. Can. J. Chem. 65, 2338 (1987).Formation of hydroxyl radicals, in relatively large quantities, by oxygen reduction due to the surface activity of asbestos in a cell-free system is demonstrated, using electron spin resonance and a spin trapping agent. The quantity of OH' produced (in general, above l o i 4 mg-I for a Canadian chrysotile) is a function of activation or passivation of the electron donor surface sites of the minerals. The role of these radicals in oxidative stress in relation to the toxic properties of these minerals is discussed.ROGER ZALMA, LIONEL BONNEAU, JOELLE GUIGNARD, HENRI PEZERAT et MARIE-CLAUDE JAURAND. Can. J. Chem. 65, 2338 (1987).En utilisant la technique des pikgeurs de radicaux et la spectroscopie en rksonance paramagnetique Clectronique on peut montrer que la rkactivitC de surface des amiantes permet, en milieu tampon, la rkduction de I'oxygbne en radicaux hydroxyles (OH'). La quantitC de radicaux formts (en gtntral supkrieure ? I lOI4 mg-I pour le chrysotile canadien) est fonction de l'activation ou de la passivation des sites de surface i caractgre Clectro-donneur. Le r61e de ces radicaux dans les phknombnes de ccstress oxydant* est discutk en relation avec les propriktts toxiques des amiantes.It is known that asbestos fibres induce chromosome damage, which might be implicated in the sequential steps of carcinogenesis. This phenomenon might be related to the formation of oxy radicals in the presence of asbestos, this formation being postulated from biochemical data concerning asbestos-induced damage to cells and lipid peroxidation (1-4), and in relation to redox properties as shown up by electron spin resonance (esr) ( 5 , 6).Weitzman and Graceffa (7) and Eberhart and RomanoFranco (8) demonstrated that asbestos catalyzes the generation of hydroxyl (OH') and superoxide (02:) radicals from hydrogen peroxide (H202) in a cell-free system. But all ironcontaining minerals (with Fe2+ or Fe3+) have approximately the same activity in such a system (9, lo), and the bioavailability of H202 is low. In our model system, with techniques similar to that of Weitzman and Graceffa (7) (esr and spin trapping agent), but taking into account the phenomenon of activation or passivation of the surface active sites of asbestos, we are able to demonstrate the formation of a high level of OH' from the reduction of oxygen, which is completely bioavailable. Our model system differs from the one employing H202 in that only part of the iron-containing minerals is active in the reduction of 02, according to the following reactions:

Increasing evidence demonstrates that emphysema in coal workers may be related to their exposure to coal dusts. The hypothesis that emphysema could be related to the production of reactive oxygen species (ROS) generated by inhaled coal dusts was examined in the present study. Using ESR, we investigated whether the interaction of different coals with dissolved oxygen in aqueous medium could generate ROS. Indeed, we found that one of the five examined French coal samples, Vouters coal, was effective in oxidizing formate anions or ethanol by a radical pathway. Inactivation of alpha 1-antitrypsin (alpha 1-AT) in vitro was then examined for all five coal filtrates. The Vouters coal filtrate, which exhibits oxidative activity, can also inactivate alpha 1-AT. When this coal filtrate was crystallized and redissolved, its oxidative activity was found to be conserved. By use of various analytical techniques, the active component of this coal filtrate was identified to be primarily ferrous sulfate. We confirmed that pure ferrous sulfate can effectively reduce oxygen to produce ROS in aqueous medium in vitro and can also inactivate alpha 1-AT. In this report, the nature of the coal-generated oxidative species, the origin of ferrous sulfate, and the stability of ferrous sulfate in the different coal samples are discussed. These results offer evidence that some inhaled coal dusts are capable of producing ROS, which may play an important role in the development of coal workers' emphysema.

Epidemiological studies have shown that a causal relationship may exist between coal dust exposure and emphysema in coal miners. Emphysema can be considered as one of the human pathologies associated with oxidative stress, resulting from oxidant-induced alpha 1-antitrypsin (alpha 1-AT) inactivation and uncontrolled proteolysis of lung tissue. We have previously reported that certain coal dusts contained hydrated ferrous sulfate (FeSO4) that inactivated alpha 1-AT [Huang et al. (1993) Chem. Res. Toxicol. 6, 452-458]. In the present study, we have shown that the FeSO4 originated from oxidation of pyrite (FeS2), which is a typical contaminant of coal dusts. The relative humidity and microenvironment around individual pyrite particles influence the formation of FeSO4 in the coal. However, the subsequent human exposure to coal dust containing FeSO4 depends on the stability of the formed FeSO4. We found that pH played the most important role in stabilizing the FeSO4, such that a final pH < 4.5 after oxidation of pyrite stabilized FeSO4, whereas at high pH the conversion of reactive Fe2+ to Fe3+ was immediate. Sulfuric acid (H2SO4), which is also produced by the oxidation of pyrite, can lower the pH, but it can also be neutralized by other minerals in coal dusts, such as calcite (CaCO3). The stability of FeSO4 in coal dust can also be influenced by the length of exposure to air. Our studies demonstrated that coal samples differed in their capacity to stabilize FeSO4. This current study strengthens our previous reported hypothesis that emphysema, which occurs irregularly in coal miners, could be directly related to exposure to coal dust containing FeSO4.

Striking differences in the prevalence of coal workers' pneumoconiosis (CWP) exist between different coal mine regions. The major factors responsible for the observed regional differences in CWP have not yet been identified. In the present study, chemical reactivity of the carbon-centered free radicals in coals and lung tissues, as well as ferrous iron in the coals, were studied by ESR techniques. The ESR spectra clearly demonstrated the presence of at least two types of carbon-centered free radical species, which might respectively attribute to the macromolecular phase and the molecular phase of coal. Grinding produced free radicals in coals. Exposure of freshly ground coal to air for 28 h induced a slight increase of free radicals for most of the coals, and a slight decrease after 4 months' exposure. The lung tissue samples of coal workers deceased of CWP showed similar ESR spectra as coal samples, and these radicals were highly stable in the lung. After incubation of coals with glutathione, hydrogen peroxide, sodium formate or oxygen, the coal sample from the Gardanne mine which has never induced CWP, and thus is the least hazardous coal, showed the most significant change in the carbon-centered free radical concentration. No significant changes were observed among other coals reported to induce CWP. On the other hand, we found that the coals released different amounts of Fe2+ in an acidic medium. Interestingly, the prevalence of CWP correlates positively with the released Fe2+ content in these coals and with the amount of oxygen radicals produced by the interaction of Fe2+ with O2 in the acidified coal filtrates. Our studies indicate that the carbon-centered free radicals may not be biologically relevant to coal dust-induced pneumoconiosis, whereas the acid soluble Fe2+, which may be dissolved in the phagolysosomes of macrophages, can then lead to Fe2+-induced oxidative stress and eventual CWP development.

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