Supplementary Data from Confirmation of Gene Expression–Based Prediction of Survival in Non–Small Cell Lung Cancer

Anecdotal reports in the press and epidemiological studies suggest that deployment to Iraq and Afghanistan may be associated with respiratory diseases and symptoms in U.S. military personnel and veterans. Exposures during military operations were complex, but virtually all service members were exposed to high levels of respirable, geogenic dust. Inhalation of other dusts has been shown to be associated with adverse health effects, but the pulmonary toxicity of ambient dust from Iraq has not been previously studied. The relative toxicity of Camp Victory dust was evaluated by comparing it to particulate matter from northern Kuwait, a standard U.S. urban dust, and crystalline silica using a single intratracheal instillation in rats. Lung histology, protein levels, and cell counts were evaluated in the bronchoalveolar lavage fluid 1-150 d later. The Iraq dust provoked an early significant, acute inflammatory response. However, the level of inflammation in response to the Iraq dust, U.S. urban dust, and Kuwait dust rapidly declined and was nearly at control levels by the end of the study At later times, animals exposed to the Iraq, U.S. urban, or Kuwait dusts showed increased small airway remodeling and emphysema compared to silica-exposed and control animals without evidence of fibrosis or premalignant changes. The severity and persistence of pulmonary toxicity of these three dusts from the Middle East resemble those of a U.S. urban dust and are less than those of silica. Therefore, Iraq dust exposure is not highly toxic, but similar to other poorly soluble low-toxicity dusts.

Nuclear transcription factor kB (NF-kB) is a multiprotein complex that regulates a variety of genes important for immunity and inflammation. The present study investigates the silica-induced activation of this transcription factor in mouse mac­ rophage cell line RAW 264.7 cells, the role of free radical reactions in the mechanism of the activation, and its possible inhibition. Tetrandrine, a benzylisoquinoline alka­ loid, which has been used as an antifibrotic drug to treat the lesions of silicosis and has been characterized as a hydroxyl radical (-OH) scavenger, inhibited the NF-kB activation induced by silica, lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). Catalase, metal chelator, deferoxamine, and the silanol group (SiOH) blocker, poly(2-vinylpyridine-N-oxide) (PVPNO), also inhibited silicainduced NF-kB activation. Electron spin resonance (ESR) spin trapping measure­ ments show that both deferoxamine and PVPNO decreased silica-mediated -OH radical generation from H20 2. It is shown that Fe(II) and not Fe(III) is able to cause NF-kB activation. The antioxidant, ascorbate, attenuated the NF-kB activation induced by silica but not by LPS. The -OH radical scavenger, sodium formate, inhibited NF-kB activation induced by silica but had only a minor effect on NF-kB * Send reprint requests to: Laurence M. Demers, Ph.D., Department o f Pathology, The Pennsylvania State University College o f Medicine, The Milton S. Hershey Medical Center, P.O. Box 850, Hershey, PA 17003 or Xianglin Shi, Ph.D., Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, 1095 Willowdale Road, Mail Stop B167; Morgantown, WV 26505.

Single-walled carbon nanotubes (SWCNT) are being developed to be used in many industrial and biomedical applications. However, SWCNT's durability and likely fibrous morphology have raised health concerns. The present investigations were focused on understanding the cellular and molecular mechanisms induced by raw SWCNT (SWCNT) in human bronchial-epithelial cells (BEAS-2B). Asbestos (crocidolite) was used as a positive control. Exposure of BEAS-2B cells to SWCNT induced apoptosis, DNA damage, and oxidative stress. The generation of hydroxyl radical (center dot OH) and increase of superoxide dismutase (SOD) activity were concentration-dependent. The increase in apoptosis was associated with activation of caspase-3, caspase-7, and poly (ADP-ribose) polymerase-1 (PARP-1). A short recovery period of 6 h of cells from SWCNT exposure resulted in reversal of caspase-3 and caspase-7, and a partial reversal of PARP-1 activation. The activation of PARP-1, caspase-3, and caspase-7 was only partially diminished after a recovery of 6 h from the exposure to crocidolite. Exposure of BEAS-2B cells to SWCNT resulted in the phosphorylation of protein p42/44 (p42/44) and protein p38 (p38). SWCNT did not induce protein serine-threonine kinase (AKT) phosphorylation. For all the above end points, crocidolite induced a greater response compared to SWCNT. SWCNT induced a significant activation of activator protein-1 (AP-1) and nuclear factor kappa B (NF-B), and the effect was inhibited by mitogen-activated protein kinase (MAPK) inhibitors. SWCNT also induced significant increase in the expression levels of c-Jun, IGH3, and CD44 genes. The results of this study show that the molecular mechanism for raw SWCNT-mediated toxicity in BEAS-2B cells is through the activation of caspase-3, caspase-7, and PARP-1. Furthermore, the mechanism of AP-1 and NF-B activation is through MAPK. This bioactivity of raw SWCNT is associated with the generation of oxidative stress and DNA damage. Considering the role of airway epithelium as a critical barrier for normal pulmonary function and focal point for tumor development, this study demonstrates that raw SWCNT activate molecular events which may be linked to adverse biological responses implicated in pulmonary diseases.

Context.—Coal worker's pneumoconiosis is a major occupational lung disease in the United States. The disease is primarily controlled through reducing dust exposure in coal mines using technological improvements and through the establishment of dust standards by regulatory means. Objective.—To determine if dust standards established in the US Federal Coal Mine Health and Safety Act of 1969 have reduced the prevalence and severity of coal worker's pneumoconiosis. Design.—The study population included materials from 6103 deceased coal miners submitted to the National Coal Workers' Autopsy Study from 1971 through 1996. Type and severity of coal worker's pneumoconiosis were classified using standardized diagnostic criteria. Results.—Among miners who worked exclusively prior to the 1969 dust standard, 82.6% had coal macules, 46.3% coal nodules, 28.2% silicotic nodules, and 10.3% progressive massive fibrosis. Lower prevalences were noted among miners exposed exclusively to post-1970 dust levels: 58.8% had coal macules, 15.0% coal nodules, 8.0% silicotic nodules, and 1.2% progressive massive fibrosis. The differences in prevalence were highly significant (P < .001) for all types of pneumoconiosis, including progressive massive fibrosis, after adjustment for age, years of mining, and smoking status. Conclusions.—The study confirms a beneficial impact of the first 25 years of the dust standard established by the 1969 act on the prevalence and severity of coal worker's pneumoconiosis in US coal miners. However, pneumoconiosis continues to occur among miners who have worked entirely within the contemporary standard, suggesting a need for further reductions in exposure to respirable coal mine dust.

Inhalation exposure to particulates such as cigarette smoke and coal dust is known to contribute to the development of chronic lung disease. The purpose of this study was to estimate the amount of elemental carbon (EC) deposits from autopsied lung samples from cigarette smokers, miners, and control subjects and explore the relationship between EC level, exposure history, and the extent of chronic lung disease. The samples comprised three subgroups representing never smokers (8), chronic cigarette smokers (26), and coal miners (6). Following the dissolution of lung tissue, the extracted EC residue was quantified using a thermal-optical transmission (TOT) carbon analyzer. Mean EC levels in the lungs of the control group were 56.68 ± 24.86 (SD) μg/g dry lung weight. Respective mean EC values in lung samples from the smokers and coal miners were 449.56 ± 320.3 μg/g and 6678.2 ± 6162 μg/g. These values were significantly higher than those obtained from the never-smoker group. EC levels in the lung and pack-years of cigarette smoking correlated significantly, as did EC levels and the severity of small airway disease. This study provides one of the first quantitative assessments of EC in human lungs from populations at high relative risk for the development of chronic lung disease.

We present a case of interstitial pulmonary fibrosis accompanied by radiographic evidence of progressive massive fibrosis in a patient who had a 15–20 year history of almost daily recreational inhalation of methamphetamine. Mineralogical analysis confirmed the presence of talc on biopsy of the area of progressive massive fibrosis. The coexistence of interstitial pulmonary fibrosis and progressive massive fibrosis suggests that prolonged recreational inhalation of methamphetamine that has been “cut” with talc can result in sufficient amount of talc being inhaled to result in interstitial pulmonary fibrosis and progressive massive fibrosis in the absence of other causes.

Introduction Oxidative stress plays an important role in the pathogenesis of diabetic nephropathy (DN). This study examined if use of N-acetylcysteine for a month in moderate doses would reduce the oxidative stress in patients with DN and reduce the proteinuria. Methods Fifteen volunteers with DN participated in the study. Participants took capsule form of N-acetylcysteine 1 gm twice a day for a month. Spot urines were collected and tested for protein/creatinine on days 1 and 30. Sera were collected on days 1, 15, 30, and 60 and tested for several oxidative stress biomarkers. Results There was no significant change in proteinuria or any of the oxidant stress markers at any point: protein-creatinine ratio (day 1, 1.6 ± 1.8; day 30, 1.3 ± 1.3), 8-isoprostane (day 1, 5.9 ± 4.2 pg/mL; day 15, 4.67 ± 2.4 pg/mL; day 30, 5.1 ± 2.8 pg/mL; and day 60, 4.7 ± 1.9 pg/mL), total antioxidant status (day 1, 1.5 ± 0.1 mM; day 15, 1.6 ± 0.2 mM; day 30, 1.5 ± 0.1 mM; and day 60, 1.5 ± 0.2 mM), aconitase (day 1, 7.9 ± 5.9 mU/mL; day 15, 10.1 ± 5.9 mU/mL; day 30, 8.9 ± 6.2 mU/mL; and day 60, 7.8 ± 5.5 mU/mL), glutathione peroxidase (day 1, 261.4 ± 56.4 mU/mL; day 15, 263.9 ± 57.2 mU/mL; day 30, 269.2 ± 66.0 mU/mL; and day 60, 257.5 ± 48.2 mU/mL), and superoxide dismutase (day 1, 242.6 ± 79.3 mU/mL; day 15, 252.1 ± 68.1 mU/mL; day 30, 262.0 ± 73.3 mU/mL; and day 60, 255.7 ± 61.5). However, 4 patients with initial high isoprostane levels showed nonsignificant decline at each subsequent time point. Conclusions N-acetylcysteine in moderate doses given over a month did not have significant effect on the overall oxidative stress in patients with DN and did not reduce proteinuria.

1 Mohamed M. Ghanem, 2 Hussein A. A. Abel-Maksoud, 3 Val Vallyathan, 3 Mark Barger, 3 Lori Battelli, 4 Joginder Nath and 3 Ann Hubbs 1 Dept. Animal Med., Fac. Vet. Med. Moshtohor, Zagazig University/Benha, Egypt 2 Dept. Biochem., Fac. Vet. Med. Moshtohor, Zagazig University/Benha, Egypt 3 National Institute for Occupational Safety and Health, Morgantown, WV USA 4 Genetics and Developmental Biology, West Virginia University, Morgantown, WV, USA ABSTRACT

Perfluorooctane sulfonate (PFOS) is a member of the perfluoroalkyl acids (PFAA) containing an eight-carbon backbone. PFOS is a man-made chemical with carbon-fluorine bonds that are among the strongest in organic chemistry, and PFOS is widely used in industry. Human occupational and environmental exposure to PFOS occurs globally. PFOS is non-biodegradable and is persistent in the human body and environment. In this study, data demonstrated that exposure of human microvascular endothelial cells (HMVEC) to PFOS induced the production of reactive oxygen species (ROS) at both high and low concentrations. Morphologically, it was found that exposure to PFOS induced actin filament remodeling and endothelial permeability changes in HMVEC. Furthermore, data demonstrated that the production of ROS plays a regulatory role in PFOS-induced actin filament remodeling and the increase in endothelial permeability. Our results indicate that the generation of ROS may play a role in PFOS-induced aberrations of the endothelial permeability barrier. The results generated from this study may provide a new insight into the potential adverse effects of PFOS exposure on humans at the cellular level.

Background: Lung cancer remains the leading cause of cancer-related deaths worldwide. The recurrence rate ranges from 35-50% among early stage non-small cell lung cancer patients. To date, there is no fully-validated and clinically applied prognostic gene signature for personalized treatment.Methodology/Principal Findings: From genome-wide mRNA expression profiles generated on 256 lung adenocarcinoma patients, a 12-gene signature was identified using combinatorial gene selection methods, and a risk score algorithm was developed with Naive Bayes. The 12-gene model generates significant patient stratification in the training cohort HLM & UM (n = 256; log-rank P = 6.96e-7) and two independent validation sets, MSK (n = 104; log-rank P = 9.88e-4) and DFCI (n = 82; log-rank P = 2.57e-4), using Kaplan-Meier analyses. This gene signature also stratifies stage I and IB lung adenocarcinoma patients into two distinct survival groups (log-rank P<0.04). The 12-gene risk score is more significant (hazard ratio = 4.19, 95% CI: [2.08, 8.46]) than other commonly used clinical factors except tumor stage (III vs. I) in multivariate Cox analyses. The 12-gene model is more accurate than previously published lung cancer gene signatures on the same datasets. Furthermore, this signature accurately predicts chemoresistance/chemosensitivity to Cisplatin, Carboplatin, Paclitaxel, Etoposide, Erlotinib, and Gefitinib in NCl-60 cancer cell lines (P<0.017). The identified 12 genes exhibit curated interactions with major lung cancer signaling hallmarks in functional pathway analysis. The expression patterns of the signature genes have been confirmed in RT-PCR analyses of independent tumor samples.Conclusions/Significance: The results demonstrate the clinical utility of the identified gene signature in prognostic categorization. With this 12-gene risk score algorithm, early stage patients at high risk for tumor recurrence could be identified for adjuvant chemotherapy; whereas stage I and II patients at low risk could be spared the toxic side effects of chemotherapeutic drugs.

Bcl‐2 is a key apoptosis regulatory protein of the mitochondrial death pathway. The oncogenic potential of Bcl‐2 is well established, with its overexpression reported in various cancers. The antiapoptotic function of Bcl‐2 is closely associated with its expression levels. Reactive oxygen and nitrogen species (ROS/RNS) are important intracellular signaling molecules that play a key role in various physiological processes including apoptosis. We have recently reported that ROS and RNS can regulate Bcl‐2 expression levels, thereby impacting its function. Superoxide anion (·O2–) plays a proapoptotic role by causing downregulation and degradation of Bcl‐2 protein through the ubiquitin‐proteasomal pathway. In contrast, nitric oxide (NO)‐mediated S‐nitrosylation of Bcl‐2 prevents its ubiquitination and subsequent proteasomal degradation, leading to inhibition of apoptosis. Interestingly, NO‐mediated S‐nitrosylation and stabilization of Bcl‐2 protein was the primary mechanism involved in the malignant transformation of nontumorigenic lung epithelial cells in response to long‐term carcinogen exposure. We describe a novel mechanism of Bcl‐2 regulation by ·O2– and NO, providing a new dimension to reactive species‐mediated Bcl‐2 stability, apoptotic cell death, and cancer development.

Carbon nanotubes (CNT), since their discovery, have become one of the most promising nanomaterials in many industrial and biomedical applications. Due to their unique physicochemical properties, interest is growing in the manufacture of CNT-based products and their subsequent marketing. Since their discovery, the prospect of possible undesirable human health effects has been a focus of many scientific studies. Although CNT possess unique physical properties that include (1) nanoscale diameter, (2) a wide length distribution ranging from tens of nanometers to several micrometers, and (3) high aspect ratio, the fibrous-like shape and durability suggest that their toxic properties may be analogous to those observed with other fibrous particles, such as asbestos. The present study provides a summary of published findings on CNT bioactivity, such as the potential of CNT, especially of multi-wall carbon nanotubes (MWCNT), to activate signaling pathways modulating transcription factor activity, induce apoptosis, induce DNA damage, and initiate biological responses. Assessment of risks to human health and adoption of appropriate exposure controls is critical for the safe and successful introduction of CNT -based products for future applications.

Hard metal or cemented carbide consists of a mixture of tungsten carbide (WC) (85%) and metallic cobalt (Co) (5–15%). WC–Co is considered to be potentially carcinogenic to humans. However, no comparison of the adverse effects of nano-sized WC–Co particles is available to date. In the present study, we compared the ability of nano- and fine-sized WC–Co particles to form free radicals and propensity to activate the transcription factors, AP-1 and NF-κB, along with stimulation of mitogen-activated protein kinase (MAPK) signaling pathways in a mouse epidermal cell line (JB6 P+). Our results demonstrated that nano-WC–Co generated a higher level of hydroxyl radicals, induced greater oxidative stress, as evidenced by a decrease of GSH levels, and caused faster JB6 P+ cell growth/proliferation than observed after exposure of cells to fine WC–Co. In addition, nano-WC–Co activated AP-1 and NF-κB more efficiently in JB6+/+ cells as compared to fine WC–Co. Experiments using AP-1-luciferase reporter transgenic mice confirmed the activation of AP-1 by nano-WC–Co. Nano- and fine-sized WC–Co particles also stimulated MAPKs, including ERKs, p38, and JNKs with significantly higher potency of nano-WC–Co. Finally, co-incubation of the JB6+/+ cells with N-acetyl-cysteine decreased AP-1 activation and phosphorylation of ERKs, p38 kinase, and JNKs, thus suggesting that oxidative stress is involved in WC–Co-induced toxicity and AP-1 activation.

RATIONALE Previous studies have shown associations between dust exposure or lung burden and emphysema in coal miners, although the separate contributions of various predictors have not been clearly demonstrated. OBJECTIVES To quantitatively evaluate the relationship between cumulative exposure to respirable coal mine dust, cigarette smoking, and other factors on emphysema severity. METHODS The study group included 722 autopsied coal miners and nonminers in the United States. Data on work history, smoking, race, and age at death were obtained from medical records and questionnaire completed by next-of-kin. Emphysema was classified and graded using a standardized schema. Job-specific mean concentrations of respirable coal mine dust were matched with work histories to estimate cumulative exposure. Relationships between various metrics of dust exposure (including cumulative exposure and lung dust burden) and emphysema severity were investigated in weighted least squares regression models. MEASUREMENTS AND MAIN RESULTS Emphysema severity was significantly elevated in coal miners compared with nonminers among ever- and never-smokers (P < 0.0001). Cumulative exposure to respirable coal mine dust or coal dust retained in the lungs were significant predictors of emphysema severity (P < 0.0001) after accounting for cigarette smoking, age at death, and race. The contributions of coal mine dust exposure and cigarette smoking were similar in predicting emphysema severity averaged over this cohort. CONCLUSIONS Coal dust exposure, cigarette smoking, age, and race are significant and additive predictors of emphysema severity in this study.