Ascorbic Acid Modifies the Surface of Asbestos: Possible Implications

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Chem. Res. Toxicol. 2003, 16, 328-335

Ascorbic Acid Modifies the Surface of Asbestos: Possible Implications in the Molecular Mechanisms of Toxicity Gianmario Martra, Maura Tomatis, Ivana Fenoglio, Salvatore Coluccia, and Bice Fubini* Dipartimento di Chimica IFM and Interdepartmental Center “G. Scansetti” for Studies on Asbestos and other Toxic Particulates, Universita` degli Studi di Torino, Via P. Giuria 7, I-10125 Torino, Italy Received June 17, 2002

Ascorbic acid is one of the major components of the antioxidants defenses of the lung lining layer where inhaled asbestos fibers are deposited. Crocidolite fibers were incubated at 37 °C in a 0.01 M aqueous solution of ascorbic acid for 25 days in order to investigate modifications in surface reactivity. Iron (820 nmol/mg) and monomeric silica (470 nmol/mg) were released in the supernatant, while ascorbic acid was consumed. The amount of iron and silicon released, respectively, 17 and 6% (in atoms) of the total fiber content, exceeded what was expected at the surface, suggesting a partial disgregation of crocidolite promoted by ascorbic acid. In the absence of ascorbic acid but at the same pH, the release of iron and monomeric silica was minimal. At time intervals, aliquots of fibers were withdrawn to evidence chemical modifications progressively taking place. Three families of Fe(II) centers, differing in coordinative unsaturation and progressively removed during incubation, have been evidenced from the FTIR spectra of NO adsorbed onto the fibers. The most uncoordinated ones are removed first. New highly uncoordinated iron sites are exposed at the fiber surface as a consequence of the erosion of the outmost layers while hydration of silica tetrahedra yields new silanol groups. The activity in the Fenton-like reaction (•OH from H2O2) decreases following surface iron depauperation. Conversely, the homolytic cleavage of the C-H bond (CO2•- from the formate ion) appears related to the small fraction of iron ions always present but easily quenched by the adsorption of ascorbic acid or its oxidation products.

1. Introduction Inhalation of asbestos fibers causes serious and fatal damage to the respiratory system, namely, asbestosis, lung cancer, and pleural mesothelioma. Despite confirmed evidence from epidemiological studies (1) and in vivo investigations (2) of the direct connection between the presence of asbestos fibers in the lung and the development, even a long time after exposure, of the above diseases, the molecular basis of pathogenicity is still partially obscure (3). It is generally accepted that fibrous habit and high biopersistence are important factors in the overall pathogenicity but that some chemical and biochemical reactionssstill to be elucidated in detailstaking place between the fiber surface and living matter play a key role in triggering the pathogenic response (4-6). Iron, present in considerable quantity as a structural component in crocidolite and amosite amphibole asbestos and as a high-level impurity in chrysotile, is believed to be responsible for the generation of free radicals, which will ultimately damage target cells (4, 7). Iron may be extracted by chelators, and this could be regarded as a possible way to diminish or even inactivate asbestos. However, iron extracted by low molecular weight endogenous chelators appears responsible for most of the DNA damage to cells (8) and could itself * To whom correspondence should be addressed. Tel: +39-011 670 7566. Fax: +39-011 670 7855. E-mail: [email protected].

contribute to the toxicity in vivo. Not all iron in the fiber appears responsible for adverse reactions but only few iron ions in well-defined redox and coordination state (912). Iron may thus act both at the surface of the fiber or after removal from the fiber. In the latter case, to attain better insight in the molecular mechanisms of toxicity, it is important to identify the surface locations where iron becomes active and to investigate the modifications in such sites that may occur in vivo, as a consequence of prolonged persistence of the fibers in the biological compartments into which they are secluded following inhalation. In the present study, we have investigated the effect of prolonged incubation of crocidolite fibers in an aqueous solution of ascorbic acid on the reactivity, and hence potential toxicity, of the fibers. The choice of ascorbic acid stems from several reasons: (i) Ascorbic acid, with glutathione, is one important component of the antioxidant defenses of the lung-lining layer (13, 14) onto which the inhaled fibers are bound to sit. Any prolonged reaction of the solid particles with it will consequently deplete the antioxidant defenses (15). (ii) Ascorbic acid has a peculiar affinity for crystalline silica, which is selectively attacked and partially solubilized in ascorbic acid solutions (16). No data are available on the reactivity of ascorbic acid with the silica framework of the various crystalline silicates, including asbestos. (iii) Ascorbic acid is a chelator for iron (17); thus, it is bound to affect the iron ions population.

10.1021/tx0200515 CCC: $25.00 © 2003 American Chemical Society Published on Web 02/14/2003

Ascorbic Acid Reactions with Crocidolite Asbestos

The effects of the interaction between ascorbic acid and crocidolite fibers have been monitored up to 25 days of incubation, measuring the amount of silicon and iron released in the solution and analyzing the changes in structure of the surface centers by IR spectroscopy, using NO as a probe molecule to test the presence of surface iron ions with different coordinative unsaturation, and of their reactivity by EPR spectroscopy, allowing, via the spin-trapping technique, the evaluation of their activity in free radical generation. The experimental conditions of the incubation are quite far from the physiological ones, as the starting ascorbic acid concentration employed (0.01 M) was ca. 1 × 104 times higher than in the bronchoalveolar lavage fluid (1.7 µM) (13, 18) and the time of incubation was incomparably shorter than the biological half-life of asbestos (decades). However, these conditions were chosen to gain experimental evidence of modifications taking place following crocidolite-ascorbic acid interaction. The results cannot be considered representative of the kinetics of the process in vivo, where many other endogenous substrates can compete for surface sites. They indicate, however, the nature of the surface reactions that may take place.

2. Experimental Section 2.1. Materials. Crocidolite asbestos employed in this work was from the same batch provided by UICC (Union Internationale Contre le Cancer) employed in previous studies (1921). High-purity NO (Matheson) and D2O (Stolher Isotope Chemicals, 98.3% D atoms) were used in FTIR experiments. They were admitted onto the samples after purification through several freeze-pump-thaw cycles. The spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), from Sigma, was purified by filtration through activated charcoal according to the method proposed by Buettner and Oberley (22). Sodium formate, hydrogen peroxide (30% w/v), and ascorbic acid were from Sigma. Phosphate buffers (mono- and disodium phosphate) were from Merck. 2.2. Incubation in Ascorbic Acid Solution. Crocidolite fibers (1 mg mL-1) were suspended (final volume, 250 mL) in 0.01 M sodium ascorbate and 0.05 M NaCl solution for 25 days at 37 °C. The suspension was continuously shaken and kept in the dark. The pH of the suspension was initially 6.9 and decreased after 7 days down to a constant value of 4.7. At time intervals, 2.50 mL of the suspension was taken and centrifuged at 10 000 rpm for 20 min to remove the asbestos. As a control, crocidolite fibers (1 mg mL-1) were suspended (final volume, 250 mL) in 0.05 M NaCl solution at 37 °C for 25 days. The pH was adjusted with HCl 0.05 M in order to reproduce the variations of pH in the experiment with ascorbate. The total amount of iron present in the supernatant was determined spectrophotometrically by addition of ferrozine and ascorbic acid (as a reducing agent), as described by Lund and Aust (17), on a Kontron Uvikon 930 dual beam spectrophotometer. The complex Fe2+-ferrozine was determined by measuring the absorbance at 562 nm (EmM ) 27.9 mM-1 cm-1). The concentration of silicon in the supernatant after incubation was determined by inductively coupled plasma atomic emission spectrometry (Libery-100, Varian). Ascorbic acid present in the supernatant was monitored by detecting the ascorbyl radical (which is always present in equilibrium with ascorbic acid) by means of EPR spectroscopy as previously reported (23). 2.3. Thermal Treatments. Outgassing at 400 °C for 45 min was carried out on samples placed in the IR cell or in a Pyrex tube connected to conventional vacuum lines (residual pressure, 1.0 × 10-6 Torr; 1 Torr ) 133.33 Pa) allowing the thermal treatments and adsorption-desorption experiments to be carried out in situ.

Chem. Res. Toxicol., Vol. 16, No. 3, 2003 329 Table 1. Amount of Iron and Monomeric Silica Released from Crocidolite Suspended in Different Solutions at 37 °C for 25 Days

entry 1 2

suspension medium 0.05 M NaCl 0.05 M NaCl + 0.01 M sodium ascorbate

amount of iron amount of monomeric released after silica released after 25 days (µmol/g) 25 days (µmol/g) 23 820

12 470

2.4. Experimental Techniques. The surface area of the samples was measured by the BET method (N2 adsorption at -196 °C) by a “Quantasorb” Quanthacrome instrument. The infrared spectra (4 cm-1 resolution) of the samples, in the form of self-supporting pellets, were obtained by a Bruker IFS88 spectrometer equipped with a MCT detector and a series of condenser mirrors placed in proximity of the sample in order to reduce the severe scattering of the fibers. The spectra of adsorbed NO are reported in absorbance, having subtracted the spectrum of the fibers before NO adsorption as background. 2.4.1. Free Radical Detection by Means of the Spin Trapping Technique. To detect the formation of radicals species in aqueous suspensions of the materials, the spintrapping technique was employed, as previously performed in our laboratory (11). The spin-trapping agent DMPO, which in aqueous medium gives stable radical adducts, was employed. The pH in all experiments was kept at 7.4 (phosphate buffer). The intensity of the EPR signal is proportional to the number of radicals in the solution. The formate ion is traditionally used as a target molecule to measure the radical-generating power of solids. Following homolytic cleavage of the C-H bond of formate ion, a carboxylate radical is formed, which gives rise, with the spin trap, to the [DMPO-COO]•- adduct, characterized by a typical EPR spectrum. The test is performed as follows: 1.0 mL of 2.0 M sodium formate in a phosphate buffer solution (1 M, pH 7.4) and 1.0 mL of 0.1 M DMPO in distilled water are added to 45 mg of fibers: (i) in their original form; (ii) withdrawn from the suspension in ascorbic acid solution, washed with distilled water, and dried at room temperature; or (iii) withdrawn from the suspension in ascorbic acid solution, washed with distilled water, dried at room temperature, and then outgassed at 400 °C for 45 min. The reaction mixture is then incubated in the dark at 37 °C under vigorous stirring, and 1.0 mL of the liquid phase is withdrawn at 30 and at 60 min, filtered through filter membranes (porosity 0.45 µm), and transferred into a quartz capillary. The corresponding EPR spectrum is recorded at room temperature 5 min after withdrawal of the aliquots. The sequence of operations in the test, with hydrogen peroxide as the target molecule, was similarly peformed, but because of the lower stability of the [DMPO-OH]• adduct, the times of incubation before withdrawal of the surnatant aliquots were shorter. A 0.5 mL amount of H2O2 0.5 M in phosphate buffer and a 1.0 mL amount of DMPO were added to 45 mg of the solid sample. The OH• formation was monitored by recording the EPR spectrum of the [DMPO-OH]• adduct after 10 and 30 min. Blanks were made by operating in the same way except that no solid particulate was introduced into the solution. All spectra were recorded on an Adani PS 100.X spectrometer at a microwave power level of 10 mW; middle of range, 3390; scan range, 100 G; and a modulation amplitude of 1 G.

3. Results and Discussion 3.1. Extraction of Iron and Silicon from Crocidolite Fibers Following Incubation with Ascorbic Acid Solution. In Table 1, the amount of iron and monomeric silica released form crocidolite fibers suspended for 25 days in the blank saline solution (entry 1) and in the saline solution also containing ascorbic acid

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(entry 2) is listed. The pH of the suspension containing ascorbic acid decreased during the time of incubation from 6.9 down to 4.7. Similar changes in pH were produced in the blank solution by adding the proper amount of HCl. It can be observed that in the case of the incubation of the fibers in the saline solution alone the release of iron and monomeric silica is minimal. By contrast, a substantial release of these species from the fibers was obtained during incubation in ascorbic acid aqueous solution, with a release of 820 nmol of iron and 470 nmol of silicon per milligram of fibers, which corresponds to 17 and 6% (in atom) of the total content of Fe and Si, respectively. At the same time, a progressive decrease of the ascorbic acid concentration was observed, and opposite to what happened in the absence of fibers, no ascorbic acid was left in the fiber suspension at the end of the experiment. The comparison with the data obtained with the blank solution indicates that the incubation in ascorbic acid yielded a significant increase of the release of iron and monomeric silica from the fibers. Furthermore, it must be considered that the measured amounts of iron and monomeric silica brought into in the aqueous phase in the presence of ascorbic acid cannot result from the removal of these species only from the surface of the fibers: taking into consideration that the specific surface area of the fibers is 8.0 m2 g-1, they would correspond to the presence of ca. 60 iron ions and ca. 35 silicon atoms per square nanometer should they be all from the surface. It can thus be concluded that ascorbic acid promotes a partial dissolution of crocidolite fibers, the mechanism and kinetics of which will be discussed in a future paper based on a parallel study devoted to the investigation of the interaction of asbestos fibers with various iron chelators (24). It may be of interest to notice that in such a study it was found that incubation in solutions of different chelators with the same procedure yielded very different results. This strongly suggests that a possible growth of microorganism, if any, in the suspending solution for long period of incubation (which should play the same role in all cases, as well as in the blank experiments) should not be responsible to a significant extent for the observed partial dissolution of the fibers. The attack of the silica tetrahedra should allow the chelation of iron ions initially in the bulk of the fibers. These processes should be responsible for the consumption of ascorbic acid as a complexing agent of extracted iron and monomeric silica. The consumption of ascorbic acid could be due to the formation of a stable complex with silicon atoms or to oxidative processes that yield several products, the most important being dehydroascorbate, which can contribute to iron extraction and solubilization. Evidence of the enhancement of the solubilization of crystalline silica in solution of ascorbic acid was recently reported by some of us (16). It can be of interest to notice that the partial dissolution of crocidolite fibers is accompanied by an increase of their specific surface area from 8.0 to 9.5 m2 g-1. This suggests that the dissolution process actually occurs as an erosion of the fibers, as proposed in a previous HRTEM study (25), with an enhancement of their surface roughness and/or the appearance of some porosity. 3.2. Modification of the Surface Structure and of the Reactivity of the Fibers by Ascorbic Acid Attack. 3.2.1. Preliminary Remarks on the Monitoring of the Coordinative State of Surface Iron Ions

Martra et al.

of Crocidolite by FTIR Spectroscopy of Adsorbed NO on Fibers Outgassed at 400 °C. Iron ions in the bulk of crocidolite fibers are in octahedral coordination, with six ligands constituted by the oxygen atoms of the double chains of silica tetrahedra that sandwich the layers containing the cations and by OH- counteranions (26). Part of these ligands is lost when iron ions are exposed at the surface and are then replaced by molecular water and hydroxyl groups resulting by the dissociation of H2O molecules when the fibers are exposed to air or put in an aqueous medium. The number of such ligands depends on the number of bonds still connecting iron ions with the surface, which can vary in dependence of the location of the ions at the surface (different local geometric structures on the extended surfaces of the fibers, edge and corner positions, etc.). Noticeably, previous studies reported in the literature proposed that such a number of bonds might affect the reactivity of iron ions at the surface of the fibers toward a variety of endogenous species present in biological media, such as H2O2, NO, and functional groups of proteins, which can replace H2O molecules and OH groups saturating the coordinative positions of surface iron ions (5, 6). The recognition of iron ions differing for the number of bonds with the surface is then a matter of interest in the elucidation of the structure and reactivity of the surface of crocidolite. Unluckily, it appears quite difficult to obtain this kind of information directly from the fibers in their native form suspended in an aqueous medium. For this reason, we adopted a method largely employed in current research on the surface features of powdered oxides and silicates, in particular, i.e., the study, by infrared spectroscopy, of the adsorption of probe molecules on the investigated surface centers (27, 28). In a previous study (20), we reported that outgassing in vacuo at 400 °C was the treatment condition allowing the highest degree of desorption of water molecules and hydroxyl groups adsorbed on iron ions at the crocidolite surface without the occurrence of a detectable change in the structure of the fibers. The coordinative unsaturation created in such a way can then be filled by suitable probe molecules, such as NO, the vibrational features of which depend on the characteristics of the surface centers where they are adsorbed. We also found that following outgassing at 400 °C, all surface iron ions detected by NO were in the 2+ oxidation state, although both Fe3+ and Fe2+ are known to be present in crocidolite (26). This is not only due to a selective presence at the surface of native fibers of Fe2+ but also to the reduction of the surface Fe3+, if any, to Fe2+ during outgassing at high temperature, as observed for other transition metal ions (29). However, this alteration of the nature of a part of the surface iron ions should not affect the effectiveness of the method employed in monitoring the presence of surface iron ions differing in their coordination to the surface and the evolution of their amount in dependence on the incubation of the fibers in ascorbic acid solution. Finally, it must be considered that outgassing at 400 °C should also result in the complete removal of ascorbic acid molecules (and/ or of their oxidation products) possibly left adsorbed on the surface of the fibers after incubation. 3.2.2. FTIR Spectra of NO Adsorbed on Original Fibers. The IR spectrum of crocidolite fibers outgassed at 400 °C and then contacted with a NO pressure allowing the saturation of adsorbing surface sites exhibits

Ascorbic Acid Reactions with Crocidolite Asbestos

Figure 1. IR spectra of NO adsorbed on crocidolite outgassed at 400 °C for 45 min. (a) In the presence of 100 Torr NO; (b) after outgassing at room temperature for 30 min.

a main peak at 1800 cm-1, slightly asymmetric on the high-frequency side, accompanied by a band at 1735 cm-1 and a weak component at 1897 cm-1 (Figure 1a). In a previous study (20), on the basis of the data reported at that time in the literature on iron ions onto amorphous silica (29, 30), the main component at 1800 cm-1 and the weak absorption at 1897 cm-1 were assigned to dinitrosylic adducts formed on highly coordinatively unsatured Fe2+ ions, while the band at 1735 cm-1 to mononitrosylic species stabilized on Fe2+ ions with a slightly lower degree of coordinative unsaturation. By decreasing the NO pressure, the component at 1800 and 1897 cm-1 disappeared, while the band at 1735 cm-1 increased in intensity (Figure 1b). In agreement with the literature data (29, 30), this behavior was interpreted as due to the conversion of dinitrosylic adducts into mononitrosylic ones, the stretching mode of which produced an absorption at a frequency very close to that of the mononitrosylic species already present at higher NO coverage. The depletion of the main component at 1800 cm-1 revealed the presence of another absorption at 1812 cm-1, assigned to mononitrosylic species adsorbed on Fe2+ ions more coordinated to the structure (29, 30). A recent IR study on the adsorption of NO on extraframework Fe2+ ions in Fe-silicalite (31), where NO adsorbed on highly homogeneous ferrous sites produced better resolved bands, allows us to reassign the couple of bands at 1897 and 1800 cm-1 to a trinitrosylic species, while the absorption at 1735 cm-1, observed at high NO coverage, can be assigned to the symmetric stretching mode of dinitrosylic species adsorbed on Fe2+ with a lower degree of coordinative unsaturation, the corresponding absorption due to the asymmetric mode, expected to occur in the 1840-1810 cm-1 range, being overlapped to the more intense component at 1812 cm-1 due to mononitrosylic species on even less coordinatively unsaturated iron ions. This new assignment leaves untouched the conclusion of the previous study about the number of different types of iron centers exposed at the surface of crocidolite fibers (20) but provides further insights on their degree of coordinative unsaturation or at least, because of the possible occurrence of surface ligands displacement reactions (31), the number of coordinative positions of surface iron ions, which can be occupied by adsorbed ligands. Anyway, the evidence of the trinitrosylic adducts corre-

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Figure 2. IR spectra of NO (100 Torr) adsorbed on crocidolite outgassed at 400 °C for 45 min. (a) Original fibers (the same as curve a in Figure 1); curves b-d: fibers previously incubated in ascorbic acid solution for (b) 5, (c) 10, and (d) 25 days (see Experimental Section).

sponds to ions, which, in the presence of adsorbed ligands, exhibit the lowest coordination to the surface. For the sake of simplicity, the surface Fe2+ able to give origin to tri-, di-, and mononitrosyls will be hereafter referred to as Fe2+A, Fe2+B, and Fe2+C, respectively. 3.2.3. IR Spectra of NO on Crocidolite Incubated with Ascorbic Acid Solution. In Figure 2, the IR spectra at high coverage of NO adsorbed on the original crocidolite and those obtained on fibers incubated with aqueous solution of ascorbic acid for different periods of time are compared. As commented on above, all samples were outgassed at 400 °C prior to NO adsorption. As reported above, the spectrum of NO adsorbed on the untreated fibers is dominated by the peak at 1800 cm-1, coupled with a very weak partner at 1897 cm-1, due to trinitrosylic species on Fe2+A sites, while at 1735 cm-1 a weaker band due to the low-frequency mode of dinitrosylic adducts on Fe2+B centers is observed (Figure 2a). The corresponding high-frequency mode and the component due to mononitrosylic species on Fe2+A sites are responsible for the asymmetry present on the highfrequency side of the peak at 1800 cm-1. The spectrum of NO adsorbed on the fibers treated in ascorbic acid exhibited a progressive decrease of the overall intensity by increasing the duration of the treatment, accompanied at each step by significant changes in relative intensity, shape, and position of the various components (Figure 2b-d). In fact, the spectral pattern of NO adsorbed on crocidolite treated with ascorbic acid for 5 days appeared dominated by a peak at 1816 cm-1 asymmetric on the low-frequency side, likely resulting from the superposition of a dominant band due to Fe2+C(NO) species and a weaker component at ca. 1800 cm-1, coupled with a very weak absorption at 1897 cm-1, produced by Fe2+A(NO)3 adducts (Figure 2b). Furthermore, it can be noticed also that the band at 1735 cm-1 due to the Fe2+B(NO)2 species appears significantly less intense. On the basis of these features, it can be inferred that the treatment in ascorbic acid for such a period resulted in a preferential removal of Fe2+B and Fe2+A ions from the surface of crocidolite fibers with respect to the Fe2+C ones. As expected, iron ions with a higher level of coordinative unsaturation toward the surface are more easily extracted by the chelating agent.

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The admission of NO on crocidolite treated with ascorbic acid for 10 days produced an even less intense spectrum, characterized by a weak absorption at 1735 cm-1, due to Fe2+B(NO)2 adducts and a main peak at 1806 cm-1, asymmetric on the high-frequency side (Figure 2c). This latter signal may originate from the superposition of a dominant band due to the asymmetric mode of Fe2+A(NO)3 adducts, coupled with the very weak component at 1897 cm-1, and a minor component, located at higher frequency, due to the Fe2+C(NO). This pattern suggests that by prolonging the time of treatment with ascorbic acid the iron removal proceeds with Fe2+B and Fe2+C surface ions while it seems to affect at a lower extent the Fe2+A ones. This behavior might appear in contradiction with the results obtained for the fibers treated with ascorbic acid for a shorter time, which evidenced a preferential removal of Fe2+A centers. It must however be taken into consideration that as reported above, ascorbic acid also promotes the dissolution of the silicate framework of the fibers. It may be proposed that the removal of silica tetrahedra results in some erosion of the structure anchoring Fe2+B and Fe2+C ions to the surface. These centers can then be transformed to surface ions with a lower number of bonds to the surface, which can be attacked by the chelating agent. Fe2+A sites newly produced in this way, and not yet removed from the surface by chelation, can then form trinitrosylic adducts, which mainly contribute to the band at 1806 cm-1. The process of dissolution of the silicate framework and removal of iron ions further occurred by prolonging the time of treatment of the fibers with ascorbic acid. In fact, the spectrum of NO adsorbed on crocidolite treated with ascorbic acid for 25 days exhibits an overall lower intensity but still appears dominated by the band due to Fe2+A(NO)3 at 1800 cm-1 (Figure 2d). Spectra taken at decreasing NO coverage (not reported) showed that these trinitrosylic species are progressively transformed into dinitrosylic ones. 3.2.4. IR Spectra of Surface Hydroxyl Groups. Outgassing at 400 °C did not remove hydroxyl groups from all of the cations exposed at the surface of the fibers. When these groups are in their original OH form, their vibrational features can be hardly observed, because they fall in the 3700-3500 cm-1 range, where they are overshadowed by the bands of the hydroxyls in the bulk (20). However, the IR signals of surface hydroxyl groups can be evidenced by exchange with D2O. In fact, only surface hydroxyls can exchange their H atoms with D atoms of D2O, which cannot reach bulk hydroxyls, and the resulting surface OD (deuterioxyls) groups exhibit distinct IR absorption bands at lower frequency than OH hydroxyls. Curve a in Figure 3 is the spectrum in the 2800-2500 cm-1 region of the original crocidolite fibers exchanged with D2O at room temperature and then outgassed at 400 °C. Three narrow bands are present at 2692 (weak), 2681, and 2669 cm-1, each assigned to deuterioxyl groups interacting with a different set of three cations, exposed at the surface in positions equivalent to the M1M3M1 ones in the bulk (20), occupied by two magnesium and one iron ion (band at 2692 cm-1), one magnesium and two iron ions (band at 2681 cm-1), and three iron ions (band at 2669 cm-1) (20). Furthermore, a weak component at 2760 cm-1 was observed, assigned to deuterated silanols, which represents the termination of the silicate framework exposed to the surface. No significant changes of this spectral pattern

Martra et al.

Figure 3. IR spectra in the 2800-2600 cm-1 range of crocidolite (a) before incubation and b) after incubation for 25 days with ascorbic acid solution. Before IR measurement, both samples were exchanged with D2O (contact with 18 Torr at room temperature for 30 min followed by outgassing at room temperature for 30 min, repeated three times (19)) and then outgassed at 400 °C for 45 min.

were observed for the fiber treated with ascorbic acid up to 10 days (spectra not reported), whereas the bands at 2681 and 2669 cm-1 appeared strongly decreased in intensity after the treatment 25 days long (Figure 3b). A slight increase in intensity of the component at 2760 cm-1 was also observed. Previous experiments on crocidolite fibers exchanged with D2O and then outgassed at 400 °C evidenced that the adsorption of NO does not produce any perturbation of the bands related to the deuterioxyl species (20), indicating that iron ions still carrying OD groups retain a complete coordination sphere. Furthermore, the fact that OD species bound to these centers resisted outgassing at 400 °C suggests that their coordination sphere is quite stable. Such a feature could result in a higher resistance also to the attack of chelators. This might account for the fact that a significant decrease in the intensity of the OD bands, likely monitoring the removal of a part of iron ions still carrying these surface ligands, was observed only after treatment for 25 days with ascorbic acid. The fact that the band at 2669 cm-1 underwent a larger decrease in intensity, with respect to the 2681 cm-1 and the 2692 cm-1 ones, suggests that removal of these ions with a stable coordination sphere is site selective. Removal is easiest from the set of surface M1M3M1 cationic sites where iron is present in all of the three positions available. Finally, the slight increase in intensity of the band due to deuterated silanol groups observed for crocidolite treated for 25 days with ascorbic acid monitors the same increase of hydroxylated terminations of the portion of the silica tetrahedra exposed to the surface of the fibers, likely resulting from the disruption of a part of the framework of the silica and subsequent partial hydration of silica tetrahedra, originally in the bulk of the fibers, newly exposed to the contact with the aqueous medium. 3.3. Modifications of the Potential for Free Radical Release Following Incubation in Ascorbic Acid. Crocidolite fibers, when in aqueous suspensions, can generate •OH radicals from hydrogen peroxide, via a Fenton-like reaction and carbon-centered radicals following homolytic rupture of a carbon-hydrogen bond in formate ions, or other target molecules such as peptides (5, 11, 21, 32-34).

Ascorbic Acid Reactions with Crocidolite Asbestos

Figure 4. Free radical release from aqueous suspensions of crocidolite fibers. Integrated intensity of the EPR spectra of the [DMPO-OH]• adduct produced by suspending in the aqueous solution of H2O2 and DMPO the original crocidolite fibers and fibers incubated for 5, 10, and 25 days in aqueous solution of ascorbic acid (details on the procedure employed are in the Experimental Section). Inset: scheme of the [DMPO-OH]• adduct and the corresponding EPR spectrum.

According to previous findings (11, 21), both reactions took place with the original crocidolite fibers. The radical yield of the two processes was differently affected upon incubation of the fibers in ascorbic acid solution. In the case of the reaction with H2O2, a decrease to ca. 20% of the yield with original fibers takes place after the first 5 days of incubation for the fibers withdrawn from the ascorbic acid solutions and then simply dried at room temperature, and from 10 days onward, the yield further decreases down to a value of ca. 10% of the original one (Figure 4). This progressive loss in the radical release activity could have a result on both the progressive adsorption of ascorbic acid (and/or its oxidation products) and the depauperation in iron of the fibers. However, similar results were obtained with the fibers withdrawn from the ascorbic acid solutions and additionally outgassed at 400 °C (data not reported), the same procedure employed for IR experiments. As commented on above, this treatment should result in the conversion of all surface iron ions in the Fe(II) form and in the complete removal of ascorbic acid molecules left adsorbed at the surface of the fibers after incubation. As these processes should occur in the same extent for all samples, the differences in the radical release activity among fibers incubated in ascorbic acid for different time and then outgassed at 400 °C should be ascribed to processes that occurr during the incubation. On this basis, the observed trend in the •OH yield appears to be related to the depauperation in iron of the fiber surface evidenced by the IR spectra of adsorbed NO (see Figure 2 and related comments). Conversely, two distinct trends in the carboncentered radical yield were observed for the fibers incubated with ascorbic acid and simply dried at room temperature (Figure 5a, black bars) and those incubated and subsequently outgassed at 400 °C (Figure 5b, gray bars). In the first case, the release of radical species is depleted already after 5 days of incubation. By contrast, the radical activity of the fibers withdrawn from the ascorbic acid solution and outgassed at 400 °C before the reaction with formate appears affected only to a limited extent by the incubation with ascorbic acid, decreasing to ca. 60% after 5 days of incubation and then progressively recovering the original value after 25 days of treatment with ascorbic acid. A possible explanation is the following: as indicated above, in the aqueous medium, the interaction with ascorbic acid, besides the extraction of a part of surface

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Figure 5. Free radical release from aqueous suspensions of crocidolite fibers. Integrated intensity of the EPR spectra of the [DMPO-CO2]•- adduct produced by suspending crocidolite in the aqueous solution of sodium formate and DMPO. (a) Black bars: intensities obtained for original fibers and fibers incubated for 5, 10, and 25 days in aqueous solution of ascorbic acid. (b) Gray bars: intensities obtained for the same samples outgassed at 400 °C for 45 min before suspension in the sodium formate and DMPO solution (details on the procedures employed are in the Experimental Section). Inset: scheme of the [DMPO-CO2]•adduct and the corresponding EPR spectrum.

iron ions, may also result in the adsorption of ascorbic acid molecules (and/or of their oxidation products). These adsorbed species are then removed by outgassing at 400 °C, which also transform all surface iron ions in the Fe(II) form. However, this latter effect cannot account for the recovery of the fiber activity in the cleavage of C-H bonds, because in the presence of ascorbic acid one would expect iron ions to be kept at the surface in the Fe(II) form. Thus, the fact that most parts of the activity of the fibers in the cleavage of C-H bonds were restored to a large extent by outgassing at 400 °C strongly suggests that the activity loss observed after incubation in ascorbic acid solution resulted from the “quenching” of iron sites active in such reactions by adsorbed ascorbic acid molecules (and/or derived products). When these species are removed from the fibers by outgassing, the activity in the cleavage of C-H bonds is restored at a level not so different from that exhibited by the original crocidolite, and this effect was observed for all of the various aliquots of fibers incubated for different times in ascorbic acid. This indicates that such a reaction is not so sensitive to the overall amount of iron ions exposed at the surface of the fibers, which, by contrast, progressively decreased by increasing the time of incubation in ascorbic acid, as reported above. It may be concluded that iron ions, which are the active sites for this reaction, correspond to only a small fraction of the overall iron ions exposed at the surface, in agreement with previous findings (19, 20). Furthermore, it can be considered that on the basis of the trend of the yield in C-H bond cleavage obtained for the fibers outgassed after incubation in ascorbic acid, the number of such active centers slightly decreased during the first 5 days of incubation and then progressively increased for longer incubation times, likely as a consequence of the erosion of the surface of the fibers, resulting in the exposure to the surface of new active sites. The IR spectra of adsorbed NO indicated that during incubation iron ions that after outgassing at 400 °C are probed as Fe2+A are initially removed from the fibers but probably some of them are newly generated in the last stages of the interaction with ascorbic acid. It might then be proposed that the few surface iron ions responsible for the cleavage of C-H bonds have to be sought among those centers.

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4. Conclusions Ascorbic acid turned out to be a very potent agent toward crocidolite fibers, even without being associated to other chelators (8). The reaction involves both the chelation of surface iron and the attack to the siliceous part of the fibers, similar to what was recently reported for crystalline silica dusts (16) and for desferrioxaminetreated crocidolite (24, 35). A significant role in the removal of iron ions from the fiber surface is played by their coordinative unsaturation, also affected by the simultaneous erosion of the silicate framework of the fibers. The present results also provide new information on the chemical aspects of asbestos toxicity. The lung-lining layer, where the crocidolite fibers are bound to sit when inhaled, is rich in surfactants, proteins, glutathione, and ascorbic acid, glutathione and ascorbic acid providing the antioxidant defenses in that biological compartment. The present data indicate specific reactions of the fiber surface with ascorbic acid, which in vivo may have a direct impact on the pathogenetic mechanisms (4). In fact, not only ascorbic acid can be consumed, via “complexation” of extracted silica monomers and iron ions, thus depriving the lung-lining layer of its natural antioxidant defenses, but removed iron may attain biomolecules and catalyze the generation of highly damaging free radicals. Such an effect would just be partially compensated by the loss of free radical yield from the fiber surface (see Figures 4 and 5). The •OH yield is in fact decreased following the interaction of crocidolite with ascorbic acid as a consequence of the depauperation in iron ions exposed at the surface of the fibers. The homolytic cleavage of the C-H bond is blunted during the incubation with ascorbic acid, but in this case, the main cause of such deactivation is the adsorption on the active sites of ascorbic acid molecules, and/or their oxidation products. If such species are removed from the surface, as one may expect to happen in in vivo conditions, this kind of radical activity can be restored, as it is due to a small fraction of iron ions, quite resistant to the extraction from the surface by ascorbic acid. Any excess of ascorbic acid in people previously exposed to asbestos has thus to be regarded as a double sword in that instead of exerting its usual antioxidant action it may promote the adverse reaction triggered by asbestos.

Acknowledgment. The research has been carried out with financial support by the Regione Piemonte. I.F. is grateful to AMIAT-Torino for the grant received.

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