Elevation of surfactant protein A in plasma and sputum in cigarette smokers.

Serum surfactant protein (SP)-A has been postulated to associate with pulmonary fibrosis, but its role in cigarette smoking-related lung diseases is undefined. SP-A levels in plasma and induced sputum in nonsmokers, smokers with respiratory symptoms (cough and/or phlegm) and symptom-free smokers were assessed using a validated EIA method. A total of 474 current smokers without any diseases or medications were enrolled and followed for 2 yrs with 111 of them succeeding in stopping. Plasma SP-A level was detectable in all subjects and elevated in smokers independently of the symptoms compared to nonsmokers (p = 0.001). After 2 yrs of follow-up, the SP-A level was higher in those who continued smoking compared to the quitters (p<0.001). Plasma SP-A levels were associated with age, smoking history and lung function. Sputum (n = 109) SP-A was nondetectable in most nonsmokers, whereas smoking and symptoms increased sputum SP-A highly significantly (p = 0.001). In conclusion, SP-A may be involved in pathogenesis of cigarette smoking-related lung diseases. Further studies are needed to elucidate the role of SP-A in chronic obstructive pulmonary disease.

Matrix metalloproteinases-7, -8, -9 and TIMP-1 in the follow-up of diisocyanate-induced asthma.

BACKGROUND: Diisocyanate-induced asthma (DIA) is known to be associated with poor prognosis. We wished to clarify if matrix metalloproteinases (MMP)-7, -8 or -9 or tissue inhibitor of matrix metalloproteinases (TIMP-1) are associated with the functional or inflammatory outcome in DIA patients. METHODS: This is a longitudinal study where 17 patients with DIA diagnosed by a specific challenge test to diisocyanates were monitored. Exposure to diisocyanates was terminated seven (mean) months before the challenge test. The studies included spirometry, histamine challenge test and bronchoscopy. MMP-7, MMP-8, TIMP-1 [Enzyme-linked immunosorbent assay (ELISA)- and immunofluorometric assay-methods], MMP-9 (ELISA and zymography), interferon-gamma, tumour necrosis factor-alpha, interleukin-6, -8, -15, -17, CXCL-5/ENA-78, monocyte chemoattractant protein-1 and macrophage inhibitory factor (MIF) (ELISA) were assayed from bronchoalveolar lavage (BAL) fluid. Inhaled steroid therapy was initiated after the examinations, which were repeated at 6 months and at 3 years during the treatment. The results were compared with those of 15 healthy controls. RESULTS: Inhaled steroid medication increased BAL levels of MMP-9 and MMP-9/TIMP-1 and decreased MMP-7 and MMP-7/TIMP-1. The increase in MMP-9 levels was associated with a decline in the TH-2 type inflammation. CONCLUSIONS: Our data suggest that reduced TH-2 type inflammation in DIA after inhaled steroid medication is reflected as elevated MMP-9 and MMP-9/TIMP-1 levels in BAL. MIF may be the inducer of MMP-9. This might point to some protective role for MMP-9 in DIA.

Real-world clinical data identifies gender-related profiles in chronic obstructive pulmonary disease.

This study aims to compare diagnostic and clinical outcomes of Chronic Obstructive Pulmonary Disease (COPD) from the gender perspective using retrospective health care data and patient reported outcomes in a real-world setting. An electronic database was constructed from complete medical records of 844 COPD patients who were recruited in Helsinki and Turku University Central Hospitals during the years 2005-07. The patients were identified from the hospital discharge registries by ICD10 code J44.8 in the age group of 18-75 years of age. The medical history was obtained from all healthcare providers who had treated these patients during the previous 5-10 years; the study intends to continue their follow-up annually for the next 10 years. Thirty-six percent (N = 266) of the participants were women. The COPD diagnosis had been made at the same age for both genders. Women, however, reported significantly less pack-years than men. Compared to the men, the women displayed less advanced airway obstruction, but more severe gas transfer impairment. Parenchymal damage when evaluated by diffusion capacity correlated significantly stronger with FEV(1) (% of predicted) in women than men. The BMI index of the women was lower than that of the men. Cardiovascular diseases, diabetes and alcoholism were significantly more common in men, but women suffered more psychiatric conditions, especially depression. This cohort showed several significant gender dependent differences in the clinical presentation that need to taken under consideration in the assessment of COPD progression and the disease management.

Thiol proteins, redox modulation and parenchymal lung disease.

The lung is a unique organ in terms of its direct exposure to high levels of oxygen and reactive compounds. Several parenchymal lung diseases (e.g. emphysema associated with smoking and a number of fibrotic lung disorders) have been proposed to be due to the exposure of the lung to exogenous irritants leading to local redox imbalance in the alveolar epithelium. The disease progression of emphysema/chronic obstructive pulmonary disease (COPD) and fibrosis share several common factors, such as the role of reactive oxygen species, disturbances of the pulmonary thiol status and activation of growth factors and tissue destructing proteases. Importantly in COPD or fibrosis, medication does not provide any significant therapeutic effect. This review concentrates on the key thiol (-SH)-regulated mechanisms leading to the development of COPD and/or pulmonary fibrosis and the major redox-regulated defense/oxidant repair mechanisms, thioredoxin/peroxiredoxin and glutaredoxin protein families in the lung. Redox-regulated proteins, both proteases and oxidant repair enzymes, undergo conformational changes during oxidative stress, a process that modulates their activation or inactivation. In addition, some of the redox-regulated proteins influence the metabolism of glutathione (GSH), a major small molecular antioxidant of human lung, and participate in the crosstalk between numbers of GSH associated enzymes functioning in the detoxification pathways of human lung. An understanding of the processes involved in oxidant-mediated lung damage may provide the key to devising interventional strategies that can actually prevent the progression of lung parenchymal disease.

Gene copy number analysis in malignant pleural mesothelioma using oligonucleotide array CGH.

Conventional cytogenetic analyses and comparative genomic hybridization have revealed a complex and even chaotic nature of chromosomal aberrations in pleural malignant mesothelioma (MM). We set out to describe the complex gene copy number changes and screen for novel genetic aberrations using a high-density oligonucleotide microarray platform for comparative genomic hybridization (aCGH) of a series of 26 well-characterized MM tumor samples. The number of copy number changes varied from zero to 40 per sample. Gene copy number losses predominated over gains, and the most frequent region of loss was 9p21.3 (17/26 cases), the locus of CDKN2A and CDKN2B, both known to be commonly lost in MM. The most recurrent minimal regions of losses were 1p31.1--> p13.2, 3p22.1-->p14.2, 6q22.1, 9p21.3, 13cen-->q14.12, 14q22.1-->qter, and 22qcen-->q12.3. Previously unreported gains included 9p13.3, 7p22.3-->p22.2, 12q13.3, and 17q21.32-->qter. The results suggest that gene copy number losses are a major mechanism of MM carcinogenesis and reveal a recurrent pattern of copy number changes in MM.

Chronic bronchitis and chronic obstructive pulmonary disease. The Finnish Action Programme, interim report.

The Finnish National Prevention and Treatment Programme for Chronic Bronchitis and COPD, launched in 1998, has, to date, been running for 6 years (2003). The goals of this action programme were to reduce the incidence of COPD and the number of moderate and severe cases of the disease, and to reduce both the number of days of hospitalisation and treatment costs. A prevalent implementation of over 250 information and training events started. Health centres and pharmacies appointed a person in charge of COPD patients. In order to improve the cooperation between primary and specialised care, two thirds of hospital districts created local COPD treatment chains. The early diagnosis of COPD by spirometric examination was activated during the programme. Number of health centres with available spirometric services increased to 95%. Before the start of the programme, approximately 5-9% of the adult population had COPD. During the whole programme, the proportion of male and female smokers decreased from 30% to 26% and from 20% to 19%, respectively. The total number of hospitalisation periods and days due to COPD decreased by 15% and 18%, respectively. Both the number of pensioners and daily sickness days due to COPD also decreased by 18%. Registered COPD induced deaths remained at their previous levels during the monitoring period, i.e. around 1000 deaths out of 5.2 millions annually. The measures recommended by the programme have been widely introduced but they need to be still more effective.

Peroxiredoxins and antioxidant enzymes in pilocytic astrocytomas.

OBJECTIVE: Peroxiredoxins are antioxidant enzymes (AOEs), which are redox-regulated thiol proteins with potential effects on the growth, invasion and drug resistance of neoplastic cells. In this study, their biology and clinical significance were examined in pilocytic astrocytomas (PAs). MATERIAL AND METHODS: The expression of peroxiredoxins (Prx I-VI) was investigated in 105 PAs by the means of immunohistochemistry and compared with the expression of selected other antioxidant enzymes, cell proliferation, angiogenesis, apoptosis, p53, histopathology and patient survival. RESULTS: Peroxiredoxins were strongly expressed in general suggesting that oxidative damage and consequent defense takes place during the progression of pilocytic astrocytomas. In agreement with this hypothesis, several other AOEs correlated with the degenerative features and angiogenesis possibly associated with reactive oxygen species-derived cellular damage. Moreover, the expression of the AOEs was associated with each other indicating a concurrent activation of the enzymes. With the exception of manganese superoxide dismutase (MnSOD), a strong expression of AOEs was generally associated with higher cell proliferation. Prx VI seemed to have a positive association with a longer recurrence-free interval while other AOEs had no association with patient survival. Many AOEs, such as MnSOD, induce chemo- and radioresistance and are highly elevated in aggressive malignancies. PAs lack this confounding factor, and these tumors are treated only by surgery. CONCLUSIONS: Taken together, the results of this study on pilocytic astrocytomas suggest that the levels of Prxs and other AOEs and their related thiol proteins are generally strongly expressed in these tumors. At least Prx VI can contribute to tumor behavior which can make it a potential prognostic factor.

Tissue MMP-2 and MMP-9 [corrected] are better prognostic factors than serum MMP-2/TIMP-2--complex or TIMP-1 [corrected] in stage [corrected] I-III lung carcinoma.

Matrix metalloproteinases (MMPs) are involved in tumor growth and spreading. Here, we investigated the tumor immunoreactive protein of MMP-2, MMP-9 and TIMP-1 as well as the levels of circulating total TIMP-1 and MMP-2/TIMP-2-complex as prognostic factors in lung cancer patients. The material included 59 patients, 30 with a squamous cell carcinoma, 21 with an adenocarcinoma and eight with other histology. Circulating antigens were measured by ELISA assay and the protein expression in primary tumors was analyzed by streptavidin-biotin immunohistochemical staining using specific monoclonal antibodies. The strong positivity for MMP-2 or MMP-9 in tumor predicted poor prognosis. The 5-year survival rates were 83 or 85% in patients negative for MMP-2 or MMP-9, respectively. Only 17% of the patients with a tumor highly positive for MMP-2 and 43% of those with a high positivity for MMP-9 survived at that time (Cox regression P=0.042 for MMP-2 and log rank P=0.046 for MMP-9). On the contrary, strong tissue positivity for TIMP-1 demonstrated a tendency for a favorable survival, although the difference did not reach statistical significance. In patients with a squamous cell carcinoma Stage I, low serum TIMP-1 (or=300 ng/ml) associated with an increased survival rate, the 5-year survival being 81 versus 34% (log rank P=0.069) in patients with high or low serum levels for MMP-2/TIMP-2-complex, respectively. Tissue MMP-2 correlated to high expression of MMP-9 immunoreactive protein (P=0.003), but the serum levels of MMP-2/TIMP-2-complex or TIMP-1 did not correlate to the immunostaining of the corresponding tumors. We conclude that in lung carcinoma the best prognostic value is achieved by using immunohistochemistry for MMP-2 and MMP-9. In early disease, however, serum TIMP-1 or MMP-2/TIMP-2-complex could offer some further prognostic value.

Oxidative/nitrosative stress and peroxiredoxin 2 are associated with grade and prognosis of human renal carcinoma.

Peroxiredoxins (Prxs) 1-6 were assessed in 138 renal cell carcinomas (RCC) using immunohistochemistry and selected samples by Western blotting analysis. Oxidative/nitrosative damage was evaluated using nitrotyrosine immunoreactivity. The expressions of Prxs were correlated with tumor grade and survival and nitrotyrosine reactivity. Non-malignant kidney tubular cells showed positivity with variable intensity for all six Prxs. In RCCs, most cases were positive for Prxs 1 and 2, while only 15-20% of tumors showed expression for Prxs 3 and 4. Prx 2 was associated with tumors of a lower grade (p=0.009) and with a lower frequency of distant metastases (p=0.046). Patients with tumors expressing Prx2 had better prognosis (p=0.027). Instead, nitrotyrosine was significantly associated with high grade tumors (p=0.001). Compared with the non-malignant kidney tubular cells, low Prx expression in the tumor cells can make them more susceptible to oxidative damage. Prx 2 was more abundantly expressed in low grade tumors, suggesting that this protein could play a role in preventing the development of oxidative damage, which in turn can lead to the activation of pathways leading to aggressive tumors.

Claudins in differential diagnosis between mesothelioma and metastatic adenocarcinoma of the pleura.

AIM: To study the expression of claudins in mesothelioma and metastatic pleural adenocarcinoma. METHODS: Immunohistochemical staining of claudins 1, 2, 3, 4, 5, and 7 was studied in 35 malignant mesotheliomas and the expression compared with 24 cases of pleural metastatic adenocarcinoma. All cases were also immunostained with calretinin. RESULTS: Claudin 1, 2, 3, 4, 5, and 7 expression was seen in 40%, 80%, 18%, 23%, 14%, and 43% of mesotheliomas, respectively, while the corresponding figures for adenocarcinoma were 100%, 88%, 90%, 100%, 50%, and 92%. Claudins 1, 3, 4, 5, and 7 were significantly less positive in mesothelioma than in metastatic adenocarcinoma, while no difference was observed for claudin 2. Claudins 1, 3, 4, 5, and 7 were also inversely associated with calretinin positivity. Sarcomatoid and biphasic mesothelioma subtypes appeared more negative for these claudins than pure epithelioid subtypes. Claudin expression was not associated with survival of patients with malignant mesotheliomas. CONCLUSIONS: The results show that malignant mesotheliomas have a lower expression of claudins 1, 3, 4, 5, and 7 than adenocarcinomas, and their expression could thus be used as an adjunct in differential diagnosis between the two. The difference was most evident for claudins 3 and 4, which were nearly as good as calretinin in mesothelioma detection. Sarcomatoid and biphasic mesotheliomas showed expression of these claudins only occasionally, which could be due to or contribute to their less epithelial appearance.

Activation and relocalization of caspase 3 during the apoptotic cascade of human mesothelioma cells.

Apoptosis plays an important role in cancer biology. We investigated the expression of caspases 3 and 8 in malignant mesothelioma and malignant mesothelioma cell lines and putative changes in their ultrastructural expression prior and after exposure to epirubicin. Further studies were conducted to compare these changes to the localization and expression of the bcl-2 group of proteins bcl-X, bax and mcl-1, and Fas-Fas ligand in the same cells. In the histological samples, caspase 3 and 8 immunoreactivity was seen in 27/37 (73%) and 16/37 (43%) of the mesotheliomas. The immunostaining was cytoplasmic diffuse, granular, and occasionally nuclear. All six mesothelioma cell lines expressed caspases 3 and 8 by immunoblotting. After exposure to epirubicin the extent of apoptosis was increased in all cell lines investigated, being weakest in the most resistant M38K cell line. Immunoelectron microscopy revealed immunogold labeling for caspases 3 and 8 in the mitochondria with the accumulation of caspase 3 in the apoptotic bodies, while the mitochondrial localization of the bcl-2 proteins appeared to be very stable. Fas receptor could be detected by flow cytometry, whereas the most resistant cell line (M38K) lacked Fas ligand when assessed by RT-PCR. These results suggest the importance of caspase 3 during the apoptotic process of mesothelioma cells and indicate that epirubicin-induced apoptosis is independent of the mitochondrial pathway.

Regulation of the acute myeloid leukemia cell line OCI/AML-2 by endothelial nitric oxide synthase under the control of a vascular endothelial growth factor signaling system.

It is generally accepted that the vascular endothelial growth factor (VEGF) signal system has no role in the maintenance of normal blood cell formation, although it obviously regulates the development of primitive hematopoiesis during an early stage of embryogenesis. The VEGF signaling pathway, however, might have some role in malignant hematopoiesis, since malignant hematopoietic cells, including acute myeloid leukemia (AML) cells, have been shown to express VEGF and its receptors. In endothelial cells, the VEGF/Flk-1/KDR signal system is a very important generator of nitric oxide (NO) through the activation of its downstream effectors phosphatidylinositol-3-OH-kinase (PI3-K), Akt kinase and endothelial NO synthase (eNOS). It is known that NO regulates hematopoiesis and modulates AML cell growth. The role of the VEGF signaling pathway in the control of AML cell growth through eNOS, however, has not been studied. By using the OCI/AML-2 cell line, which expresses VEGF receptor-2, ie Flk-1/KDR, eNOS and VEGF, as analyzed by flow cytometry, and produces VEGF into growth medium, as analyzed by ELISA, we showed that the Akt kinase and NOS activities in these cells were decreased by the inhibitors of VEGF, Flk-1/KDR and PI3-K, and NOS activity also by the direct inhibitor of NOS. The decreased NOS activity led to inhibition of clonogenic cell growth and, to some extent, induction of apoptosis. We also found that blast cells of bone marrow samples randomly taken from 14 AML patients uniformly expressed Flk-1/KDR and to varying degrees eNOS and VEGF, as analyzed by immunohistochemistry. We conclude that autocrine VEGF through Flk-1/KDR, by activating eNOS to produce NO through PI3-K/Akt kinase, maintains clonogenic cell growth in the OCI/AML-2 cell line. Since the patient samples did not express VEGF in all cases, it is possible that in vivo the regulatory connection between these two signal systems is also mediated via endocrine VEGF in addition to autocrine or paracrine VEGF.

Inducible nitric oxide synthase expression, apoptosis, and angiogenesis in in situ and invasive breast carcinomas.

In this investigation, we studied the expression of inducible nitric oxide synthase (iNOS) and its association to apoptosis and angiogenesis in 43 in situ and 68 invasive breast carcinomas. Its expression was studied immunohistochemically using a polyclonal iNOS antibody, and the staining was evaluated both in tumor and stromal cells. Apoptosis was detected by 3' end labeling of fragmented DNA (terminal deoxynucleotidyl transferase-mediated nick end labeling method). Vascularization was detected immunohistochemically using an antibody to the FVIII-related antigen, and calculated microvessel densities were determined. In addition to strong iNOS expression in stromal cells, iNOS positivity was observed in tumor cells in 46.5% of in situ and 58.8% of invasive carcinomas. In invasive carcinomas, there were more cases with iNOS positivity both in tumor and stromal cells compared to in situ carcinomas (0.007). The proportion of cases with iNOS-positive tumor cells increased in in situ carcinomas from grade I to III (20.0%, 46.2%, and 73.3%). In invasive ductal carcinomas, there were more cases with iNOS-positive tumor cells than with in situ carcinomas (P = 0.04). Carcinomas with both iNOS-positive tumor and stromal cells had a higher apoptotic index (P = 0.02) and a higher calculated microvessel densities index (P = 0.02). A high number of iNOS-positive stromal cells associated with metastatic disease (P = 0.05). The results show that breast carcinoma cells, in addition to stromal cells, express iNOS and are capable of producing NO. Carcinomas with iNOS-positive tumor and stromal cells have a higher apoptotic indices and increased vascularization, suggesting that iNOS contributes to promotion of apoptosis and angiogenesis in breast carcinoma. The association of the number of iNOS-positive stromal cells with metastatic disease might be attributable to stimulation of angiogenesis, resulting in a higher vascular density and consequently a higher probability for tumor cells to invade.

Apoptosis and expression of apoptosis regulating proteins bcl-2, mcl-1, bcl-X, and bax in malignant mesothelioma.

We investigated apoptosis and the expression of bcl-2, mcl-1, bcl-X, and bax in histological sections from 35 malignant mesotheliomas and 21 metastatic adenocarcinomas. Moreover, the expression of bcl-2, mcl-1, bcl-X, and bax were assessed by Western blotting in nonmalignant human mesothelial cells (Met5A) and seven malignant cell lines. The apoptotic index in mesotheliomas was 1.07+/-1.14%. Patients with mesotheliomas showing a high apoptotic index (> or =0.75%) had a worse prognosis (P = 0.008). bcl-2 positivity was observed in only seven cases, but bcl-X, mcl-1, and bax positivity was seen in all of them. In immunoblotting experiments, all mesothelioma cell lines were negative for bcl-2 but positive for bcl-X, mcl-1, and bax. The apoptotic index in bcl-2-negative mesotheliomas was 1.25+/-1.24% and in bcl-2-positive ones, 0.47+/-0.42% (P = 0.014). The apoptotic index did not significantly associate with bcl-X, mcl-1, or bax expression (P = 0.19, P = 0.25, and P = 0.46, respectively). No significant difference was observed in apoptosis or expression of bcl-2, bcl-X, or bax between malignant mesotheliomas and metastatic adenocarcinomas. The former, however, showed more often weak mcl-1 immunoreactivity (P = 0.01). The results show that the extent of apoptosis may influence patient prognosis. bcl-2 is inversely associated with the apoptotic index but is relatively infrequently expressed in malignant mesotheliomas. Widespread expression of bcl-X, mcl-1, and bax suggests that these proteins may also take part in apoptosis regulation in mesotheliomas.

Elevated serum levels of type I collagen degradation marker ICTP and tissue inhibitor of metalloproteinase (TIMP) 1 are associated with poor prognosis in lung cancer.

PURPOSE: The aim of this study was to evaluate the correlation between type I collagen degradation marker ICTP, MMP (matrix metalloproteinase)-9, and tissue inhibitor of metalloproteinase (TIMP)-1 and to compare their value as prognostic factors in lung cancer. EXPERIMENTAL DESIGN: From the sera of 141 lung cancer patients, we assessed markers of type I collagen synthesis (PINP and PICP) and degradation (ICTP) by radioimmunoassays, and we assessed MMP-9 and its tissue inhibitor TIMP-1 by ELISA. There were 62 squamous cell carcinomas, 42 adenocarcinomas, 14 small cell carcinomas, and 23 cases with other histology. Seventeen of these patients had advanced disease. Sixty-seven patients had been operated on, 33 had received radiation therapy, 7 had received chemotherapy, and the rest had received other treatment combinations. RESULTS: We examined the relationship between these markers and found a correlation between ICTP and MMP-9 (r = 0.201; P = 0.01) or TIMP-1 (r = 0.415; P = 0.00). Elevated serum concentrations of ICTP (>5 microg/liter) and/or TIMP-1 (>300 ng/ml) correlated with poor prognosis. In univariate regression analysis, ICTP had prognostic value (odds ratio, 1.6462; P < 0.03): the patients with elevated serum concentrations of ICTP (>5 microg/liter) had a 64% higher risk of dying from lung cancer than did patients with opposite values. CONCLUSIONS: Our results indicated that ICTP and TIMP-1 are good prognostic markers in lung cancer. The association between serum MMP-9 and ICTP suggests that MMP-9 could play a role in the degradation of the extracellular matrix producing ICTP in this pathological situation.

Widespread expression of thioredoxin and thioredoxin reductase in non-small cell lung carcinoma.

We investigated the expression of thioredoxin (Trx) and thioredoxin reductase (TrxR) in 89 non-small cell lung carcinomas. Additionally, immortalized human bronchial epithelial cells (BEAS 2B) and four human lung carcinoma cells lines (A549, SK-MES-1, CALU-6, and A427) were studied by Western blot and reverse transcription-PCR for the synthesis of Trx and TrxR protein and mRNA expression in vitro. The histological samples were also studied for immunohistochemical p53 and proliferating cell nuclear antigen expression and apoptosis. In non-neoplastic lung, Trx and TrxR expression was seen in bronchial epithelial cells and alveolar macrophages, metaplastic alveolar epithelial cells, and chondrocytes of the bronchus. In non-small cell lung carcinomas, there was a widespread expression of Trx and TrxR with only three and eight cases negative, respectively. Trx and TrxR expression was located in both cytoplasmic and nuclear compartments of the cells. There was a statistical association between cytoplasmic and nuclear Trx or TrxR expression. Grade I-II tumors showed stronger cytoplasmic and nuclear Trx and TrxR immunoreactivity than grade III tumors. No association was found between p53 and proliferating cell nuclear antigen expression and Trx or TrxR immunoreactivity. However, apoptosis was inversely associated with nuclear Trx and TrxR positivity. In the cell lines studied, both non-neoplastic BEAS 2B cells and all of the carcinoma cell lines expressed Trx and TrxR proteins and mRNA. The results show that these redox-regulating proteins are highly expressed in lung carcinomas taking part in activation of transcriptional factors and regulation of apoptosis in non-small cell lung carcinoma. In high-grade tumors, Trx and TrxR expression is diminished, suggesting loss of redox regulation in tumors with low differentiation.

Cytotoxicity of oxidants and asbestos fibers in cultured human mesothelial cells.

The authors investigated the mechanisms caused by oxidants (superoxide and hydrogen peroxide) and asbestos (amosite) fibers in human mesothelial cells. Immortalized human pleural mesothelial cells (MET 5A) were exposed in vitro to one of the following: hypoxanthine (100-200 microM) plus xanthine oxidase (10-20 mU/ml) as a superoxide-generating system, H2O2 (50 microM-5 mM); or amosite (1-100 micrograms/cm2). Cellular adenine nucleotide depletion, DNA single strand breaks, extracellular release of nucleotides, and their catabolites and lactate dehydrogenase (LDH) were assessed as markers of cell damage after 4-6 h exposure to the oxidants or fibers. The effect of intracellular antioxidant enzymes and exogenous antioxidants on cell damage were investigated during oxidant and amosite exposure. Superoxide radical and H2O2 exposure resulted in the depletion of adenine nucleotides, accumulation of the products of nucleotide catabolism, induction of DNA single strand breaks, and extracellular LDH release. Amosite exposure did not cause nucleotide depletion or induction of DNA single strand breaks. Inactivation of the intracellular antioxidant enzymes glutathione reductase or catalase augmented cell damage during H2O2 exposure but not during amosite exposure.

Neutrophil and asbestos fiber-induced cytotoxicity in cultured human mesothelial and bronchial epithelial cells.

This study investigates reactive oxygen species generation and oxidant-related cytotoxicity induced by amosite asbestos fibers and polymorphonuclear leucocytes (PMNs) in human mesothelial cells and human bronchial epithelial cells in vitro. Transformed human pleural mesothelial cells (MET 5A) and bronchial epithelial cells (BEAS 2B) were treated with amosite (2 micrograms/cm2) for 48 h. After 24 h of incubation, the cells were exposed for 1 h to nonactivated or amosite (50 micrograms) activated PMNs, washed, and incubated for another 23 h. Reactive oxygen species generation by the PMNs and the target cells was measured by chemiluminescence. Cell injury was assessed by cellular adenine nucleotide depletion, extracellular release of nucleotides, and lactate dehydrogenase (LDH). Amosite-activated (but also to a lesser degree nonactivated) PMNs released substantial amounts of reactive oxygen metabolites, whereas the chemiluminescence of amosite-exposed mesothelial cells and epithelial cells did not differ from the background. Amosite treatment (48 h) of the target cells did not change intracellular adenine nucleotides (ATP, ADP, AMP) or nucleotide catabolite products (xanthine, hypoxanthine, and uric acid). When the target cells were exposed to nonactivated PMNs, significant adenine nucleotide depletion and nucleotide catabolite accumulation was observed in mesothelial cells only. In separate experiments, when the target cells were exposed to amosite-activated PMNs, the target cell injury was further potentiated compared with the amosite treatment alone or exposure to nonactivated PMNs. In conclusion, this study suggests the importance of inflammatory cell-derived free radicals in the development of amosite-induced mesothelial cell injury.

Manganese superoxide dismutase in human pleural mesothelioma cell lines.

Mesothelioma is a malignant pleural or intraperitoneal tumor attributable to asbestos exposure in more than 80% of the cases. Manganese superoxide dismutase (MnSOD), a mitochondrial superoxide radical scavenging enzyme, is low in most tumors but is known to be induced by asbestos fibers and certain cytokines. Induction of MnSOD may be associated in asbestos-related pulmonary diseases in vivo. We investigated here MnSOD specific activity and MnSOD mRNA level using healthy human lung tissue, SV40-transformed human pleural mesothelial cells (Met5A), and six human malignant mesothelioma cell line cells. Total SOD (CuZnSOD + MnSOD) and MnSOD activities were 20.0 +/- 4.8 U/mg protein and 3.2 +/- 1.2 U/mg protein in healthy human lung tissue, and 25.6 +/- 10.7 U/mg and 3.8 +/- 1.0 U/mg in Met5A cells, respectively. In four mesothelioma cell lines MnSOD activity was significantly elevated, the highest activity (30.1 +/- 8.2 U/mg) was almost 10-fold compared to the activity in Met5A cells. The steady state mRNA level of MnSOD was low in Met5A cells and markedly higher in all mesothelioma cell lines roughly in proportion with enzyme activities. Cytotoxicity experiments, which were conducted in four cell lines, indicated that cells containing high MnSOD mRNA level and activity were resistant to the mitochondrial superoxide-producing agent menadione. In conclusion, our results suggest that human mesothelioma may express high levels of MnSOD, which is associated with high oxidant resistance of these cells.

DNA single strand breaks and adenine nucleotide depletion as indices of oxidant effects on human lung cells.

The comet assay (single cell gel electrophoresis) is a novel method to assess DNA strand breaks in single cells. We studied the oxidant sensitivity of cultured primary and transformed (MeT-5A) human pleural mesothelial cells, as well as primary and transformed (BEAS 2B) human bronchial epithelial cells, and compared the results obtained with the Comet assay to other markers of oxidant effects on cells, such as depletion of intracellular high-energy nucleotides (ATP, ADP, AMP), accumulation of products of nucleotide catabolism (xanthine, hypoxanthine, uric acid), and release of lactate dehydrogenase (LDH). The cells were exposed for 5 min to 4 h to 50-500 microM H2O2 or to 5-50 microM menadione. Significant tail moment increase, which is a marker of DNA strand breaks in the Comet assay, and intracellular nucleotide depletion occurred simultaneously in MeT-5A and BEAS 2B cells during the first 30-60 min of exposure to H2O2 and menadione. In the Comet assay variation between the individual cells could be detected. LDH release, a marker of cell injury, showed that mesothelial cells were far more sensitive than epithelial cells to oxidant-induced lytic cell injury. MeT-5A and BEAS 2B cells contained similar intracellular antioxidant enzyme activities, which may explain their similar oxidant sensitivity in the Comet assay. A significant increase (164%) in the tail moment was detectable in MeT-5A cells exposed to 50 microM H2O2 for 30 min. This returned to control level during the 4 h of continuing exposure. A 30 min exposure of 25 microM menadione caused a 61% increase in the mean tail moment but, unlike with H2O2, the change was irreversible during the following 4 h incubation. We conclude that human pleural mesothelial cells and bronchial epithelial cells show similar oxidant sensitivity when assessed by the Comet assay, but various oxidants differ in their potency in causing DNA breaks in these cells.