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B T Mossman

Publications and source records attributed to B T Mossman.

At least 91 records · Page 5Linked to original sources

In vitro studies on the biologic effects of fibers: correlation with in vivo bioassays.

In vitro studies employing organ cultures, primary cell cultures, cell lines, and bacterial systems have been used to assess the toxicity, mutagenicity, and carcinogenic potential of asbestos and nonasbestos fibers. These experiments have been useful in defining mechanisms contributing to the causation of fiber-associated lung diseases. Long (greater than 8 microns), thin asbestos fibers are more active in vitro than short (less than or equal to 2 microns) fibers or nonfibrous particles, an observation supporting the importance of fiber dimension in disease. Although in vitro bioassays cannot evaluate characteristics such as clearance and/or durability of fibers which may be critical determinants of fiber toxicity in lung, they can be used both to address dosimetry at the cellular level (i.e., number of fibers per cell that elicit a measurable biologic end point) and to evaluate preventive approaches to fiber-induced cell injury. Development of in vitro models employing target cells of the lung, i.e., mesothelial cells, tracheobronchial epithelial cells, and lung fibroblasts, as well as carefully characterized preparations of fibers and particles, will be necessary to evaluate whether in vitro bioassays are amenable to predicting the pathogenic potential of synthetic and naturally occurring fibers comparatively.

Animals↗

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.

Animals↗

Sensitivity of hamster tracheal epithelial cells to asbestiform minerals modulated by serum and by transforming growth factor beta 1.

A number of proliferative and cytotoxic responses similar to those induced by classical tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate occur in tracheal epithelial cells and organ cultures exposed to asbestiform minerals. In these studies, cloned diploid hamster tracheal epithelial cells were examined in a number of short term (3-24 h) and long term (greater than 24 h) biological assays for their responsiveness to crocidolite asbestos in comparison to other fibers (chrysotile asbestos, Code 100 fiberglass) and particulates (riebeckite, antigorite, glass beads). In addition, the influence of serum on the biological effects of crocidolite were assessed. In 10% serum-containing medium, low concentrations of crocidolite (1.0 micrograms/cm2 dish) stimulated cellular proliferation (measured as increased [3H]thymidine incorporation), and concentrations greater than 5 micrograms/cm2 dish were cytotoxic (measured by decreased colony forming efficiency). In 2% serum-containing medium, lower concentrations of fibers (0.1-0.5 micrograms/cm2 dish) caused increases in [3H]thymidine incorporation and colony forming efficiency whereas higher concentrations of fibers were required for responses equitoxic to those observed in 10% serum. Crocidolite-induced [3H]thymidine incorporation was inhibited when 2% serum-containing medium was supplemented with transforming growth factor type beta at concentrations (3 ng/ml) which give rise to altered morphology of hamster tracheal epithelial cells. Furthermore, the profiles of colony forming efficiency and 51Cr release of cells grown in the presence of transforming growth factor beta resembled those of cells grown in 10% serum. Results suggest that the phenotypic changes induced by serum factors such as transforming growth factor beta influence the sensitivity of hamster tracheal epithelial cells to crocidolite asbestos.

Animals↗

Mechanisms of asbestos-induced squamous metaplasia in tracheobronchial epithelial cells.

Within 1 to 4 weeks after exposure to asbestos, differentiated rodent and human tracheobronchial epithelial cells in organ culture undergo squamous metaplasia, a putative preneoplastic lesion characterized by conversion of mucociliary cell types to keratinizing cells. The exogenous addition of retinal acetate (RA) to culture medium of hamster tracheal organ cultures reverses preestablished, asbestos-induced squamous metaplasia, although data suggest that the effectiveness of RA decreases as the length of time between exposure to asbestos and initial application of RA increases. alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), inhibits squamous metaplasia caused by asbestos or vitamin A deficiency, whereas addition of methylglyoxal bis(guanylhydrazone) (MGBG), a structural analog of spermidine and inhibitor of S-adenosylmethionine decarboxylase, causes an enhancement of metaplasia under both circumstances. Basal cell hyperplasia and increased incorporation of 3H-thymidine by tracheal epithelial cells also are seen after addition of the polyamines, putrescine or spermidine, to tracheal organ cultures, an observation supporting the importance of polyamines in the development of this lesion. The use of retinoids and inhibitors of ODC could be promising as preventive and/or therapeutic approaches for individuals at high risk for development of asbestos-associated diseases.

Animals↗

Evidence supporting a role for active oxygen species in asbestos-induced toxicity and lung disease.

Asbestos is an important occupational and environmental toxicant that affects several cell types in the respiratory tract. In an effort to understand how asbestos causes cell injury and/or altered proliferation and differentiation of cells, this laboratory has focused on reactive oxygen species as mediators of asbestos-induced biological effects. A compendium of experimental results reported by this laboratory and others supports this hypothesis. For example, scavengers of reactive oxygen metabolites and iron chelators (i.e., desferroxamine) prevent cytotoxicity after addition of asbestos to a variety of cell lines and macrophages in vitro. DNA strand breakage associated with toxicity of crocidolite asbestos in C3H10T 1/2 cells also is ameliorated with use of desferroxamine. All types of asbestos cause lipid peroxidation in mammalian cells and artificial membranes, a phenomenon that can be prevented by removal of catalytic iron. Last, asbestos causes generation of active oxygen species after interaction with leukocytes or by reduction of oxygen on the surface of the fibers.

Antioxidants↗

Mechanisms of fibre-induced superoxide release from alveolar macrophages and induction of superoxide dismutase in the lungs of rats inhaling crocidolite.

Asbestos resembles the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), in its ability to elicit release of superoxide (O2-.) from rodent alveolar macrophages (AM) in vitro. In addition, superoxide dismutase (SOD), the antioxidant enzyme scavenging O2-, is increased in cultures of tracheobronchial epithelial cells and lung fibroblasts after exposure to either crocidolite or chrysotile asbestos. Our objectives here were to determine: (1) the chemical and physical properties of asbestos important in the generation of O2- from rat AM; and (2) the effects of O2- in comparison with asbestos on biosyntheses of collagen and non-collagen protein in rat lung fibroblasts in vitro. We were also interested in whether increased production of SOD occurred in the lungs of rats after inhalation of crocidolite asbestos. To determine whether O2- was elicited in response to a variety of asbestiform fibres, AM lavaged from Fischer 344 rat lungs were exposed in vitro to equivalent non-toxic amounts of crocidolite asbestos, erionite, Code 100 fibreglass, sepiolite, and their non-fibrous analogues, riebeckite, mordenite and glass particles. In addition, sized preparations of long (greater than 10 microns) and short (less than 2 microns) asbestos were introduced at identical concentrations to determine whether length of fibres is critical in O2- release. The amount of O2- released from AM in response to dusts was then determined by measuring SOD-inhibitable reduction of cytochrome C. All asbestiform fibres caused a significant (p less than 0.05) increase in generation of O2- from epithelial cells, whereas non-fibrous particles were less active at comparable concentrations. Experiments with long (greater than 10 microns) versus short (less than 2 microns) chrysotile showed that long fibres caused a more striking, dosage-dependent release of O2-. To determine whether O2- plays a role in the causation of fibrotic lung disease, rat lung fibroblasts were exposed to a biochemical generation system (xanthine-xanthine oxidase) for O2- before quantitation of cell-associated collagen and non-collagen protein at 24, 48 and 72 h thereafter. At the latter time periods, significant increases in total collagen per ng DNA were observed. In comparison with controls, the generation system for O2- also caused an initial decrease in synthesis of non-collagen protein followed by increases in synthesis of non-collagen protein at 48 and 72 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Mechanisms of induction of ornithine decarboxylase activity in tracheal epithelial cells by asbestiform minerals.

Asbestos induces a constellation of biological responses in cells of the respiratory tract that are similar to those of classical tumor promoters. In this regard, induction of ornithine decarboxylase (ODC) activity and increased incorporation of [3H]thymidine have been documented after addition of crocidolite and chrysotile asbestos to a hamster tracheal epithelial cell line (J. M. Landesman and B. T. Mossman, Cancer Res., 42:3669-3675, 1982). The objectives of studies here were to determine: (a) the importance of geometry, size, and/or chemical composition of asbestos fibers on induction of ODC activity; and (b) the possible involvement of calcium and/or protein kinase C in asbestos-induced ODC activity. After addition for 24 h to confluent hamster tracheal epithelial cells, fibers of crocidolite, chrysotile, and glass in medium containing fresh serum caused a significant increase in ODC activity. Stimulation of ODC was not observed when nonfibrous analogues (riebeckite, antigorite, and glass particles) were used. Sized preparations of long (greater than 10-micron length) chrysotile fibers were more potent in enhancing ODC activity than shorter (less than or equal to 2-micron length) fibers at similar concentrations. The mechanisms of ODC induction by asbestos were probed by adding the calcium channel blockers (verapamil and nifedipine) and inhibitors [10(-5) to 10(-7)M of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, of N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide, of TMB-8, and of palmitoyl carnitine] of protein kinase C simultaneously with chrysotile asbestos. These agents inhibited ODC activity by chrysotile in a dosage-dependent fashion. Results suggest that the fibrous geometry and length of asbestos fibers are critical in initiating ODC activity in airway epithelial cells. Moreover, they implicate the importance of calcium and protein kinase C in asbestos-induced mitogenic responses.

Animals↗

Generation of superoxide (O2-.) from alveolar macrophages exposed to asbestiform and nonfibrous particles.

Active oxygen species are implicated causally in tumor promotion by 12-O-tetradecanoylphorbol-13-acetate and in cell damage by asbestos, a putative tumor promoter of the respiratory tract. To determine the properties of asbestos important in generation of the oxygen free radical, superoxide (O2-.), hamster and rat alveolar macrophages were exposed in vitro to nontoxic concentrations of fibrous (crocidolite, erionite, Code 100 fiberglass, sepiolite) and nonfibrous (riebeckite, mordenite, glass) particulates. The amount of O2-. released by cells in response to dusts was determined by measuring the reduction of cytochrome c in the presence and absence of superoxide dismutase. Results showed that all fibrous (defined as a greater than 3:1 length:diameter ratio) dusts caused a significant increase in both release of O2-. from rat macrophages and enhancement of zymosan-triggered O2-. from hamster macrophages. Nonfibrous particles were less active than fibers at comparable concentrations. These results suggest that the geometry of particulates is of critical importance in the generation of O2-. from cells of the respiratory tract.

Animals↗

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(.).

Animals↗

A possible mechanism of chrysotile asbestos toxicity.

In vitro evaluations of heat treated and/or irradiated chrysotile asbestos indicate the importance of electron transfer in determining the biological outcome of asbestos exposure. Regardless of the assay system used, all show the same trends. Namely, heat treatment reduces cytotoxicity while heat treatment in combination with irradiation increases activity over heat treatment alone. Furthermore, studies of BSA and DNA binding consistently indicate a lower molecular adsorption for heated asbestos. BSA and DNA adsorption are similar for untreated, irradiated and heated or irradiated samples. Physical and chemical analyses indicate that the size and elemental composition are unaffected by the treatment processes. X-ray diffraction indicates no change in the crystal structure of chrysotile asbestos. IR spectroscopy suggests that heat treatment affects the external hydroxyl group population while heat treatment and irradiation may repopulate this functional group. A proposed mechanism consistent with the biological and physical characterization is based upon electron transfer from the chrysotile asbestos matrix to biological receptors.

Adsorption↗

Comparative interactions of streptozotocin and chlorozotocin with DNA of an insulin-secreting cell line (RINr).

Both streptozotocin and chlorozotocin, the 2-chloroethyl analogue of streptozotocin, are diabetogenic chemicals in rodents. Although these chemicals are similar structurally, they appear to act on pancreatic B cells via different mechanisms. In studies here, damage and repair of DNA after exposure of an insulin-secreting cell line to streptozotocin and chlorozotocin were assessed by nucleoid sedimentation and alkaline elution. Equitoxic concentrations of streptozotocin and chlorozotocin caused significant single-strand breakage of DNA (p less than 0.005). These lesions were repaired in a time-dependent manner, with most repair completed by 24-h post-exposure to chemicals. Additionally, chlorozotocin caused DNA-DNA and DNA-protein crosslinks in insulinoma cells. When proteinase K was included in the crosslinking assay, a substantial proportion of the chlorozotocin-associated crosslinks proved to be DNA interstrand in nature. Analysis of the amount of interstrand crosslinking in insulinoma cells after exposure to chlorozotocin for 1 h showed that formation of interstrand crosslinks was slow. Increasing amounts appeared over a 24-h period. These results suggest that the formation of irreversible DNA interstrand crosslinks may be a critical factor in cytotoxicity and diabetogenicity caused by chlorozotocin.

Alkylation↗

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.

Animals↗

Alteration of superoxide dismutase activity in tracheal epithelial cells by asbestos and inhibition of cytotoxicity by antioxidants.

We report here the inhibition of asbestos-induced cytotoxicity in a hamster tracheal epithelial cell line by superoxide dismutase, a scavenger of superoxide (O2-.), and by mannitol and dimethylthiourea, scavengers of the hydroxyl radical (OH.). By using these agents, cell damage was ameliorated in cultures exposed to long (greater than 10 microns in length) fibers of chrysotile and crocidolite asbestos. In contrast, injury to epithelial cells by short (less than or equal to 2 microns) chrysotile or glass fibers was not prevented by scavengers of O2-., OH., H2O2 or 1O2 (singlet oxygen). These results implicate active oxygen species as mediators of injury by long asbestos fibers to cells of the respiratory tract. By using immunocytochemical and biochemical techniques, we detected appreciable amounts of copper-zinc superoxide dismutase in hamster tracheobronchial epithelial cells and alveolar macrophages in vitro and in histologic sections of rat and human respiratory tract. Activity of total endogenous superoxide dismutase (copper-zinc and manganese forms) increased in tracheal epithelial cells exposed for several days in vitro to either crocidolite or chrysotile asbestos but was unchanged in untreated cells and those exposed to comparable amounts of glass fibers. After inhalation of asbestos by rats, or exposure of cells in culture to asbestos, long fibers were observed protruding from both epithelial cells and alveolar macrophages. The unsuccessful phagocytosis of long fibers of asbestos coupled with generation of oxygen free radicals might explain the increased pathogenic potential of long fibers in asbestos-associated diseases of the respiratory tract.

Animals↗