Sung Gu Han

Acute pulmonary response of mice to multi-wall carbon nanotubes

Widespread use of carbon nanotubes is predicted for future and concerns have been raised about their potential health effects. The present study determined the pulmonary response of mice to multi-wall carbon nanotubes (MWCNTs). The MWCNT suspension in sterile phosphate-buffered saline (PBS) was introduced into mice lungs by oropharyngeal aspiration. Female C57Bl mice were treated with either 20 or 40 μg of MWCNTs in 40 μl PBS and control groups received equal volume of PBS. From each group, half of the mice were euthanized at day 1 and the remaining half at day 7 post treatment. Bronchoalveolar lavage (BAL) fluids, serum, and lung tissue samples were analyzed for inflammatory and oxidative stress markers. The results showed significant cellular influx by a single exposure to MWCNTs. Yields of total cells and the number of polymorphonuclear leukocytes in BAL cells were significantly elevated in MWCNT-treated mice post-treatment days 1 and 7. Analysis of cell-free BAL fluids showed significantly increased levels of total proteins, lactate dehydrogenase, tumor necrosis factor-α, interleukin-1β, mucin, and surfactant protein-D (SP-D) in MWCNT-treated mice at day 1 post treatment. However, these biomarkers returned to basal levels by day 7 post exposure except mucin and SP-D. An increase in the urinary level of 8-hydroxy-2’-deoxyguanosine in mice treated with MWCNT suggested systemic oxidative stress. Western analysis of lung tissue showed decreased levels of extracellular superoxide dismutase (SOD) protein in MWCNT-treated mice but copper/zinc and manganese SOD remained unchanged. It is concluded that a single treatment of MWCNT is capable of inducing cytotoxic and inflammatory response in the lungs of mice.

Acute pulmonary effects of combined exposure to carbon nanotubes and ozone in mice.

Ozone (O3) is a well-investigated gaseous air pollutant known to produce acute and chronic toxicity in the respiratory system. Whether prior exposure to nanoparticles influences the toxicity of O3 has not been well investigated. To determine if there are toxicological interactions between particulate and gas exposures, we examined acute pulmonary effects of a 3-h ozone exposure (0.5 ppm) in female C57Bl mice that had been preexposed to a single dose of 20 μ g multiwall carbon nanotubes (CNT) by pharyngeal aspiration 12 h earlier. A total of four groups were compared: (1) PBS/air-control, (2) PBS/O3, (3) CNT/air, and (4) CNT/O3. Analyses of the bronchoalveolar lavage fluid (BALF) and lung tissue samples collected at 5 and 24 h post O3 exposure were performed for various markers of cytotoxicity and inflammation using standard enzyme-linked immunosorbent assay (ELISA) and immunoblot procedures. The results showed a pronounced cellular response and increase in various cytotoxicity/inflammatory markers in the lungs of CNT-exposed mice. Ozone by itself produced minimal effects, but in CNT-exposed animals there was a significant increase in total brochoalveolar lavage (BAL) cells and polymorphonuclear leukocytes. Additionally, protein, lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and mucin levels in BALF at 5 and 24 h were higher in CNT-exposed animals than in corresponding air-exposed controls or animals exposed to O3 alone. A comparable increase over the controls was also observed in the CNT/O3 group, but neither an additive nor a synergistic interaction was observed in mice that received sequential exposure to CNT and ozone. In fact, some CNT-induced cytotoxic/inflammatory responses were attenuated in mice following exposure to both CNT and low levels of ozone. These results are contrary to enhanced responses that were anticipated and may represent the development of “cross-tolerance” reported by others for some sequentially administered pollutants.

Atherogenic and pulmonary responses of ApoE- and LDL receptor-deficient mice to sidestream cigarette smoke

Plasma lipoproteins play important roles in the development and progression of atherosclerosis. Two widely used mouse models of experimental atherosclerosis, apolipoprotein E-deficient (ApoE -/-) and LDL receptor-deficient (LDLr -/-) mice, have major differences in lipoprotein characteristics. These include differences in lipoprotein cholesterol distribution, lipoprotein compositions, apoliporoteins distribution, and susceptibility to oxidation. In the present study, we compared pulmonary and cardiovascular responses of ApoE -/- and LDLr -/- mice to sidestream cigarette smoke (SSCS) exposure to determine if strain differences influence their predisposition to SSCS-mediated promotion of atherosclerosis. Female ApoE -/- and LDLr -/- mice were maintained on a saturated fat enriched diet and exposed to SSCS in whole body exposure chambers for 15 weeks (4h/day, 5 days/week). At terminations, the levels of pulmonary injury markers in bronchoalveolar lavage (BAL) fluids from 6 mice per group and atherosclerotic lesion formation in 14 mice per group were analyzed. Total BAL cells and polymorphonuclear leukocytes were not significantly altered by SSCS exposure in both mouse models. Total protein, LDH, and cytokine concentrations in cell-free BAL fluids were also not significantly affected by chronic SSCS exposure in either mouse strain. SSCS significantly reduced surfactant protein D levels in both strains to a similar extent. However, SSCS exposure increased significantly the percent atherosclerotic lesion areas covering aortic intimal surfaces of ApoE -/- (control-25.3±1.52 vs. SSCS-31.9±2.02, p=0.012) as well as in LDLr -/- (control-30.97±1.1 vs. SSCS-36.61±1.7, p=0.028) mice. In contrast, the serum cholesterol concentrations of SSCS-exposed ApoE -/- mice were similar to that of controls (control-1255±85 vs. SSCS-1190±61mg/dl, p=0.552) but increased significantly in SSCS-exposed LDLr -/- mice (control-998±114 vs. SSCS-1577±142mg/dl, p=0.008). These results showing different effects of identical SSCS exposure on plasma cholesterol concentrations in these two mouse models suggest a role of multiple mechanisms in SSCS-induced atherosclerosis.

Enhanced platelet reactivity and thrombosis in Apoe-/- mice exposed to cigarette smoke is attenuated by P2Y12 antagonism

INTRODUCTION Smoking increases the risk of acute arterial thrombosis, including myocardial infarction, likely due to multi-factorial effects on the vasculature. Heightened platelet reactivity may be among the adverse effects of smoke exposure. METHODS To examine the effects of smoke exposure on platelet function in an atherosclerotic environment, Apoe-deficient female mice, maintained on a Western diet, were exposed (4 hrs/d, 5 d/wk) to sidestream cigarette smoke in a whole-body exposure chamber for 12 weeks. A separate group of wild type C57BL/6J mice were also exposed to smoke in an identical fashion. RESULTS In comparison to control Apoe-/- mice exposed to filtered ambient air, smoke-exposed Apoe-/- mice displayed a 1.8±0.3 fold enhanced ADP-induced fibrinogen binding ex vivo (P<0.001) and had a shorter time to thrombotic occlusion following ferric chloride injury of the carotid artery (median time to thrombosis of 8 vs. 13 min; P=0.015). Administration of the direct-acting P2Y12 antagonist cangrelor blunted ex vivo fibrinogen binding and attenuated thrombosis (median time 20 min) in Apoe-/- mice exposed to sidestream smoke. The effects of smoke exposure required a proatherosclerotic background, as wild-type C57Bl/6J mice exposed to smoke displayed similar fibrinogen binding and thrombotic occlusion times as did control mice. CONCLUSIONS Our results demonstrate that exposure to smoke heightens platelet reactivity and thrombosis in Apoe-/- mice and implicate signaling through platelet P2Y12 receptor as a mediator of the adverse consequence of smoke exposure. These results may partially explain the recent observations that smokers derive greater clinical benefit from the P2Y12 antagonist clopidogrel than do non-smokers.

Effect of Dietary Selenium and Cigarette Smoke on Pulmonary Cell Proliferation in Mice

The objective of this study was to determine if dietary selenium could inhibit pulmonary cell proliferation in control and cigarette smoke-exposed female A/J mice. Selenium in the form of sodium selenite was supplemented to purified diets similar to the AIN-93M diet to yield 0.15, 0.5, or 2.0 mg selenium/kg diet. After 3 weeks, mice in each dietary group were divided into two subgroups; one used as control, whereas the other was exposed to cigarette smoke for five consecutive days. Mice from both groups were euthanized 3 days later. Mice were administered bromodeoxyuridine in the drinking water starting 5 days before the initiation of the smoke exposure and continuing until they were euthanized. After euthanasia, the left lung lobe was processed for histology and cell proliferation analysis. Cigarette smoke increased cell proliferation in the terminal bronchioles and large airways, but not in alveoli. High-selenium diets inhibited cell proliferation in the alveoli, terminal bronchioles and large airways areas in both control and smoke-exposed mice. Increasing the dietary selenium level led to increased selenium levels in the blood and lung, and increased glutathione peroxidase (GPx) activity in the lung. Cytochrome P-450 1A1 protein levels in the lung were increased by cigarette smoke but were not affected by dietary selenium. It is concluded that dietary selenium inhibits pulmonary cell proliferation in both control and cigarette smoke-exposed mice, indicating that selenium is inhibiting cell proliferation independently of smoke exposure, and that this inhibition may be related to selenium concentration and GPx activity in the lung.

Exposure of Human Lung Cells to Tobacco Smoke Condensate Inhibits the Nucleotide Excision Repair Pathway.

Exposure to tobacco smoke is the number one risk factor for lung cancer. Although the DNA damaging properties of tobacco smoke have been well documented, relatively few studies have examined its effect on DNA repair pathways. This is especially true for the nucleotide excision repair (NER) pathway which recognizes and removes many structurally diverse DNA lesions, including those introduced by chemical carcinogens present in tobacco smoke. The aim of the present study was to investigate the effect of tobacco smoke on NER in human lung cells. We studied the effect of cigarette smoke condensate (CSC), a surrogate for tobacco smoke, on the NER pathway in two different human lung cell lines; IMR-90 lung fibroblasts and BEAS-2B bronchial epithelial cells. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6–4 photoproducts and cyclobutane pyrimidine dimers. We find a dose-dependent inhibition of 6–4 photoproduct repair in both cell lines treated with CSC. Additionally, the impact of CSC on the abundance of various NER proteins and their respective RNAs was investigated. The abundance of XPC protein, which is required for functional NER, is significantly reduced by treatment with CSC while the abundance of XPA protein, also required for NER, is unaffected. Both XPC and XPA RNA levels are modestly reduced by CSC treatment. Finally, treatment of cells with MG-132 abrogates the reduction in the abundance of XPC protein produced by treatment with CSC, suggesting that CSC enhances proteasome-dependent turnover of the protein that is mediated by ubiquitination. Together, these findings indicate that tobacco smoke can inhibit the same DNA repair pathway that is also essential for the removal of some of the carcinogenic DNA damage introduced by smoke itself, increasing the DNA damage burden of cells exposed to tobacco smoke.

Dietary selenium fails to influence cigarette smoke-induced lung tumorigenesis in A/J mice

The goal of the study was to determine if dietary selenium inhibited the induction of lung tumorigenesis by cigarette smoke in A/J mice. Purified diets containing 0.15, 0.5, or 2.0mg/kg selenium in the form of sodium selenite were fed to female A/J mice. Half of the mice in each dietary group were exposed to cigarette smoke 6h/day, 5days/week for five months followed by a four month recovery period in ambient air, while the other half were used as controls. After the recovery period, the mice were euthanized, and their lungs were removed for further analysis. Mice exposed to smoke had a higher tumor incidence and a higher tumor multiplicity, whereas dietary Se did not affect either the tumor incidence or tumor multiplicity. An increase in dietary selenium led to increased levels of selenium in the lung as well as GPx protein levels, but dietary Se did not affect lung SOD protein levels. In conclusion, these data confirm the carcinogenic activity of cigarette smoke in mice but show that dietary Se provided as sodium selenite does not affect smoke-induced carcinogenesis in this model.

Inorganic arsenic inhibits the nucleotide excision repair pathway and reduces the expression of XPC

Chronic exposure to arsenic, most often through contaminated drinking water, has been linked to several types of cancer in humans, including skin and lung cancer. However, the mechanisms underlying its role in causing cancer are not well understood. There is evidence that exposure to arsenic can enhance the carcinogenicity of UV light in inducing skin cancers and may enhance the carcinogenicity of tobacco smoke in inducing lung cancers. The nucleotide excision repair (NER) pathway removes different types of DNA damage including those produced by UV light and components of tobacco smoke. The aim of the present study was to investigate the effect of sodium arsenite on the NER pathway in human lung fibroblasts (IMR-90 cells) and primary mouse keratinocytes. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6-4 photoproducts (6-4 PP) and cyclobutane pyrimidine dimers (CPDs). We find a concentration-dependent inhibition of the removal of 6-4 PPs and CPDs in both cell types treated with arsenite. Treatment of both cell types with arsenite resulted in a significant reduction in the abundance of XPC, a protein that is critical for DNA damage recognition in NER. The abundance of RNA expressed from several key NER genes was also significantly reduced by treatment of IMR-90 cells with arsenite. Finally, treatment of IMR-90 cells with MG-132 abrogated the reduction in XPC protein, suggesting an involvement of the proteasome in the reduction of XPC protein produced by treatment of cells with arsenic. The inhibition of NER by arsenic may reflect one mechanism underlying the role of arsenic exposure in enhancing cigarette smoke-induced lung carcinogenesis and UV light-induced skin cancer, and it may provide some insights into the emergence of arsenic trioxide as a chemotherapeutic agent.

Bhas 42 cell transformation activity of cigarette smoke condensate is modulated by selenium and arsenic

Cigarette smoking remains a major health risk worldwide. Development of newer tobacco products requires the use of quantitative toxicological assays. Recently, v‐Ha‐ras transfected BALB/c3T3 (Bhas 42) cell transformation assay was established that simulates the two‐stage animal tumorigenesis model and measures tumor initiating and promoting activities of chemicals. The present study was performed to assess the feasibility of using this Bhas 42 cell transformation assay to determine the initiation and promotion activities of cigarette smoke condensate (CSC) and its water soluble fraction. Further, the modulating effects of selenium and arsenic on cigarette smoke‐induced cell transformation were investigated. Dimethyl sulfoxide (DMSO) and water extracts of CSC (CSC‐D and CSC‐W, respectively) were tested at concentrations of 2.5–40 µg mL−1 in the initiation or promotion assay formats. Initiation protocol of the Bhas 42 assay showed a 3.5‐fold increase in transformed foci at 40 µg mL−1 of CSC‐D but not CSC‐W. The promotion phase of the assay yielded a robust dose response with CSC‐D (2.5–40 µg mL−1) and CSC‐W (20–40 µg mL−1). Preincubation of cells with selenium (100 nM) significantly reduced CSC‐induced increase in cell transformation in initiation assay. Co‐treatment of cells with a sub‐toxic dose of arsenic significantly enhanced cell transformation activity of CSC‐D in promotion assay. The results suggest a presence of both water soluble and insoluble tumor promoters in CSC, a role of oxidative stress in CSC‐induced cell transformation, and usefulness of Bhas 42 cell transformation assay in comparing tobacco product toxicities and in studying the mechanisms of tobacco carcinogenesis. Environ. Mol. Mutagen. 57:220–228, 2016.

In Utero Tobacco Smoke Exposure Alters Pulmonary Responses of Newborn Rats to Ozone

Prenatal tobacco smoke (TS) exposure has been implicated in various adverse health outcomes in the offspring, including poor development of lung and immune system, which in turn can alter the response of neonates to environmental challenges. This study was performed to determine whether in utero exposure to TS influences the pulmonary response of newborn rat pups to ozone (O3). Timed pregnant Sprague-Dawley (SD) rats were exposed to TS or air for 3 h/d from gestation d 7 through 21. The pulmonary response of pups was assessed following a single 3-h exposure to air or 0.6 ppm O3 on d 13 after birth. In all, 4 exposure groups were evaluated: (1) Air/Air (in utero air and postnatal air), (2) Air/O3 (in utero air and postnatal O3), (3) TS/Air (in utero TS and postnatal air), and (4) TS/O3 (in utero TS and postnatal O3). Bronchoalveolar lavage (BAL) was performed, and BAL cells and fluid were analyzed. Data revealed a significant increase in polymorphonuclear leukocytes (PMN) and total BALF protein in the Air/O3 group compared to the Air/Air control, reflecting the inflammatory and cytotoxic effects of O3. However, in utero exposure to TS attenuated PMN infiltration into the air spaces for recovery in the BAL of TS/O3 pups. Lung tissue myeloperoxidase activity significantly increased only in the TS/O3 group but not in Air/O3 pups, thus suggesting that PMN are sequestered in the lung tissue and that the in utero TS likely inhibits O3-mediated influx of PMN into the air spaces. Lung tissue analyses further showed a significant rise in manganese superoxide dismutase (SOD) protein and a decrease in extracellular SOD protein in the Air/O3 group, suggesting oxidative effects of O3. Interestingly, in utero TS exposure again suppressed these effects in the TS/O3 group. Overall, results suggest that in utero exposure to TS alone produced minimal acute pulmonary effects in newborn rats, but modulated adverse effects of postnatal O3 exposure. The results are contrary to the interactive toxic responses predicted for sequential exposures to TS and O3, and may represent the development of “cross-tolerance.”