Joanne P. Marsh

Prevention of asbestos induced cell death in rat lung fibroblasts and alveolar macrophages by scavengers of active oxygen species

Cell injury and inflammation caused by asbestos are critical to the pathogenesis of pulmonary fibrosis (asbestosis). Our goal in studies here was to investigate the possible modulation of asbestos-related cell death using antioxidants in both target and effector cells of asbestosis. After exposure to crocidolite asbestos at a range of concentrations (2.5-25 micrograms/cm2 dish), the viability of a normal rat lung fibroblast line (RL-82) and freshly isolated alveolar macrophages (AM) was determined by exclusion of trypan blue and nigrosin, respectively. In comparison to fibroblasts, AM were more resistant to the cytotoxic effects of asbestos. Cytotoxic concentrations of asbestos then were added to both cell types in combination with the antioxidants, superoxide dismutase (SOD), a scavenger of superoxide (O2-.), and catalase, an enzyme scavenging H2O2. Dimethylthiourea (DMTU), a scavenger of the hydroxyl radical (OH.) and deferoxamine, an iron chelator, also were evaluated in similar studies. Results showed significant dosage-dependent reduction (P less than 0.001) of asbestos-associated cell death with all agents. In contrast, asbestos-induced toxicity was not ameliorated after addition of chemically inactivated SOD and catalase or bovine serum albumin. Results above suggest asbestos-induced cell damage is mediated by active oxygen species. In this regard, the iron associated with the fiber and/or its interaction with cell membranes might be critical in driving a modified Haber-Weiss (Fenton-type) reaction resulting in production of OH(.).

Increased expression of manganese-containing superoxide dismutase in rat lungs after inhalation of inflammatory and fibrogenic minerals

Steady-state mRNA levels and immunoreactive protein for manganese-containing superoxide dismutase (MnSOD) were assayed in rat lungs after subchronic inhalation of the fibrogenic silicon dioxide, cristobalite, or preparations of titanium dioxide (TiO2) of different inflammatory and fibrogenic potential. Total and differential cell counts recoverable by bronchoalveolar lavage (BAL) were also measured to ascertain whether induction of certain antioxidant enzymes (AOE) correlated with inflammatory responses. Inhalation of cristobalite and ultra-fine TiO2, a particle causing pulmonary inflammation and fibrosis, caused dramatic increases in MnSOD mRNA levels in rat lung which correlated with increases in MnSOD immunoreactive protein. Increases in gene expression of other AOE [catalase, glutathione peroxidase (GPX), copper-zinc containing superoxide dismutase (CuZnSOD)] were less striking and did not correlate precisely with inflammatory potential of minerals. Inflammatory changes in BAL correlated directly with steady-state MnSOD mRNA levels in lung. Inhalation of TiO2-F, a noninflammatory, nonfibrogenic mineral, failed to induce MnSOD or mRNAs for other AOE. Our data suggest that particles causing inflammation and pulmonary fibrosis increase expression of AOE in lung, most notably MnSOD. Thus, elevations of MnSOD mRNA levels in lung or BAL may be predictive of lung disease.

Transfection of a manganese-containing superoxide dismutase gene into hamster tracheal epithelial cells ameliorates asbestos-mediated cytotoxicity

To determine if overexpression of manganese-containing SOD (MnSOD) alters cell sensitivity to asbestos, an expression cassette containing murine MnSOD cDNA was cotransfected with pSV2neo, a plasmid conferring resistance to the antibiotic G418, into a diploid cell line of hamster tracheal epithelial (HTE) cells. Pools of G418-resistant transfectants were characterized by Southern and Northern blot analyses and enzyme activity assays. Although increases in MnSOD gene copies in individual cell pools ranged from approximately 7- to 86-fold in comparison to cells transfected with pSV2neo alone, steady-state levels of MnSOD mRNA were increased only by 1.4-to 2.3-fold. Despite modest increases in MnSOD mRNA, significant elevations in MnSOD enzyme activity were observed in pools of G418-resistant cells. MnSOD-transfected cell lines were more resistant to the cytotoxic effects of crocidolite asbestos using a sensitive colony-forming efficiency (CFE) assay. These data show that MnSOD has a direct role in cell defense against asbestos-induced cytotoxicity, an oxidant-dependent process.

Approaches to prevention of asbestos-induced lung disease using polyethylene glycol (PEG)-conjugated catalase.

Asbestos-associated damage to cells of the respiratory tract in vitro can be prevented by the simultaneous addition of scavengers of active oxygen species to cultures. To determine if administration of scavenger enzymes to animals and humans is a plausible approach to the prevention of asbestos-induced lung disease, osmotic pumps were filled with various concentrations of PEG-coupled catalase and implanted subcutaneously into Fischer 344 rats over a 28-day period. At 3, 14, and 28 days after implantation of the pumps, the animals were evaluated for levels of catalase in serum and lung. In addition, lung tissue and lavage fluids were examined at 28 days for biochemical and morphologic indications of cell injury, inflammation, and fibrotic lung disease. At all time points examined, the administration of PEG-catalase caused a dosage-dependent increase in serum levels of catalase. The levels of lung catalase were evaluated at 28 days but not at earlier time periods. In comparison to control rats, the amounts of enzymes (lactic dehydrogenase, alkaline phosphatase), protein, and cells in lavage fluids from treated animals were unaltered. Moreover, the lungs showed no evidence of inflammation or fibrotic disease as determined by differential cell counts in lavage and measurement of hydroxyproline. These studies suggest that administration of PEG-catalase does not cause injury or other alterations in lung tissue and can be pursued as a feasible approach to prevention of asbestosis.

Increases in endogenous antioxidant enzymes during asbestos inhalation in rats

Although the pathogenesis of asbestos-induced pulmonary damage is still not completely understood, an important role has been attributed to active oxygen species. In the present paper we present results of a study investigating the effect of crocidolite asbestos inhalation on different lung antioxidant enzymes in rats. During the development of pulmonary fibrosis induced by crocidolite asbestos, lung superoxide dismutase, catalase and selenium-dependent glutathione peroxidase activities increased, indicating an adaptive response to increased pulmonary oxidant stress. However, this adaptive response obviously is not sufficient to protect the lung from asbestos-induced pulmonary damage. Considering the role of active oxygen species in both the fibrotic process and tumor promotion, it is hypothesized that antioxidants may also protect the lung from chronic asbestos-induced pulmonary damage such as bronchogenic carcinoma.

Mechanisms of Toxic Injury by Asbestos Fibers: Role of Oxygen-Free Radicals

In comparison to a variety of innocuous and/or “nuisance” dusts, asbestos is unique because of its association with an increased risk in man of pulmonary fibrosis, pleural and peritoneal mesothelioma, and bronchogenic carcinoma (Craighead and Mossman 1982). All of these diseases occur following an initial inflammatory reaction to asbestos in the lung and peritoneum. Increased proliferation and abnormal differentiation of the affected cell types then ensue - phenomena culminating in disease (Fig. 1).

Effects of Aramid, a high Strength Synthetic Fiber, on Respiratory Cells in Vitro

Industry continues to develop synthetic fibers for new technologies and as replacements for asbestos, a toxic and carcinogenic fiber. To determine whether the in vitro effects of the aromatic polyamide fiber, Aramid (Kevlar, Twaron), resembled those induced by asbestos, fibers were surveyed for (1) cytotoxicity as measured by total cell protein, and (2) proliferative capacity as measured by [3H]thymidine incorporation, colony forming efficiency (CFE), and ornithine decarboxylase (ODC) activity in two target cells of mineral dust-induced lung damage, hamster tracheal epithelial (HTE) cells and rat lung (RL90) fibroblasts. Results of cytotoxicity tests indicated that Aramid was as toxic to HTE and RL90 cells as were crocidolite and chrysotile asbestos when expressed on both an equal mass and equal fiber number basis. In HTE cells, Aramid caused a statistically significant increase in [3H]thymidine incorporation and CFE and produced a dose-dependent induction of ODC enzyme activity. Proliferative effects by asbestos or Aramid were not observed in RL90 fibroblasts. Thus, when tested over a respirable size range, Aramid exhibited many of the same effects on epithelial cells in vitro as did asbestos, including increased radiolabeled nucleotide incorporation into DNA and induction of ODC enzyme activity.

Investigations into the Mechanisms of Asbestos Toxicity

Asbestos has been widely used in North America for insulation, corrosion resistance, material filling, and filtration. Of particular economic importance is chrysotile asbestos which is principally produced for western trading in Canada and Rhodesia. Interestingly, the first use of asbestos is thought to be 2,000 years ago when the Romans used asbestos cloth for wrapping their dead. Presently, attention is directed toward asbestos as a human toxicant and respiratory tract carcinogen. Although asbestos is a well-documented lung fibrogen and carcinogen, little is known about the mechanism of action.

Activation of Early Cellular Responses by Asbestos: Induction of c-FOS and c-JUN Protooncogene Expression in Rat Pleural Mesothelial Cells

Asbestos fibers belong to a family of ubiquitous mineral silicates. Two types of malignancies have been documented in respiratory tract and pleura following inhalation of asbestos. Bronchogenic carcinoma, arising in the tracheobronchialepithelium, is a tumor found in individuals exposed occupationally to asbestos. Since this tumor is rarely found in non-smoking asbestos workers, asbestos is thought to act mainly as a co-carcinogen or tumor promoter in the development of this disease (Mossman et al. 1990). However, the development of malignant mesothelioma in the pleura is not associated with cigarette smoking. The generally accepted view is that amphibole asbestos acts as a complete carcinogen in the development of mesothelioma (Mossman et al.1990).

Effects of Asbestos on Specific Binding of Phorbol Ester Tumor Promoter and Protein Kinase C Activity in Hamster Tracheal Epithelial (HTE) Cells

Crocidolite asbestos causes proliferative changes in tracheobronchial epithelial cells that are similar to those observed after exposure of a number of cell types to phorbol esters, i.e. classical tumor promoters in skin and other organs (reviewed in Mossman et al., 1985). These biological changes, which include increased uptake of 3H-thymidine (Landesman and Mossman, 1982), increased activity of ornithine decarboxylase (ODC), i.e. a rate-limiting enzyme in the biosynthesis of polyamines (Marsh and Mossman, 1988), and the development of squamous metaplasia (Mossman et al., 1980a), may be important in the pathogenesis of bronchogenic carcinoma, a tumor of high risk in asbestos workers who smoke. In an attempt to understand how asbestos initiates alterations in cell proliferation and differentiation, we focused here on whether crocidolite asbestos causes activation of protein kinase C (PKC), a calcium- and phospholipid-dependent enzyme which phosphorylates several intracellular protein substrates and plays a crucial role in growth control and tumor promotion (Nishizuka, 1986). The potent phorbol tumor promoter, 12-0-tetradecanoylphorbol-13-acetate (TPA), and related compounds activate PKC by structurally resembling diacylglycerol.