Cobalt-induced oxidative stress contributes to alveolar/bronchiolar carcinogenesis in B6C3F1/N mice.

Rodent alveolar/bronchiolar carcinomas (ABC) that arise either spontaneously or due to chemical exposure are similar to a subtype of lung adenocarcinomas in humans. B6C3F1/N mice and F344/NTac rats exposed to cobalt metal dust (CMD) by inhalation developed ABCs in a dose dependent manner. In CMD-exposed mice, the incidence of Kras mutations in ABCs was 67% with 80% of those being G to T transversions on codon 12 suggesting a role of oxidative stress in the pathogenesis. In vitro studies, such as DMPO (5,5-dimethyl-1-pyrroline N-oxide) immune-spin trapping assay, and dihydroethidium (DHE) fluorescence assay on A549 and BEAS-2B cells demonstrated increased oxidative stress due to cobalt exposure. In addition, significantly increased 8-oxo-dG adducts were demonstrated by immunohistochemistry in lungs from mice exposed to CMD for 90 days. Furthermore, transcriptomic analysis on ABCs arising spontaneously or due to chronic CMD-exposure demonstrated significant alterations in canonical pathways related to MAPK signaling (IL-8, ErbB, Integrin, and PAK pathway) and oxidative stress (PI3K/AKT and Melatonin pathway) in ABCs from CMD-exposed mice. Oxidative stress can stimulate PI3K/AKT and MAPK signaling pathways. Nox4 was significantly upregulated only in CMD-exposed ABCs and NOX4 activation of PI3K/AKT can lead to increased ROS levels in human cancer cells. The gene encoding Ereg was markedly up-regulated in CMD-exposed mice. Oncogenic KRAS mutations have been shown to induce EREG overexpression. Collectively, all these data suggest that oxidative stress plays a significant role in CMD-induced pulmonary carcinogenesis in rodents and these findings may also be relevant in the context of human lung cancers.

Persistent effects of Libby amphibole and amosite asbestos following subchronic inhalation in rats.

BACKGROUND: Human exposure to Libby amphibole (LA) asbestos increases risk of lung cancer, mesothelioma, and non-malignant respiratory disease. This study evaluated potency and time-course effects of LA and positive control amosite (AM) asbestos fibers in male F344 rats following nose-only inhalation exposure. METHODS: Rats were exposed to air, LA (0.5, 3.5, or 25.0 mg/m(3) targets), or AM (3.5 mg/m(3) target) for 10 days and assessed for markers of lung inflammation, injury, and cell proliferation. Short-term results guided concentration levels for a stop-exposure study in which rats were exposed to air, LA (1.0, 3.3, or 10.0 mg/m(3)), or AM (3.3 mg/m(3)) 6 h/day, 5 days/week for 13 weeks, and assessed 1 day, 1, 3, and 18 months post-exposure. Fibers were relatively short; for 10 mg/m(3) LA, mean length of all structures was 3.7 µm and 1% were longer than 20 µm. RESULTS: Ten days exposure to 25.0 mg/m(3) LA resulted in significantly increased lung inflammation, fibrosis, bronchiolar epithelial cell proliferation and hyperplasia, and inflammatory cytokine gene expression compared to air. Exposure to 3.5 mg/m(3) LA resulted in modestly higher markers of acute lung injury and inflammation compared to AM. Following 13 weeks exposure, lung fiber burdens correlated with exposure mass concentrations, declining gradually over 18 months. LA (3.3 and 10.0 mg/m(3)) and AM produced significantly higher bronchoalveolar lavage markers of inflammation and lung tissue cytokines, Akt, and MAPK/ERK pathway components compared to air control from 1 day to 3 months post-exposure. Histopathology showed alveolar inflammation and interstitial fibrosis in all fiber-exposed groups up to 18 months post-exposure. Positive dose trends for incidence of alveolar epithelial hyperplasia and bronchiolar/alveolar adenoma or carcinoma were observed among LA groups. CONCLUSIONS: Inhalation of relatively short LA fibers produced inflammatory, fibrogenic, and tumorigenic effects in rats which replicate essential attributes of asbestos-related disease in exposed humans. Fiber burden, inflammation, and activation of growth factor pathways may persist and contribute to lung tumorigenesis long after initial LA exposure. Fiber burden data are being used to develop a dosimetry model for LA fibers, which may provide insights on mode of action for hazard assessment.

Use of high-throughput RT-qPCR to assess modulations of gene expression profiles related to genomic stability and interactions by cadmium.

Predictive test systems to assess the mode of action of chemical carcinogens are urgently required. Within the present study, we applied the Fluidigm dynamic array on the BioMark™ HD System for quantitative high-throughput RT-qPCR analysis of 95 genes and 96 samples in parallel, selecting genes crucial for maintaining genomic stability, including stress response as well as DNA repair, cell cycle control, apoptosis and mitotic signaling. The specificity of each individually designed sequence-specific primer pair and their respective target amplicons were evaluated via melting curve analysis as part of qPCR and size verification via agarose gel electrophoresis. For each gene, calibration curves displayed high efficiencies and correlation coefficients in the identified linear dynamic range as well as low intra-assay variations. Data were processed via Fluidigm real-time PCR analysis and GenEx software, and results were depicted as relative gene expression according to the ΔΔC q method. Subsequently, gene expression analyses were conducted in cadmium-treated adenocarcinoma A549 and epithelial bronchial BEAS-2B cells. They revealed distinct dose- and time-dependent and also cell-type-specific gene expression patterns, including the induction of genes coding for metallothioneins, the oxidative stress response, cell cycle control, mitotic signaling and apoptosis. Interestingly, while genes coding for the DNA damage response were induced, distinct DNA repair genes were down-regulated at the transcriptional level. Thus, this approach provided a comprehensive overview on the interaction by cadmium with distinct signaling pathways, also reflecting molecular modes of action in cadmium-induced carcinogenicity. Therefore, the test system appears to be a promising tool for toxicological risk assessment.

Kras, Egfr, and Tp53 Mutations in B6C3F1/N Mouse and F344/NTac Rat Alveolar/Bronchiolar Carcinomas Resulting from Chronic Inhalation Exposure to Cobalt Metal.

Rodent lung tumors are morphologically similar to a subtype of human lung adenocarcinomas. The objective of this study was to evaluate Kirsten rat sarcoma oncogene homolog (Kras), epidermal growth factor receptor (Egfr), and tumor protein 53 (Tp53) mutations, which are relevant to human lung cancer, in cobalt metal dust (CMD)-induced alveolar/bronchiolar tumors of B6C3F1/N mice and F344/NTac rats. Kras mutations were detected in 67% (mice) and 31% (rats) of CMD-induced lung tumors and were predominantly exon 1 codon 12 G to T transversions (80% in mice and 57% in rats). Egfr mutations were detected in 17% (both mice and rats) of CMD-induced lung tumors and were predominantly in exon 20 with 50% G to A transitions (mice and rats). Tp53 mutations were detected in 19% (mice) and 23% (rats) of CMD-induced lung tumors and were predominant in exon 5 (mice, 69% transversions) and exon 6 (rats, all transitions). No mutations were observed for these genes in spontaneous lung tumors or normal lungs from untreated controls. Ames assay indicated that CMD is mutagenic in the absence but not in the presence of S9 mix. Thus, the mutation data (G to T transversions) and Ames assay results suggest that oxidative damage to DNA may be a contributing factor in CMD-induced pulmonary carcinogenesis in rodents.

Subchronic toxicity and carcinogenicity studies of 1,2-dichloropropane inhalation to mice.

The subchronic toxicity and carcinogenicity of 1,2-dichloropropane (DCP) in male and female B6D2F1 mice exposed to DCP by inhalation for 13 weeks or for 2 years was investigated. The DCP concentrations used were 50, 100, 200, 300 or 400 ppm (v/v) in the 13-week study, and 32, 80 or 200 ppm (v/v) in the 2-year study. Thirteen weeks inhalation exposure of mice to DCP caused death in the mice exposed to 300 ppm and above, and was found to induce hemolytic anemia and lesions of the liver, forestomach and heart. Two years exposure to DCP significantly increased the combined incidence of bronchiolo-alveolar adenomas and carcinomas in females and marginally increased the incidence of Harderian gland adenomas in males. As non-neoplastic lesion, atrophy and respiratory metaplasia in the olfactory epithelium, and respiratory metaplasia in the submucosal gland of the nasal cavity were increased. Thus, two years inhalation exposure to DCP is carcinogenic in female mice and there is a marginal evidence of carcinogenicity in males.

Chronic exposure to particles caused bronchioloalveolar carcinoma in a patient with cryptogenic organizing pneumonia evaluated by elemental analysis.

An 81-year-old Japanese man had organizing pneumonia (OP), and he had worked as a painter and had a history of exposure of various paints over 20 years. The major features on computed tomography (CT) in patients were cryptogenic organizing pneumonia (COP) showing airspace consolidation, and air bronchograms were consistent finding in consolidation in right lung of S¹°. Such parenchymal abnormalities were clinically and pathologically diagnosed COP and the lesion was improved by corticosteroid therapy. About 1.5 years later, similar shadows emerged in new locations of right S4 and left S8, and these were bronchioloalveolar carcinoma (BAC) classified as adenocarcinoma. BAC causes similar X-ray changes to COP and inflammation accompanying BAC can also respond to corticosteroids, which may lead to delay in the diagnosis of BAC associated with COP. These radiological features lead to difficulty in making a diagnosis of new parenchymal diseases. The present patient had been painter, and metals of carcinogens were proven in both tissue of COP and BAC. Here, we reported a painter with COP and new-onset BAC who had been exposed to particles proven by elemental analysis. The combination of COP with BAC is considered uncommon, but the risk of BAC may increase when there is a history of particle inhalation.

Multisite carcinogenicity and respiratory toxicity of inhaled 1-bromopropane in rats and mice.

Two-year 1-bromopropane (1-BP) inhalation studies were conducted because of the potential for widespread exposure, the lack of chronic toxicity and carcinogenicity data, and the known carcinogenicity of structurally related compounds. Male and female F344/N rats and B6C3F1/N mice were exposed by inhalation to 0, 62.5 (mice only), 125, 250, or 500 (rats only) ppm 1-BP for 6 hr/day, 5 days/week for 105 weeks. Exposure of male and female rats to 1-BP resulted in significantly increased incidences of adenomas of the large intestine and skin neoplasms. In male rats, the incidence of malignant mesothelioma of the epididymis was statistically significantly increased at 500 ppm, but the biological significance of this common lesion is unclear. Incidences of pancreatic islet adenoma in male rats were significantly increased at all concentrations relative to concurrent controls but were within the historical control range for inhalation studies. There was no evidence of carcinogenic activity of 1-BP in male B6C3F1 mice; however, significantly increased incidences of alveolar/bronchiolar neoplasms of the lung were present in female mice. Exposure to 1-BP also resulted in increased incidences of nonneoplastic lesions in the nose of rats and mice, the larynx of rats and male mice, the trachea of female rats and male and female mice, and the lungs of mice. Inflammatory lesions with Splendore Hoeppli (S-H) material were present primarily in the nose and skin of exposed male and female rats, indicating that 1-BP caused immunosuppression.

FTY720 treatment in experimentally urethane-induced lung tumors.

FTY720 has been shown to prevent cancer development in experimental models but there is no report whether this beneficial effect is associated with the time point of the drug administration. Lung adenoma was induced in mice by urethane injection followed by different periods of FTY720 administration in order to evaluate lung tumor development. BALB/c mice received two doses (1, 5 g/kg) of urethane intraperitoneally and were submitted to five daily doses of FTY720 (1 mg/kg/day) starting just after urethane injection (G2 n=5), 4 weeks after urethane injection (G3 n=10), 8 weeks after urethane injection (G4 n=10) and no FTY720 administration (G1 n=5). Twenty-four weeks after urethane administration mice were evaluated for leukocyte numbers in blood, lymphocytes in spleen, and lungs were evaluated for changes in histology and PCNA expression. Lung nodules were present in higher numbers both in non treated (G1; 0.0-7.0) and FTY720 treated 8 weeks after urethane injection (G4; 0.0-6.0). G4 Group also presented the highest number of papillary nodules. There was a decrease in PCNA staining in early time FTY720 treated mice. Therefore, our data suggest that FTY720 treatment in early periods after lung tumor induction is beneficial and impairs adenoma development.

Gene expression studies demonstrate that the K-ras/Erk MAP kinase signal transduction pathway and other novel pathways contribute to the pathogenesis of cumene-induced lung tumors.

National Toxicology Program (NTP) inhalation studies demonstrated that cumene significantly increased the incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. Cumene or isopropylbenzene is a component of crude oil used primarily in the production of phenol and acetone. The authors performed global gene expression analysis to distinguish patterns of gene regulation between cumene-induced tumors and normal lung tissue and to look for patterns based on the presence or absence of K-ras and p53 mutations in the tumors. Principal component analysis segregated the carcinomas into groups with and without K-ras mutations, but failed to separate the tumors based on p53 mutation status. Expression of genes associated with the Erk MAP kinase signaling pathway was significantly altered in carcinomas with K-ras mutations compared to tumors without K-ras mutations or normal lung. Gene expression analysis also suggested that cumene-induced carcinomas with K-ras mutations have greater malignant potential than those without mutations. In addition, significance analysis of function and expression (SAFE) demonstrated expression changes of genes regulated by histone modification in carcinomas with K-ras mutations. The gene expression analysis suggested the formation of alveolar/bronchiolar carcinomas in cumene-exposed mice typically involves mutation of K-ras, which results in increased Erk MAP kinase signaling and modification of histones.

Genetic alterations in K-ras and p53 cancer genes in lung neoplasms from B6C3F1 mice exposed to cumene.

The incidences of alveolar/bronchiolar adenomas and carcinomas in cumene-treated B6C3F1 mice were significantly greater than those of the control animals. We evaluated these lung neoplasms for point mutations in the K-ras and p53 genes that are often mutated in humans. K-ras and p53 mutations were detected by cycle sequencing of PCR-amplified DNA isolated from paraffin-embedded neoplasms. K-ras mutations were detected in 87% of cumene-induced lung neoplasms, and the predominant mutations were exon 1 codon 12 G to T transversions and exon 2 codon 61 A to G transitions. P53 protein expression was detected by immunohistochemistry in 56% of cumene-induced neoplasms, and mutations were detected in 52% of neoplasms. The predominant mutations were exon 5, codon 155 G to A transitions, and codon 133 C to T transitions. No p53 mutations and one of seven (14%) K-ras mutations were detected in spontaneous neoplasms. Cumene-induced lung carcinomas showed loss of heterozygosity (LOH) on chromosome 4 near the p16 gene (13%) and on chromosome 6 near the K-ras gene (12%). No LOH was observed in spontaneous carcinomas or normal lung tissues examined. The pattern of mutations identified in the lung tumors suggests that DNA damage and genomic instability may be contributing factors to the mutation profile and development of lung cancer in mice exposed to cumene.

Carcinogenicity of 5-nitrofurans and related compounds with amino-heterocyclic substituents.

Carcinogenicity of eight 5-nitrofurans with heterocyclic substituents at the 2-position of the furan ring was investigated by feeding the chemicals to Sprague-Dawley female rats. N-[5-(5-nitro-2-furyl)-1,3,4-thiadiazol-2-yl]acetamide induced in 30 rats the highest incidence of tumors with the greatest number of tissues involved: forestomach squamous cell tumors (22), kidney pelvis transitional cell carcinomas (15), pulmonary alveolar cell carcinomas (16), hemangioendothelialsarcomas (20) of the intestine, mesentery, liver, lung, and pancreas, and a few tumors of other tissues. 2-Amino-5-(5-nitro-2-furyl)-1,3,4-thiadiazole, 2-amino-5-(5-nitro-2furyl)-1,3,4-oxadiazole, and trans-2-[dimethylamino)methylimino]-5-[2-(5-nitro-2-furyl)vinyl]-1,3,4-oxadiazole produced high incidences of mammary tumors. The other four 5-nitrofurans tested: N-[4-(5-NITRO-2-FURYL)-2-THIAZOLYL]ACETAMIDE;2,3,4-TRIFLUORO-N-[4-(5-NITRO-2-furyl)-2-thiazoly]acetamide;5-(5-nitro-2-furyl)-1,3,4-oxadiazol-2-ol; and N-( [3-(5-nitro-2-furyl)-1,2,4-oxadiazol-5-yl]methyl)acetamide were associated with tumor incidences of 40-60%. Two other chemicals were also tested: 2-Amino-5-nitrothiazole caused a low incidence of breast and kidney pelvis tumors, and 2-amino-4-(p-nitrophenyl)thiazole induced a high incidence of breast and salivary gland adenocarcinomas and lymphomas.

Bronchiolitis obliterans organizing pneumonia simulating progression in bronchioloalveolar carcinoma.

Bronchiolitis obliterans organizing pneumonia (BOOP) is a clinicopathologic syndrome with characteristic features. The diagnosis of BOOP requires the presence of a combination of pathological, clinical, and radiological features. We report the case of a lung cancer patient with bronquiloalveolar carcinoma (BAC) presenting with BOOP after chemotherapy with docetaxel and gemcitabine producing severe respiratory insufficiency, and simulating a progression of the tumor.

Induction of Lung Tumors in Mice with Urethane.

In this protocol, urethane (ethyl carbamate) is used to induce lung tumors in mice. The use of urethane as an experimental carcinogen is especially attractive as it is inexpensive, relatively safe to handle, stable, and water soluble, and the protocol involves simple intraperitoneal (i.p.) injections in young mice. Urethane typically induces bronchioalveolar adenomas and, to a lesser extent, adenocarcinomas that resemble the adenocarcinoma subtype of non-small cell lung carcinoma. On a sensitive genetic background such as A/J, mice develop multiple adenomas visible on the lung surface by 25 wk, followed by the appearance of adenocarcinomas by 40 wk. Less-sensitive strains such as B6/129 develop tumors with a longer latency.

Carcinogenesis of nitrosomethylundecylamine in Fischer rats.

Nitrosomethylundecylamine was synthesized and administered to Fischer rats by gavage in olive oil solution, at a dose of 46 mg/animal/week for 30 weeks. There were high incidences of hepatocellular carcinomas and cholangiocarcinomas in the liver and of squamous cell carcinomas and alveolar cell adenocarcinomas in the lung. In contrast with the activity of the next higher homolog, nitrosomethyldodecylamine, no bladder tumors were induced.

Lung tumors induced by chronic inhalation of 1,2-dibromo-3-chloropropane in B6C3F1 mice.

1,2-Dibromo-3-chloropropane (DBCP) was inhaled by B6C3F1 mice of each sex at concentrations of 0.6 or 3.0 ppm for 6 h/day, 5 days/week, for 103 weeks. Untreated controls consisted of 50 mice of each sex. All surviving animals were killed at 104 weeks. Survival in the high-dose groups of female mice was significantly shorter than that in the corresponding controls. Alveolar-bronchiolar adenomas or carcinomas occurred in both male and female mice with dose-related trends (P < 0.007) in males and (P < 0.002) in females. Papillary carcinomas in the lung occurred in low-dose female mice at an increased incidence compared with controls (P < 0.006). Many induced tumors were morphologically different than those in controls.

Inhalation of tetranitromethane causes nasal passage irritation and pulmonary carcinogenesis in rodents.

Fischer 344 rats and B6C3F1 mice were exposed for 2 years to vapors of tetranitromethane at concentrations below (0.5 ppm) and slightly above (2 or 5 ppm) the current U.S. recommended occupational exposure limit. Under the conditions of exposure of 6 h/day, 5 days/week, tetranitromethane was found to cause mild irritation and hyperplastic lesions in the nasal passages, but not nasal cavity neoplasms were observed. In contrast, nearly all animals exposed to the higher TNM concentrations, and the majority of animals exposed to the lower concentrations developed alveolar/bronchiolar adenoma or carcinoma; squamous cell neoplasms of the lung also occurred in exposed rats. The extent of the lung tumor response, and the low concentrations of tetranitromethane required for this response, are unprecedented in National Toxicology Program (NTP) studies.

Increased expression of cyclooxygenase-2 protein in rat lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine.

Expression of cyclooxygenase (COX)-2 protein in preneoplastic and neoplastic lung lesions induced by the administration of 2000 ppm of N-nitrosobis(2-hydroxypropyl)amine (BHP) in the drinking water to Wistar male rats, was examined immunohistochemically. The majority of alveolar/bronchiolar adenomas (ADs) and all adenocarcinomas (ADCs) examined, stained positive or strongly positive for COX-2. In contrast, only a minority of alveolar/bronchiolar hyperplasias demonstrated immunoreactivity and half of the squamous cell carcinomas examined, were only weakly positive. Western blotting analysis also revealed expression of COX-2 protein in the resected ADs and ADCs. These results clearly indicate up-regulated expression of COX-2 in lung neoplastic lesions, particularly ADs and ADCs, induced by BHP in rats.

Increased susceptibility to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in transgenic mice overexpressing c-myc and epidermal growth factor in alveolar type II cells.

PURPOSE: As previously described, SPC/myc transgenic mice developed bronchioloalveolar adenocarcinomas derived from alveolar type II (AT II) cells within 10-14 months, whereas SPC/IgEGF transgenic mice developed hyperplasias. Our purpose was to determine the potential interplay of environmental and genetic factors in lung tumorigenesis. MATERIALS AND METHODS: Six-week-old SPC/myc and SPC/IgEGF transgenic mice, overexpressing c-myc and a secretable form of the epidermal growth factor (IgEGF) under the control of the surfactant protein C (SPC) promoter, were treated with a single dose of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). As control groups, SPC/myc and SPC/IgEGF transgenic mice were treated with NaCl and non-transgenic littermates were treated with NNK or NaCl, respectively. RESULTS: After 6 months, none of the NaCl-treated transgenic littermates showed bronchioloalveolar hyperplasia and adenocarcinoma formation, whereas 100% of the NNK-treated SPC/myc transgenic mice did. The effect of NNK on SPC/IgEGF transgenic mice was less pronounced, inducing hyperplasia in the lung in only 16.7% of them. In 90% of the NNK-treated non-transgenic littermates no neoplastic changes were detected in the lung. CONCLUSIONS: These results demonstrate that the progression of pulmonary bronchioloalveolar adenocarcinomas, induced by expression of c-myc as a transgene, was accelerated by NNK, suggesting that c-myc cooperates with NNK-induced mutations.

Lung tumor induction by inhalation exposure to molybdenum trioxide in rats and mice.

Inhalation studies of molybdenum trioxide (MoO3) were conducted because of its wide use in industry, human exposure, and lack of data on carcinogenicity. Groups of 50 male and 50 female F344/N rats and B6C3F1 mice were exposed to MoO3 by inhalation at 0, 10, 30, or 100 mg/m3, 6 h/day, 5 days/week, for 2 years. In both rats and mice, survival and mean body weights of exposed groups of males and females were similar to those of their respective controls. There were significant exposure-dependent increases in blood molybdenum concentration in exposed rats and mice. There were no toxicological differences in bone density or curvature between exposed animals and their respective controls. In rats, dose-dependent increases in incidence of hyaline degeneration in the nasal olfactory epithelium and squamous metaplasia of the epithelium lining the base of the epiglottis were observed. The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) was marginally increased in males but not in females compared with controls. In mice, the incidences of squamous metaplasia of the epithelium lining the base of the epiglottis, hyperplasia of the laryngeal epithelium, and metaplasia of the alveolar epithelium were significantly increased in all exposed males and females compared with controls. The incidence of alveolar/bronchiolar adenoma or carcinoma (combined) in exposed groups of males and females was significantly greater than that in the control groups.

Morphology of spontaneous and induced tumors in the bronchiolo-alveolar region of F344 rats.

The morphology of spontaneous and chemically-induced metastasizing carcinomas and adenomas in the bronchiolo-alveolar region of F-344 rats was studied. Histologically, the tumors were tubulo-papillary. Ultra-structurally, they consisted of cells which formed and secreted osmiophilic lamellated inclusion bodies, a marker of alveolar type II cells. Mitotic tumor cells also demonstrated such bodies. No cells of bronchial or bronchiolar origin were found in the tumors. We conclude that in F344 rats, lung tumors located in the bronchioloalveolar region consist of alveolar type II cells exclusively and are, therefore, alveolar cell adenomas and carcinomas, respectively.