The Potential Contribution of Hexavalent Chromium to the Carcinogenicity of Chrysotile Asbestos

Abstract

Chrysotile asbestos is a carcinogenic mineral that has abundantly been used in industrial and consumer applications. The carcinogenicity of the fibers is partly governed by reactive Fe surface sites that catalyze the generation of highly toxic hydroxyl radicals (HO^•) from extracellular hydrogen peroxide (H₂O₂). Chrysotile also contains Cr, typically in the low mass permille range. In this study, we examined the leaching of Cr from fibers at the physiological lung pH of 7.4 in the presence and absence of H₂O₂. Furthermore, we investigated the potential of cells from typical asbestos-burdened tissues and cancers to take up Cr leached from chrysotile in PCR expression, immunoblot, and cellular Cr uptake experiments. Finally, the contribution of Cr to fiber-mediated H₂O₂ decomposition and HO^• generation was studied. Chromium readily dissolved from chrysotile fibers in its genotoxic and carcinogenic hexavalent redox state upon oxidation by H₂O₂. Lung epithelial, mesothelial, lung carcinoma, and mesothelioma cells expressed membrane-bound Cr(VI) transporters and accumulated Cr up to 10-fold relative to the Cr(VI) concentration in the spiked medium. Conversely, anion transporter inhibitors decreased cellular Cr(VI) uptake up to 45-fold. Finally, chromium associated with chrysotile neither decomposed H₂O₂ nor contributed to fiber-mediated HO^• generation. Altogether, our results support the hypothesis that Cr may leach from inhaled chrysotile in its hexavalent state and subsequently accumulate in cells of typically asbestos-burdened tissues, which could contribute to the carcinogenicity of chrysotile fibers. However, unlike Fe, Cr did not significantly contribute to the adverse radical production of chrysotile.

Figure 1

Cr concentrations (in μmol L^–1) mobilized from 1 g L^–1 pristine fibers at pH 7.4 by 1 mmol L^–1 of the synthetic chelators DTPA and EDTA and the siderophore DFOB and in the absence of ligands (blank). Error bars indicate standard deviations (n = 2). Data presented in this figure are reported in Table S2.

Figure 2

Cr concentrations (in μmol L^–1) mobilized from 1 g L^–1 pristine (panel a), blank-altered (panel b), and DFOB-altered chrysotile fibers (panel c) as a function of time at pH 7.4, either in the presence or absence of 3.3 g L^–1 H₂O₂ (∼100 mmol L^–1) (starting concentration). Error bars indicate standard deviations (n = 2). Data presented in this figure are reported in Table S3.

Figure 3

Cr redox speciation of selected samples from the leaching experiments (all at an ionic strength of 300 mmol L^–1) determined by LC-ICP-MS. For the chromatographic conditions, see the experimental section. The Cr(III) retention time was <4 min, whereas the Cr(VI) retention time was >5 min. (a) Cr(III) (left peak) and Cr(VI) (right peak) standard, both at 50 ppb, used as upper limit for the calibration of the LC-ICP-MS speciation method. (b) Leachate collected after incubation of 1 g L^–1 chrysotile at pH 7.4 for 168 h at an initial H₂O₂ concentration of 3.3 g L^–1 (∼100 mmol L^–1) (sample was diluted 10 times). (c) Leachate collected after incubation of 1 g L^–1 chrysotile at pH 3.0 for 168 h (sample was diluted 20 times). The corresponding Cr redox species concentrations in the leachates for which the chromatograms are presented in panels (b) and (c) are reported in Table S4; full chromatograms up to approximately 15 min retention time are presented in Figure S7.

Figure 4

(a) Expression of SLC4A1 and SLC26A1 in mesothelial (MeT-5A, NP1, NP2), mesothelioma (P31, MM05, SPC212, VMC23, VMC40), lung epithelial (BEAS-2B), and lung carcinoma (A549) cells assessed by qPCR. All expression values were calculated as 2^–dCT × 10⁶ relative to two housekeeping genes (β-actin and GAPDH). Medians for each category (horizontal lines) and expression levels of individual cell lines are shown. (b) Expression levels of SLC4A1 and SLC26A1 (two oligos, v1 and v1) in a panel of mesothelial (n = 2) and mesothelioma (n = 35) cells extracted from Agilent 44 K microarray data, shown as raw hybridization signal. Horizontal lines indicate mean values of the presented data. (c) Immunoblots of the SLC4A1 and SLC26A1 proteins in MeT-5A, P31, BEAS-2B, and A549 cells. β-actin was used as loading control. (d) Cr(VI) uptake in MeT-5A, P31, BEAS-2B, and A549 cells when either 0.2, 2, 20, 200, and 2000 μmol L^–1 Cr(VI) were spiked into the cell incubation media. Mean background protein normalized intracellular Cr concentrations ranged from 18 (P31) to 57 (MeT-5A) nmol g^–1 (Table S7). (e) Cr(VI) uptake in MeT-5A, P31, BEAS-2B, and A549 cells in the presence and absence of 200 μmol L^–1 of the anion transporter inhibitor DIDS when the cell incubation media had initially been spiked with 2 μmol L^–1 of Cr(VI)). (f) Accumulation of Cr(VI) in the intracellular compartment of MeT-5A, P31, BEAS-2B, and A549 cells as compared to measured Cr(VI) concentrations in the cell media alone (”Medium” replicates) when 0.2, 2, 20, 200, and 200 μmol L^–1 Cr(VI) were spiked into the cell media. Measured intracellular Cr contents [μmol L^–1] in panels (d) and (e) were normalized to the cellular protein content [g L^–1]. Data to this Figure are presented in Table S7 (the plotted data) and Table S1 (list of used cell lines, their type and source). **p < 0.005, ***p < 0.001 DIDS versus no DIDS. Error bars in panels (d) and (e) represent standard deviations (n = 3–5).

Figure 5

(a) H₂O₂ degradation at pH 7.4 in the absence of fibers (“MOPS buffer”), by pristine fibers and by fibers that had been preconditioned for 336 h at pH 7.4 in the presence of 1 mmol L^–1 EDTA, DTPA or DFOB. The starting H₂O₂ concentration was 3.3 g L^–1 (∼100 mmol L^–1). The data were collected in two separate experiments; hence, for both experiments, the MOPS control treatment is reported (the MOPS (DTPA & EDTA) and MOPS (DFOB & pristine) columns). (b) Hydroxyl radical generation by fibers that were preconditioned in the absence of ligands or with DTPA, EDTA, and DFOB. A hydroxyl radical yield of 100% corresponds to the radical generation by pristine fibers (no preconditioning). Error bars indicate standard deviations (n = 2 in panel (a) and n = 4 in panel (b)). Data from the pristine fiber and DFOB treatment and the corresponding MOPS buffer control in panel (a) were taken from Walter et al. Data presented in this figure is reported in Table S8.