Effect of cigarette smoke exposure and mutant Kras overexpression on pancreatic cell proliferation.

Pancreatic cancer is the fourth leading cause of cancer-associated mortality. The major risk factor for pancreatic cancer is cigarette smoking. Kras mutations are commonly observed in human pancreatic cancers. The present study examined the hypothesis that exposure to cigarette smoke and overexpression of a mutant Kras gene in the pancreas affects pancreatic cell proliferation in mice. Mice overexpressing the mutant Kras gene (KRasG12D) in the pancreas as well as wild-type mice were exposed to environmental tobacco smoke for 2 weeks. Overexpression of mutant Kras increased cell proliferation in pancreatic ductal, acinar and islet cells. Notably, cigarette smoke exposure decreased cell proliferation in pancreatic ductal and acinar cells, and had no effect in islet cells. Cigarette smoke did not affect pancreatic protein levels of tumor necrosis factor (TNF)α, p53, or cyclin D1, but mutant Kras overexpression slightly decreased TNFα and p53 protein levels. Therefore, pancreatic cell proliferation in mice overexpressing mutant Kras is associated with the later development of pancreatic tumors, but effects of cigarette smoke on pancreatic cell proliferation do not provide a good model for human pancreatic carcinogenesis.

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.

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.

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.

Impact of apigenin and kaempferol on human head and neck squamous cell carcinoma.

OBJECTIVE: Apigenin and kaempferol are plant flavonoids with reported chemopreventive activities. This study aimed to determine the effect of apigenin and kaempferol on cell viability in cultured cells derived from the pharynx (FaDu cell line), an oral cavity carcinoma (PCI-13 cell line), and a metastatic lymph node (PCI-15B cell line) and in explanted FaDu cells. STUDY DESIGN: The in vitro viability of FaDu, PCI-13, and PCI-15B cells treated with apigenin and kaempferol was determined. Tumor growth of FaDu explants was evaluated in athymic mice that were gavaged with either apigenin or kaempferol. RESULTS: Although apigenin and kaempferol treatment decreased viability of cells in vitro, cell-type-dependent differences in responsiveness were observed. In vivo apigenin treatment significantly increased the tumor size of FaDu explants. Results obtained using kaempferol were similar. CONCLUSIONS: The in vitro decrease in FaDu cell viability by apigenin and kaempferol was not observed in in vivo tumor explants using the conditions described in this study.

Adenomatous polyposis coli-mediated accumulation of abasic DNA lesions lead to cigarette smoke condensate-induced neoplastic transformation of normal breast epithelial cells.

Adenomatous polyposis coli (APC) is a multifunctional protein having diverse cellular functions including cell migration, cell-cell adhesion, cell cycle control, chromosomal segregation, and apoptosis. Recently, we found a new role of APC in base excision repair (BER) and showed that it interacts with DNA polymerase ß and 5'-flap endonuclease 1 and interferes in BER. Previously, we have also reported that cigarette smoke condensate (CSC) increases expression of APC and enhances the growth of normal human breast epithelial (MCF10A) cells in vitro. In the present study, using APC overexpression and knockdown systems, we have examined the molecular mechanisms by which CSC and its major component, Benzo[α]pyrene, enhances APC-mediated accumulation of abasic DNA lesions, which is cytotoxic and mutagenic in nature, leading to enhanced neoplastic transformation of MCF10A cells in an orthotopic xenograft model.

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.

Oxidative DNA adducts detected in vitro from redox activity of cigarette smoke constituents.

Cigarette smoke contains a variety of carcinogens, cocarcinogens, mutagens, and tumor promoters. In addition to polycyclic aromatic carcinogens and tobacco-specific nitrosamines, cigarette smoke also contains an abundance of catechols, aldehydes, and other constituents, which are DNA damaging directly or indirectly; therefore, they can also contribute to cigarette smoke-mediated carcinogenicity. In this study, we investigated the potential of cigarette smoke constituents to induce oxidative damage to DNA through their capacity to redox cycle. When DNA (300 µg/mL) was incubated with cigarette smoke condensate (0.2 mg of tobacco particulate matter/mL) and CuCl(2) as a catalyst (50-100 µM), a variety of oxidative DNA adducts were detected by (32)P-postlabeling/TLC. Of the total adduct burden (2114 ± 419 adducts/10(6) nucleotides), over 40% of all adducts were attributed to the benchmark oxidative DNA lesion, 8-oxodeoxyguanosine (8-oxodG). Adducts were formed dose dependently. Essentially, similar adduct profiles were obtained when cigarette smoke condensate was substituted with ortho- and para-dihydroxybenzenes. Vehicle treatment with Cu(2+) or CSC alone did not induce any significant amount of oxidative DNA damage. Furthermore, coincubation of cigarette smoke condensate and ortho-dihydroxybenzene with DNA resulted in a higher amount of oxidative DNA adducts than obtained with the individual entity, suggesting that adducts presumably originated from catechols or catechol-like compounds in cigarette smoke condensate. Adducts resulting from both cigarette smoke condensate and pure dihydroxybenzenes were chromatographically identical to adducts formed by reaction of DNA with H(2)O(2), which is known to produce 8-oxodG, and many other oxidative DNA adducts. When the cigarette smoke condensate-DNA reaction was performed in the presence of ellagic acid, a known antioxidant, the adduct formation was inhibited dose dependently, further suggesting that adducts originated from oxidative pathway. Our data thus provide evidence of the capacity of catechols or catechol-like constituents in cigarette smoke to produce oxidative DNA damage, which may contribute to the tumor-promoting activity of cigarette smoke.

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.

Cigarette smoke condensate-induced oxidative DNA damage and its removal in human cervical cancer cells.

Exposure to cigarette smoke is well documented to increase oxidative stress and could account for higher risk of cervical cancer in smokers. Cervical pre-cancerous lesions that are initiated by human papillomavirus (HPV) infection generally regress in the absence of known risk factors such as smoking. 8-oxodeoxyguanosine (8-oxodG) is a highly mutagenic oxidative DNA lesion that is formed by the oxidation of deoxyguanosine. In the present study, we examined: a) the effect of cigarette smoke condensate (CSC) on 8-oxodG formation in and its removal from HPV-transfected (ECT1/E6 E7), HPV-positive (CaSki) and HPV-negative (C33A) human cervical cancer cells, and b) the cell cycle progression and apoptosis in CSC-treated ECT1/E6 E7 cells. CSC induced 8-oxodG in a dose- (p=0.03) and time (p=0.002)-dependent fashion in ECT1/E6 E7 cells as determined by flow cytometry. A 2.4-fold higher level of 8-oxodG was observed in HPV-positive compared with HPV-negative cells. However, 8-oxodG lesions were almost completely removed 72 h post-exposure in all cell lines as determined by ImageStream analysis. This observation correlates with the 2- and 5-fold increase in the p53 levels in ECT1/E6 E7 and CaSki cells with no significant change in C33A cells. We conclude that: a) cigarette smoke constituents induce oxidative stress with higher burden in HPV-positive cervical cancer cells and b) the significant increase observed in p53 levels in wild-type cervical cells (ECT1/E6 E7 and CaSki) may be attributed to the p53-dependent DNA repair pathway while a p53-independent pathway in C33A cells cannot be ruled out.

Decreased cytochrome c oxidase IV expression reduces steroidogenesis.

Steroidogenic acute regulatory protein facilitates the translocation of cholesterol to the inner mitochondrial membrane, thereby initiating steroidogenesis. At the inner mitochondrial membrane, cytochrome P450 side-chain cleavage enzyme converts cholesterol to pregnenolone, an oxidative process requiring electrons from NADPH. Pregnenolone then serves as the substrate for the formation of progesterone or dehydroepiandrosterone by downstream enzymes. Studies have shown that cigarette smoke (CS) influences steroid hormone levels. To better understand the underlying mechanisms, we used a mouse model to study the effects of chronic CS exposure on steroidogenesis. Through radioimmunoassay and metabolic conversion assays, we found that CS reduced progesterone and dehydroepiandrosterone without affecting cytochrome P450 side-chain cleavage enzyme or 3ß-hydroxysteroid dehydrogenase 2 expression. However, CS did reduce expression of cytochrome c oxidase IV (COX IV), a component of the mitochondrial complex that serves as the last enzyme in the electron transport chain. Small interfering RNA-mediated COX IV knockdown indeed decreased progesterone synthesis in steroidogenic cells. In summary, COX IV likely plays a role in steroidogenesis, and passive smoking may negatively affect steroidogenesis by disrupting the electron transport chain.

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."

Effects of cigarette smoke on the activation of oxidative stress-related transcription factors in female A/J mouse lung.

Cigarette smoke contains a high concentration of free radicals and induces oxidative stress in the lung and other tissues. Several transcription factors are known to be activated by oxidative stress, including nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1), and hypoxia-inducible factor (HIF). Studies were therefore undertaken to examine whether cigarette smoke could activate these transcription factors, as well as other transcription factors that may be important in lung carcinogenesis. Female A/J mice were exposed to cigarette smoke for 2, 5, 10, 15, 20, 42, or 56 d (6 hr/d, 5 d/wk). Cigarette smoke did not increase NF-kappaB activation at any of these times, but NF-kappaB DNA binding activity was lower after 15 d and 56 d of smoke exposure. The DNA binding activity of AP-1 was lower after 10 d and 56 d but was not changed after 42 d of smoke exposure. The DNA binding activity of HIF was quantitatively increased after 42 d of smoke exposure but decreased after 56 d. Whether the activation of other transcription factors in the lung could be altered after exposure to cigarette smoke was subsequently examined. The DNA binding activities of FoxF2, myc-CF1, RORE, and p53 were examined after 10 d of smoke exposure. The DNA binding activities of FoxF2 and p53 were quantitatively increased, but those of myc-CF1 and RORE were unaffected. These studies show that cigarette smoke exposure leads to quantitative increases in DNA binding activities of FoxF2 and p53, while the activations of NF-kappaB, AP-1, and HIF are largely unaffected or reduced.

Early changes in gene expression induced by tobacco smoke: Evidence for the importance of estrogen within lung tissue.

Lung cancer is the leading cause of cancer deaths in the United States, surpassing breast cancer as the primary cause of cancer-related mortality in women. The goal of the present study was to identify early molecular changes in the lung induced by exposure to tobacco smoke and thus identify potential targets for chemoprevention. Female A/J mice were exposed to either tobacco smoke or HEPA-filtered air via a whole-body exposure chamber (6 h/d, 5 d/wk for 3, 8, and 20 weeks). Gene expression profiles of lung tissue from control and smoke-exposed animals were established using a 15K cDNA microarray. Cytochrome P450 1b1, a phase I enzyme involved in both the metabolism of xenobiotics and the 4-hydroxylation of 17beta-estradiol (E(2)), was modulated to the greatest extent following smoke exposure. A panel of 10 genes were found to be differentially expressed in control and smoke-exposed lung tissues at 3, 8, and 20 weeks (P < 0.001). The interaction network of these differentially expressed genes revealed new pathways modulated by short-term smoke exposure, including estrogen metabolism. In addition, E(2) was detected within murine lung tissue by gas chromatography-coupled mass spectrometry and immunohistochemistry. Identification of the early molecular events that contribute to lung tumor formation is anticipated to lead to the development of promising targeted chemopreventive therapies. In conclusion, the presence of E(2) within lung tissue when combined with the modulation of cytochrome P450 1b1 and other estrogen metabolism genes by tobacco smoke provides novel insight into a possible role for estrogens in lung cancer.

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.

Dietary coenzyme Q10 does not protect against cigarette smoke-augmented atherosclerosis in apoE-deficient mice.

Dietary coenzyme Q10 reduces spontaneous atherosclerosis in the apoE-deficient mouse model of experimental atherosclerosis. We have shown previously that exposure to sidestream cigarette smoke (SSCS) enhances atherosclerotic lesion formation in apoE-deficient mice. The aim of the present study was to determine if CoQ10 protected against SSCS-mediated atherosclerosis. Female apoE-deficient mice were fed a saturated fat-enriched diet (SFD) alone, or supplemented with 1% wt/wt coenzyme Q10 (SFD-Q10). Mice in each diet group were exposed to SSCS for 4hrs/day, 5days/week in a whole-body exposure chamber maintained at 35+/-4mg smoke particulates/m(3). Mice kept in filtered ambient air served as controls. Mice were euthanized after either 6 or 15weeks of SSCS exposure and following measurements were performed: i) lung 7-ethoxyresorufin-O-deethylase (EROD) activity; ii) plasma cholesterol and CoQ10 concentrations; iii) aortic intimal area covered by atherosclerotic lesions; and, iv) pathological characterization of lesions. Lung EROD activity increased in SSCS mice of both diet groups, confirming SSCS exposure. Plasma concentrations of CoQ10 in SFD-Q10-fed mice were increased markedly in comparison to SFD-fed mice. Plasma cholesterol concentrations and distributions of cholesterol in lipoprotein fractions were unaffected by SSCS exposure. Dietary supplementation with CoQ10 significantly reduced atherosclerotic lesions in control mice. As reported previously, exposure to SSCS increased the size of lesions in apoE-/- mice at both time points. However, dietary supplementation with CoQ10 had no effect on atherosclerotic lesions augmented by SSCS exposure. The results suggest a role of oxidative processes in smoke-augmented atherosclerosis that are different than those mitigated by CoQ10.

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 microg of MWCNTs in 40 microl 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-alpha, interleukin-1beta, 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.

Cigarette smoke-induced DNA damage and repair detected by the comet assay in HPV-transformed cervical cells.

Human papillomavirus (HPV) is the causative factor in the development and progression of cervical cancers in >97% of the cases, although insufficient. Epidemiological studies suggest an elevated risk of cervical cancer for cigarette smokers; therefore, we examined cigarette smoke-induced DNA damage and repair in HPV16-transformed human ectocervical cells (ECT1/E6 E7). Cells were treated with cigarette smoke condensate (CSC) for 72 h to assess the formation of single- and double-strand DNA breaks, measured by alkaline and neutral single cell gel electrophoresis assays, respectively. The mean tail length of cells with single-strand breaks was increased by 1.8-, 2.7- and 3.7-fold (p<0.001) after treatment with 4, 8 and 12 microg/ml CSC, respectively. The tail length with double-strand breaks was also increased dose-dependently. These results were further supported by measurement of the mean tail moment: the increase in both single- and double-strand breaks were much more pronounced with increasing concentration of CSC, by up to 23.5-fold (p<0.0001 for both assays). To examine the DNA repair, cells were treated with CSC for 72 h, followed by CSC withdrawal and re-incubation of the cells with fresh medium for 24, 48, or 72 h. Both single- and double-strand DNA breaks were removed during the initial 24 h but no further removal of the damage was observed. Up to 80% of residual single- and double-strand DNA breaks (p<0.05) were found to persist at all CSC concentrations examined. Ellagic acid, a known antioxidant and free-radical scavenger, was found to significantly inhibit DNA breaks induced by CSC. Thus, free radicals may be a plausible source of CSC-induced DNA damage. These data show that CSC-mediated DNA strand breaks are highly persistent, and suggest that persistence of cigarette smoke-associated DNA damage in the presence of HPV infection may lead to increased mutations in cervical cells and ultimately higher cancer risk.

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.

Naphthoquinones and bioactive compounds from tobacco as modulators of neuronal nitric oxide synthase activity.

Studies were conducted with extracts of several varieties of tobacco in search of neuronal nitric oxide synthase (nNOS) inhibitors which may be of value in the treatment of stroke. Current therapies do not directly exploit modulation of nNOS activity due to poor selectivity of the currently available nNOS inhibitors. The properties of a potentially novel nNOS inhibitor(s) derived from tobacco extracts, and the concentration-dependent, modulatory effects of the tobacco-derived naphthoquinone compound, 2,3,6-trimethyl-1,4-naphthoquinone (TMN), on nNOS activity were investigated, using 2-methyl-1,4-naphthoquinone (menadione) as a control. Up to 31 microM, both TMN and menadione stimulated nNOS-catalysed L-citrulline production. However, at higher concentrations of TMN (62.5-500 microM), the stimulation was lost in a concentration-dependent manner. With TMN, the loss of stimulation did not decrease beyond the control activity. With menadione (62.5-500 microM), the loss of stimulation surpassed that of the control (78+/-0.01% of control activity), indicating a true inhibition of nNOS activity. This study suggests that potential nNOS inhibitors are present in tobacco, most of which remain to be identified.