a duration of 7 days, followed by a 7-day exposure to Microcystin-LR. Our findings reveal that α-LA pre-treatment significantly mitigated hepatic pathologies in Microcystin-LR-exposed mice. Furthermore, α-LA administration led to a notable elevation in the activities and expression levels of nuclear factor erythroid 2-related factor 2, superoxide dismutase, glutathione peroxidase, glutathione S-transferase, and glutathione-indicative of its antioxidative capacity. Concurrently, a significant decrease was observed in the activities and expression levels of malondialdehyde and cytochrome P450 2E1. Consequently, α-LA emerges as a promising therapeutic candidate for the amelioration of liver oxidative damage subsequent to Microcystin-LR exposure.

Dust fall PM2.5-induced lung inflammation in rats is associated with hypermethylation of the IFN-γ gene promoter via the PI3K-Akt-DNMT3b pathway.

Inflammation is one of the major adverse effects of fine particulate matter (PM2.5) on the lung system; however, its mechanisms remain unclear. Rats were exposed to different concentrations of PM2.5 to investigate the mechanism of short-term exposure-induced lung inflammation. The regulation of PI3K-Akt and DNA methyltransferase 3b (DNMT3b) was assessed by using a PI3K inhibitor and a DNA methyltransferase inhibitor. We found that PM2.5 could decrease interferon-γ (IFN-γ) levels and increase interleukin 4 (IL-4), IL-5 and IL-13 levels in bronchoalveolar lavage fluid (BALF) to promote eosinophil infiltration and eventually lead to allergic pulmonary inflammation. Moreover, the CpG island methylation rate of the IFN-γ promoter and the protein expression of DNMT3b, PI3K and p-Akt were increased in lung tissues after PM2.5 exposure. Both inhibitors reversed the CpG island hypermethylation of IFN-γ. In conclusion, in PM2.5-induced lung injury, the activated PI3K-Akt pathway, via an increase in DNMT3b expression, is involved in CpG hypermethylation of the IFN-γ gene promoter.

ZWZ-3, a Fluorescent Probe Targeting Mitochondria for Melanoma Imaging and Therapy.

The increased drug resistance and metastasis of melanoma resulted in poor prognosis of patients. Here, we designed and synthesized a novel hemicyanine-based fluorescent probe ZWZ-3, and investigated its application for melanoma imaging and treatment both in vitro and in vivo. ZWZ-3 preferentially accumulated in melanoma cells via a process that depended on the organic anion-transporting polypeptide (OATP), which targeted mitochondria on the hemicyanine cationic nitrogen. In addition, we investigated the effect and molecular mechanism of ZWZ-3 in melanoma. In vitro studies showed that ZWZ-3 promoted the generation of reactive oxygen species and induced mitochondrial-mediated cell apoptosis by upregulating Bax and activating caspase-3, caspase-9, and PARP. Importantly, ZWZ-3 also induced autophagy by upregulating LC-3II and Atg5 and downregulating P62. It significantly suppressed tumor growth of A375 xenograft tumor in mice without notable side effects. Histological and immunohistochemical analyses revealed that ZWZ-3 induced apoptosis and inhibited tumor cell proliferation. Thus, ZWZ-3 represents a novel theranostic agent that can be used to effectively targeting, detecting, and treating melanoma. It could also help monitoring disease progression and response to treatment.

CircRNA_0026344 via exosomal miR-21 regulation of Smad7 is involved in aberrant cross-talk of epithelium-fibroblasts during cigarette smoke-induced pulmonary fibrosis.

For smoking-induced pulmonary fibrosis (PF), a serious disease endangering human health, there is no effective clinical treatment. Aberrant epithelium-fibroblast cross-talk is involved in formation of the excessive extracellular matrix (ECM) that contributes to PF. Circular RNAs have been associated with various pulmonary diseases. However, the mechanisms of circRNAs in PF are not clear. Herein, our goals were to investigate the involvement of circRNA_0026344 in the aberrant epithelium-fibroblast cross-talk induced by cigarette smoke (CS) and to define its mechanism. Chronic exposure (16 weeks) of BALB/c mice to 500 mg/m3 CS induced lung injury and fibrosis in lung tissues. From HBE cells, circRNA_0026344 was selected by microarray analysis and verified as that with the most severe down-regulation caused by cigarette smoke extract (CSE). The regulatory relationship between circRNA_0026344 and miR-21 was assessed by use of bioinformatics, RNA pull-down assays, and qRT-PCR. We found that miR-21 binding sites were present in circRNA_0026344 and, in HBE cells, it could act as a sponge for miR-21. When pcDNA3.0-circRNA_0026344, a high expression plasmid of circRNA_0026344, was transfected into HBE cells, the CSE-induced up-regulation of miR-21 levels was reversed. In MRC-5 cells, HBE-secreted exosomal miR-21 decreased levels of Smad7 and activated the TGF-ß1/Smad3 pathway. By using the Targetscan database, the presence of species-conserved miR-21 binding sites in the Smad7 3'UTR region were predicted. We verified, by use of a luciferase reporter gene, that miR-21 bound to the 3'UTR region of Smad7 mRNA to inhibit its transcription. In conclusion, the results reveal that, in CS-induced pulmonary fibrosis, circRNA_0026344, via exosomal miR-21 regulation of Smad7, is involved in aberrant cross-talk of epithelium-fibroblasts. These results will be useful for the discovery of early biomarkers and for providing therapeutic targets for smoking-induced pulmonary fibrosis.

Long-term instillation to four natural representative chrysotile of China induce the inactivation of P53 and P16 and the activation of C-JUN and C-FOS in the lung tissues of Wistar rats.

Chrysotile is the only type of asbestos still widely exploited, and all kinds of asbestos including chrysotile was classified as a group I carcinogen by the IARC. There is a wealth of evidence that chrysotile can cause a range of cancers, including cancer of the lung, larynx, ovary, and mesothelioma. As the second largest chrysotile producer, China is at great risk of occupational exposure. Moreover, our previous experiment and some other studies have shown that the toxicity of mineral fibre from various mining areas may be different. To explore the oncogenic potential of chrysotile from different mining areas of China, Wistar rats were administered 0.5 mL chrysotile asbestos suspension of 2.0 mg/mL (from Akesai, Gansu; Mangnai, Qinghai; XinKang, Sichuan; and Shannan, Shaanxi) dissolved in saline by intratracheal instillation once-monthly and were sacrificed at 1 mo, 6 mo, and 12 mo. Our results found that chrysotile caused lung inflammation and lung tissue damage. Moreover, prolonged exposure of chrysotile can induce inactivation of the tumor suppressor gene P53 and P16 and activation of the protooncogene C-JUN and C-FOS both in the messenger RNA and protein level. In addition, chrysotile from Shannan and XinKang has a stronger effect which may link to cancer than that from Akesai and Mangnai.

Oxidative effects of lungs in Wistar rats caused by long-term exposure to four kinds of China representative chrysotile.

Chrysotile accounts for some 90% to 95% of all the asbestos used worldwide. Scientific evidences have shown that asbestos (including chrysotile) exposure is associated with increased rates of lung cancer, asbestosis, and mesothelioma. However, molecular mechanisms underlying the toxicity effects of chrysotile are not clear. This study evaluated the oxidative stress in chronic lung toxicity caused by the intratracheal instillation (IT) of four kinds China representative chrysotile once a month for 12 months in Wistar rats. These results indicated that chrysotile exposure led to an obvious increase in lung mass and slowed the growth of body mass. Inflammation and fibrosis were observed by hematoxylin-eosin (HE) staining. Exposure to chrysotile significantly increased the accumulation of reactive oxygen species (ROS) and the level of lipid peroxidation and decreased antioxidant capacity in lung tissues. Furthermore, 1-6-month chrysotile exposure activated heme oxygenase-1 (HO-1) and heat shock protein 70 (HSP70) expression, whereas 12-month exposure caused significant decreases of two-factor expression levels in XK and MN groups when compared to negative control group. Therefore, our results suggested that chronic chrysotile pulmonary injury in Wistar rats is triggered by oxidative damage. Meanwhile, the oxidative damage of MN and XK was stronger than that of SSX and AKS, and the difference of oxidative damage in four chrysotile could have been brought by its properties, morphology, chemical composition, and particle size. With all the above mentioned in view, we hope that the revealed data in the experiment could contribute to the progress of further researches on the toxicity and mechanism of chrysotile.

Chrysotile effects on the expression of anti-oncogene P53 and P16 and oncogene C-jun and C-fos in Wistar rats' lung tissues.

Chrysotile is the most widely used form of asbestos worldwide. China is the world's largest consumer and second largest producer of chrysotile. The carcinogenicity of chrysotile has been extensively documented, and accumulative evidence has shown that chrysotile is capable of causing lung cancer and other forms of cancer. However, molecular mechanisms underlying the tumorigenic effects of chrysotile remained poorly understood. To explore the carcinogenicity of chrysotile, Wistar rats were administered by intratracheal instillation (by an artificial route of administration) for 0, 0.5, 2, or 8 mg/ml of natural chrysotile (from Mangnai, Qinghai, China) dissolved in saline, repeated once a month for 6 months (a repeated high-dose exposure which may have little bearing on the effects following human exposure). The lung tissues were analyzed for viscera coefficients and histopathological alterations. Expression of P53, P16, C-JUN, and C-FOS was measured by western blotting and qRT-PCR. Our results found that chrysotile exposure leads the body weight to grow slowly and lung viscera coefficients to increase in a dose-dependent manner. General sample showed white nodules, punctiform asbestos spots, and irregular atrophy; moreover, HE staining revealed inflammatory infiltration, damage of alveolar structures, agglomerations, and pulmonary fibrosis. In addition, chrysotile can induce inactivation of the anti-oncogene P53 and P16 and activation of the proto-oncogenes C-JUN and C-FOS both in the messenger RNA and protein level. In conclusion, chrysotile induced an imbalanced expression of cancer-related genes in rats' lung tissue. These results contribute to our understanding of the carcinogenic mechanism of chrysotile.

Chrysotile and rock wool fibers induce chromosome aberrations and DNA damage in V79 lung fibroblast cells.

According to global estimates, at least 107,000 people die each year from asbestos-related lung cancer, mesothelioma, and asbestosis resulting from occupational exposure. Chrysotile accounts for approximately 90% of asbestos used worldwide. Artificial substitutes can also be cytotoxic to the same degree as chrysotile. But only a few researchers focused on their genetic effects and mutagenicity information which is useful in evaluating the carcinogenicity of chemicals. In this study, chrysotile from Mangnai, Qinghai, China, and an artificial substitute, rock wool fiber were prepared as suspensions and were tested at concentrations of 50, 100, and 200 µg/ml in V79 lung fibroblasts. Chromosome aberrations were detected by micronucleus assay after exposure for 24 h, and DNA damage were estimated by single cell gel electrophoresis after exposure for 12, 24, or 48 h. According to the results, chrysotile and rock wool fibers caused micronuclei to form in a dose-dependent manner in V79 cells; olive tail moment values increased in a dose- and time-dependent manner. When V79 cells were exposed to a concentration of 200 µg/ml, the degree of DNA damage induced by chrysotile fibers was greater than rock wool fibers. Our study suggests that both chrysotile and rock wool fibers could induce chromosome aberrations and DNA damage. These materials are worthy of further study.

In vitro genotoxicity of asbestos substitutes induced by coupled stimulation of dissolved high-valence ions and oxide radicals.

The wide use of asbestos and its substitutes has given rise to studies on their possible harmful effects on human health and environment. However, their toxic effects remain unclear. The present study was aimed to disclose the coupled effects of dissolved high-valence ions and oxide radicals using the in vitro cytotoxicity and genotoxicity of chrysotile (CA), nano-SiO2 (NS), ceramic fiber (CF), glass fiber (GF), and rock wool (RW) on Chinese hamster lung cells V79. All samples induced cell mortality correlated well with the chemical SiO2 content of asbestos substitutes and the amount of dissolved Si. Alkali or alkaline earth metal elements relieved mortality of V79 cells; Al2O3 reinforced toxicity of materials. Asbestos substitutes generated lasting, increasing amount of acellular ·OH which formed at the fiber surface at sites with loose/unsaturated bonds, as well as by catalytic reaction through dissolved iron. Accumulated mechanical and radical stimulation induced the intracellular reactive oxygen species (ROS) elevation, morphology change, and deviating trans-membrane ion flux. The cellular ROS appeared as NS > GF > CF ≈ CA > RW, consistent with cell mortality rather than with acellular ·OH generation. Chromosomal and DNA lesions in V79 cells were not directly associated with the cellular ROS, while influenced by dissolved high-valence irons in the co-culture medium. In conclusion, ions from short-time dissolution of dust samples and the generation of extracellular ·OH presented combined effects in the elevation of intracellular ROS, which further synergistically induced cytotoxicity and genotoxicity.

Lung injury and expression of p53 and p16 in Wistar rats induced by respirable chrysotile fiber dust from four primary areas of China.

Chrysotile products were widely used in daily life, and a large amount of respirable dust was produced in the process of production and application. At present, there was seldom research on the safety of chrysotile fiber dust, and whether its long-term inhalation can lead to lung cancer was unknown. In order to determine whether respirable chrysotile fiber dust of China caused lung cancer, four major chrysotile-producing mine areas in China were selected for this study. Chrysotile fibers were prepared into respirable dust. Particle size was measured by laser particle analysis, morphology was observed by scanning electron microscope, chrysotile fiber phase was analyzed by X-ray diffraction, trace chemical elements were identified by X-ray fluorescence, and the structure and the active groups of the dust were determined after grinding by Fourier transform infrared spectroscopy. Male Wistar rats were exposed to non-exposed intratracheal instillation with different concentrations of chrysotile fiber dust. The rats were weighed after 1, 3, and 6 months, then the lung tissues were separated, the lung morphology was observed, and the pulmonary index was calculated. Pathological changes in lung tissues were observed by optical microscope after the HE staining of tissues, and the gene expression of p53 and p16 was determined by reverse transcription polymerase chain reaction. First, the results showed that the particle sizes of the four fibers were less than 10 µm. Four primary areas of chrysotile had similar fibrous structure, arranged in fascicles, or mixed with thin chunks of material. Second, the elementary composition of the four fibers was mainly chrysotile, and the structure and the active groups of the grinding dust were not damaged. Third, the weights of the treated rats were obviously lower, and the lung weights and the pulmonary index increased significantly (P < 0.05). Fourth, the treated Wistar rat lung tissues revealed different degrees of congestion, edema, inflammatory cell infiltration, and mild fibrosis. Fifth, the p53 and p16 genes decreased in the Mangnai group after 1 month of exposure, and the other groups increased. The expression of p53 and p16 in each group decreased significantly after 6 months (P < 0.05). In conclusion, the respirable chrysotile fiber dust from the four primary areas of China had the risk of causing lung injury, and these changes may be related to the physical and chemical characteristics of chrysotile from different production areas.