Asbestos-associated chromosomal changes in human mesothelial cells.

Abstract

Replicative cultures of human pleural mesothelial cells were established from noncancerous adult donors. The cells exhibited normal mesothelial cell characteristics including keratin, hyaluronic acid mucin, and long branched microvilli, and they retained the normal human karyotype until senescence. The mesothelial cells were 10 and 100 times more sensitive to the cytotoxic effects of asbestos fibers than normal human bronchial epithelial or fibroblastic cells, respectively. In addition, cultures of mesothelial cells that survived two cytotoxic exposures of amosite fibers were aneuploid with consistent specific chromosomal losses indicative of clonal origin. These aneuploid cells exhibit both altered growth control properties and a population doubling potential of >50 divisions beyond the culture life span (30 doublings) of the control cells.Epidemiological studies have established that exposure to asbestos fibers is the primary cause of mesothelioma in the industrialized world (1-3). The latency period for this disease ranges from 15 to >40 years (4) and because of the high use of asbestos during and since World War II, the mortality rate from mesothelioma has doubled since 1967. Projections indicate that the incidence will continue to increase until the year 2007 (5). Carcinogenesis studies with animals have shown that mesothelioma can be caused by either intrapleural or intraperitoneal injections of asbestos (6, 7). In addition, phagocytosis of chrysotile asbestos by rat mesothelial cells in culture has been investigated (8). However, studies with human mesothelial cells have not been reported previously. In addition, the mechanisms by which asbestos causes mesothelioma remain obscure. Thus, we elected to investigate both short-and long-term effects of asbestos fibers on replicative cultures of normal human mesothelial cells. MATERIALS AND METHODSCell Culture and Growth Medium. Cultures of mesothelial cells were initiated from pleural effusions obtained from noncancerous donors who had medical indications of thoracentesis (e.g., congestive heart failure). The fluid was initially centrifuged at 125 x g for 5 min. The pelleted cells were suspended in growth medium, washed by centrifugation, resuspended in growth medium, and inoculated into surface-coated 10-cm culture dishes at a ratio of 1 dish per 50 ml of pleural fluid. Semiconfluent cultures were dissociated by using trypsin (9) and either expanded by inoculating 200,000 cells per 10-cm culture dish, cryopreserved (9), or used according to experimental protocols.Growth medium was prepared by supplementing medium M199 with hydrocortisone (0.4 ,AM), zinc-free insulin (0.87 ,uM), epidermal growth factor (3.3 nM), Hepes (20 mM), trace elements (9), fetal bovine serum (5%), and gentamicin (50 Ag/ml). Hydrocortisone was purchased from Steraloids (Wilton, NH); insulin and epidermal growth factor were obtained from Collaborative Research (Waltham, MA). Lux culture dish surfaces were coated (10) with a mixture of human fibronectin (10 pug/ml), collagen (Vitro...

Alterations in Gene Expression in Human Mesothelial Cells Correlate with Mineral Pathogenicity

“…Models of transcript profiling for discrimination of toxic and nontoxic compounds in liver and other organs have also been developed in rodents (18), confirming the hypothesis that predictive modeling for classification of toxic agents and carcinogens is feasible. Here we used toxicogenomic approaches in human mesothelial cells, a cell type exquisitely sensitive to asbestos (19) and human contact-inhibited ovarian epithelial cells, a cell type not linked to carcinogenesis by asbestos, to determine whether the magnitude of altered gene expression by insoluble particulates correlated with their toxicity to cells and documented pathogenicity in humans. Although a recent study has examined gene expression profiles comparatively in crocidolite asbestos-exposed human lung adenocarcinoma (A549) and SV40-immortalized bronchial (BEAS-2B) or pleural mesothelial cell lines (MET5A) by cluster analysis (20), our studies are the first to examine gene expression changes by asbestos in comparison to other well-characterized particles in a human cell line that exhibits features of normal mesothelial cells (5).…”

Mechanisms of fiber-induced genotoxicity.

“…In a study of mitotic disturbances in SHE cells, Dopp et al (81) observed kinetochore staining in the micronucleus formed after asbestos treatment, indicating that whole chromosomes have been lost. Numerical chromosome abnormalities have been observed in human (91) and rat mesothelial cells (84) Table 9. The feeding of mice with asbestos did not result in chromosome abnormalities in germinal cells (106).…”

Dielectric changes in membrane properties and cell interiors of human mesothelial cells in vitro after crocidolite asbestos exposure.

“…Conventional dielectrophoresis and ROT measurements have revealed that significant differences exist in the dielectric properties of cells in different physiologic states (14, (40), induction of damage to chromosomes (41) and DNA (40), transformation (42), and apoptosis (43). To date, almost nothing is known about early surface property changes of the cellular membrane and interior dielectric changes of asbestostreated cells.…”