Regular articleLong and short fiber amosite asbestos alters at a different extent the redox metabolism in human lung epithelial cells
Introduction
Asbestos fibers, such as crocidolite, amosite, and chrysotile, when inhaled can induce pulmonary fibrosis, lung cancer, and a variety of pleural diseases, including malignant mesothelioma (Hodgson and Darnton, 2000). Despite the increasing body of studies performed in this field, it is still unclear what determines asbestos-related diseases Kamp and Weitzman, 1999, Robledo and Mossman, 1999. The fibrous habit has been the first characteristic considered as a cause of asbestos toxicity (Stanton and Wrench, 1972). Afterward, other physicochemical properties have been also reported to play a key role in the molecular mechanism of pathogenicity, such as chemical composition Kamp and Weitzman, 1999, Robledo and Mossman, 1999 and surface reactivity (Fubini and Otero-Aréan, 1999). Fiber shape and dimensions do play an important role in asbestos toxicity: indeed, in the distal respiratory airways, longer fibers are less easily phagocytized and cleared, showing a prolonged biopersistence, and may interfere with cell growth and division more efficiently than shorter ones (Kinnula, 1999). However it is nowadays accepted that both form and chemical composition of the fibers contribute to their pathogenic potential (Kane, 1996). In order to provide evidence about the role played by fiber size in pathogenicity, years ago a batch of short amosite fibers was prepared by prolonged milling of long fibers and the in vitro and in vivo activity of these “short” and “long” fiber samples was thoroughly investigated at the Institute of Occupational Medicine (Edinburgh, UK) by Davis and coworkers Davis et al., 1986, Donaldson et al., 1989. Compared with the short fiber sample, long amosite caused more mesotheliomas and pulmonary tumors in exposed animals (Davis et al., 1986), was more inflammogenic (Donaldson et al., 1989), caused more epithelial detachment injury in vitro (Donaldson et al., 1993), elicited more release of superoxide anion (Hill et al., 1995) and tumor necrosis factor from macrophages (Donaldson et al., 1992), and induced more chromosomal aberrations in Chinese hamster ovary cells (Donaldson and Golyasnya, 1994). When checked for their potential to release iron in solution and to damage DNA, short and long fiber amosite (SFA and LFA, respectively) behaved differently, suggesting differences in surface reactivity of the two fiber specimens Gilmour et al., 1997, Graham et al., 1999. We have thus revisited this historical set of fibers, in order to relate their physico-chemical properties to their different biological activity.
The oxidative step of pentose phosphate pathway (PPP), converting glucose 6-phosphate into ribulose 5-phosphate and CO2, leads to the synthesis of NADPH, a redox cofactor for many antioxidant enzymes. The metabolic flux through PPP represents a very sensitive index of the cell exposure to oxidant molecules, as its rate-limiting enzyme, glucose 6-phosphate dehydrogenase (G6PD), is activated by any oxidative stress via a decrease of the NADPH/NADP+ ratio. We have recently demonstrated that crocidolite, another type of amphibole asbestos, inhibits the oxidative step of PPP and the activity of G6PD in human lung epithelial cells (Riganti et al., 2002). Subsequently, having found that amosite exhibits a similar effect, we used the Edinburgh set of long and short amosite fibers, where the latter is obtained by prolonged grinding of the former, to elucidate whether the asbestos effect on cell redox metabolism is influenced by fiber size and milling treatment. During these experiments, we tried also to provide more information on the potential of fibers to initiate radical reactions and on the mechanism by which asbestos may decrease the cell reducing potential and impair the cell ability to oppose oxidative stress.
Section snippets
Fibers
Samples of SFA and LFA were kindly supplied by Dr. Lang Tran from the Institute of Occupational Medicine (Edinburgh, UK). These samples were prepared from the same batch of South African amosite: the SFA was obtained by grinding LFA in a ceramic ball-milled system (agata mortar) followed by sedimentation in water (Davis et al., 1986). The resulting material was so fine that only 37% of particles could be regarded as fibers following the WHO definition (i.e., having an aspect ratio > 3:1) (Davis
Results
SFA did not inhibit significantly the PPP activity of A549 cells, except at the higher concentration used, which decreased the menadione-stimulated PPP only (Fig. 1); on the other hand, every concentration of LFA significantly inhibited both basal and menadione-stimulated PPP (Fig. 1). PPP flux is mainly regulated by the activity of G6PD: similarly to PPP experiments, LFA significantly inhibited G6PD activity at each concentration used, while SFA was effective only at the higher dose and with
Discussion
Amosite, like crocidolite, contains very high amounts of iron (about 28%) Fubini and Otero-Aréan, 1999, Hardy and Aust, 1995, generates hydroxyl radicals in the presence of H2O2 via a Fenton-like reaction, and in some circumstances cleaves C–H bonds leaving a carbon centered radical (as reviewed in Hardy and Aust, 1995, Fubini and Otero-Aréan, 1999, Kamp and Weitzman, 1999; amosite is likely to evoke a cellular oxidative stress, as it lowers reduced glutathione in rat alveolar macrophages
Acknowledgements
This research has been carried out with the financial support of Regione Piemonte. Elisabetta Aldieri and Maura Tomatis are recipients of post-doc fellowships funded by Regione Piemonte and Università di Torino. We are grateful to Dr. Lang Tran (Institute of Occupational Medicine, Edinburgh, UK) for having supplied the samples of short and long amosite fibers.
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