Chem. Res. Toxicol. 2000, 13, 913-921
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Using in Vitro Iron Deposition on Asbestos To Model Asbestos Bodies Formed in Human Lung Zhihua Shen,† Dirk Bosbach,‡,§ Michael F. Hochella, Jr.,‡ David L. Bish,| M. Glenn Williams, Jr.,⊥ Ronald F. Dodson,⊥ and Ann E. Aust*,† Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, Department of Geological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0240, Earth and Environmental Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, and Department of Cell Biology and Environmental Sciences, University of Texas Health Center at Tyler, P.O. Box 2003, Tyler, Texas 75710 Received February 10, 2000
Recent studies have shown that iron is an important factor in the chemical activity of asbestos and may play a key role in its biological effects. The most carcinogenic forms of asbestos, crocidolite and amosite, contain up to 27% iron by weight as part of their crystal structure. These minerals can acquire more iron after being inhaled, thereby forming asbestos bodies. Reported here is a method for depositing iron on asbestos fibers in vitro which produced iron deposits of the same form as observed on asbestos bodies removed from human lungs. Crocidolite and amosite were incubated in either FeCl2 or FeCl3 solutions for 2 h. To assess the effect of longer-term binding, crocidolite was incubated in FeCl2 or FeCl3 and amosite in FeCl3 for 14 days. The amount of iron bound by the fibers was determined by measuring the amount remaining in the incubation solution using an iron assay with the chelator ferrozine. After iron loading had been carried out, the fibers were also examined for the presence of an increased amount of surface iron using X-ray photoelectron spectroscopy (XPS). XPS analysis showed an increased amount of surface iron on both Fe(II)- and Fe(III)-loaded crocidolite and only on Fe(III)-loaded amosite. In addition, atomic force microscopy revealed that the topography of amosite, incubated in 1 mM FeCl3 solutions for 2 h, was very rough compared with that of the untreated fibers, further evidence of Fe(III) accumulation on the fiber surfaces. Analysis of long-term Fe(III)-loaded crocidolite and amosite using X-ray diffraction (XRD) suggested that ferrihydrite, a poorly crystallized hydrous ferric iron oxide, had formed. XRD also showed that ferrihydrite was present in amosite-core asbestos bodies taken from human lung. Auger electron spectroscopy (AES) confirmed that Fe and O were the only constituent elements present on the surface of the asbestos bodies, although H cannot be detected by AES and is presumably also present. Taken together for all samples, the data reported here suggest that Fe(II) binding may result from ion exchange, possibly with Na, on the fiber surfaces, whereas Fe(III) binding forms ferrihydrite on the fibers under the conditions used in this study. Therefore, fibers carefully loaded with Fe(III) in vitro may be a particularly appropriate and useful model for the study of chemical characteristics associated with asbestos bodies and their potential for interactions in a biosystem.
Introduction Exposure to asbestos is associated with a variety of diseases, including pulmonary interstitial fibrosis, mesothelioma of the pleura, pericardium, and peritoneum, and carcinoma of the lung, esophagus, and stomach (1-5). It was not until the late 1950s that asbestos was recognized as a human carcinogen. Since that time, the mechanism by which asbestos causes cancer has been intensively studied. However, it is still not well-understood. Some * To whom correspondence should be addressed: Department of Chemistry and Biochemistry, Utah State University, Logan, UT 843220300. Phone: (435) 797-1629. Fax: (435) 797-3390. E-mail: AAUST@ cc.usu.edu. † Utah State University. ‡ Virginia Polytechnic Institute and State University. § Present address: Institut fu ¨ r Mineralogie, Universitaet Muenster, Corrensstr. 24, 48149 Muenster, Germany. | Los Alamos National Laboratory. ⊥ University of Texas Health Center at Tyler.
physical properties have been shown to be important, such as fiber size (6) and durability in the body (7). Recent studies have indicated that iron may be responsible for some of the biological activities and may be involved in the carcinogenicity of asbestos (8). The most carcinogenic forms of asbestos are crocidolite and amosite, which contain about 27% iron by weight (9). Research has shown that asbestos catalyzes many of the same reactions as iron does, such as generation of reactive oxygen species (10-12), lipid peroxidation (1315), and DNA damage (16, 17). Iron mobilized from asbestos by a chelator in vitro has been shown to result in crocidolite-dependent oxygen consumption (18), hydroxyl radical generation (12), and DNA single-strand break formation (16). It has also been reported that iron was mobilized from crocidolite intracellularly (19). The amount of iron mobilized into a low-molecular weight fraction in human lung epithelial cells correlated well with the cytotoxicity of the fibers.
10.1021/tx000025b CCC: $19.00 © 2000 American Chemical Society Published on Web 08/23/2000
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In addition to mobilization, asbestos can acquire iron from the environment under certain conditions. It has been shown that asbestos fibers, such as crocidolite and amosite, which persist in the lung for decades, can bind iron on their surfaces (7). An accumulation of large amounts of endogenous iron on the surface of the longer (>8 µm) fibers produces ferruginous bodies, or asbestos bodies if the cores are asbestos. Under normal physiological conditions, iron is controlled by the iron storage protein ferritin and the iron transport protein transferrin. The entry and long-term residence of asbestos in the body may disrupt normal iron metabolism, leading to the deposition of iron onto asbestos which forms asbestos bodies. This iron has been proposed to come from ferritin or hemosiderin in macrophages (20). The iron coating may be variably segmented into spherical or rectangular units spaced along the fiber (21). The ends of coated fibers are frequently knobbed, although sheathlike coats are also common. Gloyne (22) originally described a series of different coat structures that he believed comprised a cycle of formation and dissolution of the asbestos body. More recently, Botham and Holt (23) have confirmed this in an animal model, observing that the earliest form of asbestos body has a sheathlike coat that with time becomes segmented. Results from studies on asbestos bodies, performed by Pooley (24), using transmission electron microscopy and X-ray diffraction, suggest that asbestos bodies are coated with a crystalline iron oxide material with a composition similar to the iron oxide core of ferritin, the iron storage protein. Later studies of the iron oxide present in the core of ferritin identified the iron core as ferrihydrite (25). However, a direct comparison between a ferrihydrite standard and the iron coating of asbestos bodies has not been made. In addition, the mechanisms by which iron binds to asbestos have not been previously determined, making it difficult to generate synthetic asbestos bodies for research studies in the laboratory. Ghio et al. (26, 27) investigated the effect of Fe(III) loading on the biological reactivities of asbestos fibers, as well as of other silicate mineral dusts. They observed that crocidolite and silicate mineral dusts bound iron, not only from various concentrations of FeCl3 up to 1 mM but also when implanted in rat lungs. They showed that the bound iron was responsible for the biological activities of asbestos and the silicate mineral dusts (26). They also observed more strand breaks in DNA exposed to crocidolite, amosite, or chrysotile asbestos, which had been incubated in a solution containing 1 mM FeCl3, than by exposure to the untreated fibers (27). Hardy and Aust (28) also reported more strand breaks in DNA exposed to Fe(II)-loaded crocidolite than by untreated crocidolite, and they observed 58% more iron mobilization by citrate in 24 h from the loaded crocidolite than from its untreated form. Lund et al. (29) observed that iron on amosite-core asbestos bodies, isolated from human lungs, was responsible for catalyzing single-strand breaks in DNA to a greater extent than for an equal number of uncoated amosite fibers of equal length. These results suggest that the iron deposited on amosite fibers in vivo may be reactive, potentially increasing the level of damage to biomolecules, such as DNA, above that of the uncoated fibers. It is evident from the studies briefly discussed above that iron bound to asbestos, either as a constituent of the crystal or added to its surface, may very likely be
Shen et al.
involved in the pathological effects of the fibers. The purpose of this study was to develop a method for depositing iron, Fe(II) or Fe(III), on asbestos in vitro that would produce iron deposits of the same form as observed on asbestos bodies removed from human lungs. The form of iron on the surface of fibers, loaded using the method described here, was compared with that on asbestos bodies recovered from human lung tissue. In this study, iron was bound to crocidolite or amosite asbestos after incubating the fibers in either FeCl2 or FeCl3 solutions for up to 14 days. Iron uptake was monitored via loss of iron from incubation solutions as well as with fiber surface compositional analysis, using X-ray photoelectron spectroscopy (XPS).1 Fiber surface topography was monitored using both atomic force spectroscopy (AFM) and scanning electron microscopy (SEM). Fibers prepared in vitro were compared with asbestos bodies recovered from the human lung tissue by micropowder X-ray diffraction (XRD) and Auger electron spectroscopy (AES). Results indicate that iron was deposited on asbestos fibers as ferrihydrite, the form of iron also observed on asbestos bodies removed from human lungs. Ultimately, work such as this may aid in understanding how asbestos bodies are formed in the lung after inhalation of the fibers and certainly will allow future work to study the reactivity of iron in the form found on asbestos bodies.
Materials and Methods Asbestos and Chemicals. Crocidolite and amosite asbestos fibers, containing 27.3 and 28.6% iron by weight, respectively (9), were obtained from R. Griesemer (NIEHS/NTP, Research Triangle Park, NC). Dry asbestos was handled in a Multihazard Glovebox (Labconco, Kansas City, MO) to prevent inhalation of the fibers. The average dimensions of crocidolite and amosite were 10 µm × 0.27 µm and 41 µm × 0.53 µm, respectively (9). The mean surface area was 10.1 m2/g for crocidolite and 3.8 m2/g for amosite (9). Amosite asbestos bodies were obtained from the lung tissue of a deceased 68-year-old insulator who had adenocarcinoma. The asbestos bodies were isolated as previously described (30). Briefly, the tissue, fixed in 10% formalin, was rinsed in deionized water, dehydrated overnight in 95% ethanol, rinsed a second time in water, and digested sequentially in 2% potassium permanganate, 8% oxalic acid, and then 9.2% sodium hypochlorite (Wright Bleach, Wright Inc., Ft. Worth, TX). The synthetic ferrihydrite sample (#S22-1) was a gift of D. G. Schulze from the Department of Agronomy at Purdue University (West Lafayette, IN). The iron chelator, 3-(2-pyridyl)5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid, monosodium salt monohydrate (ferrozine), was obtained from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals were purchased at the highest purity available. Preparation of Iron Solutions. The solutions of FeCl2 and FeCl3 were created at low (100 µM) and high (500 µM FeCl2 or 1 mM FeCl3) concentrations. The low concentrations were typical for iron binding experiments (28, 31), and the higher concentrations were used to obtain a greater level of iron binding so that better resolution could be achieved with the surface techniques used to quantify and characterize the surface iron. As will be addressed in the Results, the amount of Fe(II) loaded from a 500 µM FeCl2 solution was about the same as the amount of Fe(III) loaded from a 1 mM FeCl3 solution in a 2 h exposure period. These loading conditions were used to allow comparison of fibers loaded with equal amounts of either Fe(II) or Fe(III). 1 Abbreviations: XPS, X-ray photoelectron spectroscopy; AFM, atomic force microscopy; SEM, scanning electron microscopy; XRD, X-ray diffraction; AES, Auger electron spectroscopy; ddH2O, distilled, deionized water; SD, standard deviation.
Modeling of Asbestos Bodies Ferrous chloride solutions (100 or 500 µM) were made as follows. Distilled deionized water (ddH2O) was purged with N2 for at least 15 min before adding FeCl2 to prevent the oxidation of Fe(II) by O2. The solution was purged continuously with N2, while the pH of the FeCl2 solution was adjusted to 7.5 and until the binding studies were completed. Ferric chloride solutions (100 µM or 1 mM) were prepared and used under red lights to prevent photochemical reduction of Fe(III) (32). The pH of the ddH2O was adjusted to 3.5 before addition of FeCl3 to prevent the excessive formation of iron oxy/ hydroxides (33). To monitor the rate of formation of ferric oxy/hydroxides, 100 µM and 1 mM FeCl3 solutions were made in ddH2O, without pH preadjustment. Aliquots were removed at time intervals to determine the concentration of Fe(III), using the total iron assay (34). The concentration of the ferric oxy/hydroxide was calculated as the change in the Fe(III) concentration (micromolar) by subtracting the concentration at each time point from the starting concentration. All of the iron solutions were prepared immediately before use. Preparation of Iron-Loaded Asbestos. Crocidolite and amosite were treated with FeCl2 or FeCl3 solutions for shortterm (2 h) and long-term (14 days) loading. Short-term ironloaded fibers were incubated in the iron solutions for 2 h as follows. Crocidolite or amosite (1 mg/mL) was incubated in either FeCl2 (100 or 500 µM) or FeCl3 (100 µM or 1 mM) for 2 h at room temperature (25 °C). The FeCl2 suspensions of asbestos were purged with N2 throughout the experiment, and the FeCl3 suspensions were placed on a wrist-action shaker in the dark. Controls containing only the appropriate form of asbestos in ddH2O at pH 3.5 or 7.5 were incubated for identical times. At 1 and 2 h, aliquots were removed and centrifuged to remove the fibers. The concentration of Fe(III) in the supernatant was determined using the total iron assay (34), and the concentration of Fe(II) was determined using the modified total iron assay (28). The amount of iron removed from the solution was determined by calculating the difference between the concentration of Fe(II) or Fe(III) in the supernatant and the concentration of the corresponding starting solution. The amount of iron bound to the fibers was calculated using the amount of iron removed from solution and was expressed as nanomoles of iron per milligram of fibers. At the end of the 2 h incubation, the fibers were centrifuged at 3750 rpm for 20 min and washed three times with ddH2O. The fibers were then dried on a watch glass in the Multihazard Glovebox for at least 48 h, and then stored dry until they were used. Long-term iron-loaded fibers were exposed for 14 days to the appropriate iron solution for 2 h per day, followed by washing, as described in the previous paragraph. After a 2 h incubation each day, the suspension was centrifuged to remove the supernatant for iron concentration analysis and the fibers were exposed to the air until loading began again the next day. This was repeated each day for 14 days for a total of 28 h of exposure to iron-containing solutions. Long-term iron-loaded crocidolite was treated with either 500 µM FeCl2 or 1 mM FeCl3, and long-term iron-loaded amosite was treated with only 1 mM FeCl3. Determination of Iron Concentrations. The Fe(III) concentrations were determined using the total iron assay with ferrozine and ascorbate, as described previously (34). When it was necessary to determine only the amount of Fe(II), a modified total iron assay without ascorbate was used to determine the amount of the Fe(II)-ferrozine complex, which was determined spectrophotometrically at 562 nm with a Shimadzu UV 160U recording spectrophotometer (28). XPS Analysis. Near-surface chemical compositions of the crocidolite and amosite fibers used for the in vitro experiments were determined using XPS (35). The measurements, taken before and after iron loading, were performed with a PerkinElmer 5400 system. XPS was not performed on the asbestos bodies recovered from the human lung tissue because of the extremely small amounts available for study.
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 915 Samples for XPS analysis were mounted on a 1 in. diameter aluminum stub. A colloidal carbon in isopropyl alcohol suspension was used as the mounting cement. Non-monochromatic Al KR X-rays (1486.6 eV) were used to analyze a 3 mm2 area on the mat of the fibers. Due to the nature and objectives of the experiments in this study, the most important near-surface compositional variable is the Fe/Si ratio and its change before and after the loading experiments. To obtain this ratio, the intensities of the Si(2p) and Fe(3p) photolines were used for all analyses. These particular photolines were chosen because of their similar binding energies and, thus, similar kinetic energies. Using peaks with similar kinetic energies is important because the electrons that make up these peaks originate from a similar depth in the sample, typically on the order of a few to several nanometers. The intensities of the measured photolines were then divided by a photoionization cross section, in this case, derived by adjusting the photoline intensities of the untreated fibers to give the correct composition. This treatment results in an Fe/Si atomic concentration ratio. The Fe/Si ratios reported in this paper should be considered to be semiquantitative which is typical for carefully performed XPS analyses (35). The standard deviation (SD) of this type of measurement is about 0.03, as reported previously (36). The Fe/Si ratio is also useful because the XPS data can be directly compared from sample to sample even though the sampling volume analyzed in each run varies due to exact sample mounting and instrument conditions. AES Experiments. Auger measurements were performed on a Perkin-Elmer 610 scanning Auger microprobe using a primary electron beam energy of 5 kV. The beam current and angle of primary beam incidence were varied to eliminate charging that would ordinarily preclude collecting useable Auger spectra on an insulating surface (35). The Auger spectra in this study result in the assessment of surface composition to a depth of only approximately 2 nm, 2-5 times shallower than XPS analysis, but the results are more qualitative than XPS results. However, AES, with a lateral resolution of about 1 µm in this study, was particularly useful in that it allowed for the surface compositional analysis of individual fibers. This was important for the analysis of the asbestos bodies recovered from the human lung tissue, as far too few of these fibers were available for conventional XPS analysis. AFM Imaging. Both crocidolite and amosite fibers used in the in vitro experiments were examined before and after iron loading with a Digital Instruments Nanoscope IIIa atomic force microscope in air. All images in this study were collected in tapping mode using Si ultrasharp tips supplied by Digital Instruments. The sharp tips minimize edge artifacts in AFM imaging, although they are still clearly apparent when imaging very narrow cylindrical particles such as silicate mineral fibers. The fibers were deposited on muscovite sheets in methanol, providing a nearly perfectly flat support. Highly dispersed mounts were prepared so that surrounding fibers did not interfere with the fiber that was being imaged. No cement was used to secure the fibers. The residue from the evaporated methanol, together with the very small lateral forces imparted to the sample by tapping mode, allowed the fibers to remain stable while they were being imaged. SEM Studies. Secondary electron images were obtained using an ISI SX-40 scanning electron microscope. Samples were dispersed on stainless steel mounting plates in methanol. No cement was used. Samples were coated with approximately 100 Å (10 nm) of gold before being imaged. Images were collected with a primary beam energy of either 20 or 30 kV at magnifications up to 10000×. XRD Measurements. X-ray powder diffraction data were obtained using a Siemens D500 diffractometer with incidentand diffracted-beam Soller slits, a Kevex energy-dispersive Si(Li) detector, and Cu KR radiation generated at 45 kV and 35 mA. The instrument was calibrated with NIST SRM 640b Si powder. The Si(Li) detector used a 300 eV energy window to eliminate contributions from fluorescent and Kβ radiation. For
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Figure 1. Increase in the amount of undetectable Fe(III) in solution vs time in solutions prepared by dissolving FeCl3 in ddH2O with no pH adjustment. The concentration of Fe(III) at the indicated times was determined using the total iron assay. The concentration of ferric oxy/hydroxide that could not be detected by ferrozine binding in the total iron assay was calculated by subtracting the concentration of detectable Fe(III) at the indicated times from the original concentration that was prepared. The results are expressed as the amount of undetected Fe(III) vs time and represent the average of two experiments. The vertical bars associated with the data points represent the standard error of the mean. If no vertical bars are visible, this means that the bar is contained within the symbol. these samples, this energy discrimination was vital due to the strong fluorescence of Fe stimulated by Cu radiation. The asbestos body sample recovered from the human lung tissue, weighing just 0.37 mg, was mounted as a slurry in isopropyl alcohol on an off-axis cut single-crystal quartz plate. Use of this “zero-background” mount eliminates any contribution from the sample mount to the diffraction pattern, a crucial step in the analysis of very small samples. Data for the lung tissue sample were obtained by step scanning from 2 to 70° for 2θ, counting for 245 s every 0.02° for 2θ. The long-term Fe(III)loaded amosite and crocidolite samples were mounted, after gently crushing under acetone, in cavities machined in Ti plates. Data for these samples were obtained by step scanning from 2 to 70° for 2θ, counting for 65 s every 0.02° for 2θ.
Results Rate of Formation of Ferric Oxy/Hydroxides in FeCl3 Solutions. Ferric chloride solutions, made without preadjusting the pH of ddH2O to 3.5 before dissolution of FeCl3, showed a dramatic decrease in detectable Fe(III) concentrations over the 2 h incubation, using the ferrozine total iron assay. Centrifugation of the solutions did not result in any visible precipitation from 100 µM FeCl3, suggesting that the Fe(III) was still in solution, just not in a form that could react with ferrozine in the total iron assay. However, with 1 mM FeCl3, there was sometimes a precipitate observed after centrifugation. As shown in Figure 1, more than 90% of the Fe(III) in 100 µM FeCl3 was undetected using the total iron assay at the end of the 2 h period, whereas about 35% of the Fe(III) in 1 mM FeCl3 was undetected (Figure 1). When the pH of the ddH2O was adjusted to 3.5 prior to dissolution of the FeCl3, the total amount of iron detected in the solution remained constant for the 2 h period. These results suggest that ferric oxy/hydroxides were formed in the solutions when the pH of ddH2O was not preadjusted and that these forms of iron could not be detected by the total iron assay. Thus, the subsequent Fe(III) loading experiments were carried out with Fe(III) solutions made by preadjusting the pH of ddH2O to 3.5 prior to FeCl3 dissolution. Although the formation of ferric oxy/
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Figure 2. Fe(II) binding to asbestos. Asbestos (1 mg/mL) was suspended in either 100 or 500 µM FeCl2 (pH 3.5) and incubated at room temperature for 2 h. At the indicated times, an aliquot was centrifuged to remove the fibers. The supernatant was assayed for Fe(II) concentration using the modified total iron assay. The amount of Fe(II) bound was calculated by subtracting the amount of Fe(II) at the indicated times from the amount of Fe(II) in the original solution. The results are presented as the mean ( SD (n ) 3). The absence of standard deviation bars indicates that the SD is contained within the symbol.
Figure 3. Fe(III) binding to asbestos. Asbestos (1 mg/mL) was suspended in either 100 µM or 1 mM FeCl3 (pH 3.5) and incubated at room temperature for 2 h. At the indicated times, an aliquot was centrifuged to remove the fibers. The supernatant was assayed for Fe(III) concentration using the total iron assay. The amount of Fe(III) bound was calculated by subtracting the amount of Fe(III) at the indicated times from the amount of Fe(III) in the original solution. The results are presented as the mean ( SD (n ) 3). The absence of standard deviation bars indicates that the SD is contained within the symbol.
hydroxides begins as low as pH 1 (33), it is apparent that ferric iron, made in ddH2O preadjusted to pH 3.5, is detectable. Therefore, this ensured that the iron remained in a detectable form for Fe(III) loading. Iron Binding to Asbestos. As shown in Figure 2, more Fe(II) was bound from solution by crocidolite than by amosite in both 100 and 500 µM FeCl2 solutions in the short-term loading. The amount of Fe(II) bound to crocidolite was dependent upon the concentration of Fe(II) in the incubation solution. Amosite was not able to bind Fe(II) well, and the binding was independent of the FeCl2 concentration (Figure 2). If corrections are made for the surface areas of the fibers (9), more Fe(II) was bound by crocidolite [6.7 × 10-10 and 2.1 × 10-9 mol of Fe(II)/cm2 of fibers in 100 and 500 µM FeCl2, respectively] than by amosite [6.3 × 10-10 and 5.8 × 10-10 mol of Fe(II)/cm2 of fibers, respectively]. In contrast, Fe(III) binding from solution in the 2 h incubation period was more similar for crocidolite and amosite on a weight basis (Figure 3). More Fe(III) was bound by crocidolite than by amosite throughout the 2 h period for both 100 µM and 1 mM
Modeling of Asbestos Bodies
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Figure 4. Long-term iron binding on asbestos fibers. Fibers (1 mg/mL) were incubated in either 1 mM FeCl3 or 500 µM FeCl2 solutions (pH 3.5) at room temperature for 2 h a day, consecutively for 14 days for a total exposure of 28 h, as described in Materials and Methods. Each day, the supernatant removed from the centrifuged suspension was analyzed for iron concentration. The data are presented as the mean ( SD (n ) 3). The absence of standard deviation bars indicates that the SD is contained within the symbol.
FeCl3 incubations. However, if surface area was taken into account, almost twice as much Fe(III) was bound by amosite [7.4 × 10-10 and 3.7 × 10-9 mol of Fe(III)/cm2 of fibers in 100 µM and 1 mM FeCl3, respectively] than by crocidolite [4.8 × 10-10 and 2.0 × 10-9 mol of Fe(III)/cm2 of fibers, respectively]. At the lower concentration (100 µM FeCl3), the extent of binding appeared to reach a plateau, whereas at the higher concentration (1 mM FeCl3), the extent of binding increased with incubation time (Figure 3). The amount of Fe(III) bound to crocidolite from the 1 mM FeCl3 solution was very similar to the amount of Fe(II) bound from the 500 µM FeCl2 solution (Figures 2 and 3). Solutions containing only FeCl2 at 100 and 500 µM or FeCl3 at 100 µM and 1 mM showed no change in the iron concentrations over the 2 h incubation period (data not shown). Control experiments with the fibers incubated in pH 7.5 ddH2O did not show any iron mobilization from the fibers, while controls at pH 3.5 showed a very small amount of iron mobilized (
