Reductive activation of hexavalent chromium by human lung epithelial cells: generation of Cr(V) and Cr(V)-thiol species

Abstract

Chromium(VI) compounds (e.g. chromates) are cytotoxic, mutagenic, and potentially carcinogenic. The reduction of Cr(VI) can yield reactive intermediates such as Cr(V) and reactive oxygen species. Bronchial epithelial cells are the primary site of pulmonary exposure to inhaled Cr(VI) and are the primary cells from which Cr(VI)-associated human cancers arise. BEAS-2B cells were used here as a model of normal human bronchial epithelium for studies on the reductive activation of Cr(VI). Cells incubated with Na(2)CrO(4) exhibited two Cr(V) ESR signals, g=1.979 and 1.985, which persisted for at least 1h. The g=1.979 signal is similar to that generated in vitro by human microsomes and by proteoliposomes containing P450 reductase and cytochrome b(5). Unlike many cells in culture, these cells continued to express P450 reductase and cytochrome b(5). Studies with the non-selective thiol oxidant diamide indicated that the g=1.985 signal was thiol-dependent whereas the g=1.979 signal was not. Pretreatment with phenazine methosulfate eliminated both Cr(V) signals suggesting that Cr(V) generation is largely NAD(P)H-dependent. ESR spectra indicated that a portion of the Cr(VI) was rapidly reduced to Cr(III). Cells incubated with an insoluble chromate, ZnCrO(4), also generated both Cr(V) signals, whereas Cr(V) was not detected with insoluble PbCrO(4). In clonogenic assays, the cells were very sensitive to Na(2)CrO(4) and ZnCrO(4), but considerably less sensitive to PbCrO(4).

Fig. 1

DMEM-HEPES medium incubated with 0.4 mM Na₂CrO₄ at 37°C under room air generates Cr(V) (left), whereas LHC-9 medium does not (right). The times of incubation with Na₂CrO₄ are indicated. Representative ESR spectra obtained at room temperature are shown. Instrument settings were: 5 G modulation amplitude, 50 mW microwave power, 6.32 × 10⁴ receiver gain, 40.96 msec time constant, 9.76 GHz microwave frequency, sweep width = 200 G, field set = 3510 G, modulation frequency = 100 kHz, scan time = 42 sec; number of scans, 9.

Fig. 2

LHC-9-grown BEAS-2B cells incubated with Na₂CrO₄ for 5 min at 37°C in LHC-9 medium under room air generate Cr(V). Representative ESR spectra (obtained at room temperature) for Cr(V) are shown for: (A) 400 µM Na₂CrO₄ plus 1 × 10⁷ cells.; (B) 400 µM Na₂CrO₄ plus 4.1 × 10⁶ cells; (C) 200 µM Na₂CrO₄ plus 1 × 10⁷ cells. For each, the total reaction volume was 0.25 ml. Instrument settings were the same as for Fig. 1.

Fig. 3

Representative Cr(V) signals from LHC-9-grown BEAS-2B cells exposed under room air to Na₂CrO₄ for 5 min at 37°C in LHC-9 medium. Spectra were recorded at room temperature. (A) 400 µM Na₂CrO₄ plus 4.1 × 10⁶ cells, 9 scans. (B) 400 µM isotopically pure Na₂⁵³CrO₄ plus 4.9 × 10⁶ cells, 9 scans. (C) The same sample as B except 36 scans were collected. The reaction volume and instrument settings were the same as those in Fig. 1.

Fig. 4

Representative ESR spectra of Cr(V) obtained following incubation of LHC-9-grown BEAS-2B cells with Na₂CrO₄. Cells were grown in LHC-9 medium, harvested by scraping, washed in pre-warmed LHC-9 medium, and resuspended in a small volume of LHC-9. Aliquots of the cell suspension containing the indicated number of cells were incubated with Na₂CrO₄ for the indicated times at 37°C. For each, the total reaction volume was 0.25 ml. Instrument settings were the same as for Fig. 1.

Fig. 5

Representative ESR spectra of Cr(V) obtained following incubation of DMEM-grown BEAS-2B cells with Na₂CrO₄. Cells were grown in DMEM-HEPES medium with 10% FBS, harvested by scraping, washed in pre-warmed LHC-9 medium, and resuspended in a small volume of LHC-9. Aliquots of the cell suspension containing the indicated number of cells were incubated with Na₂CrO₄ for the indicated times at 37°C. Controls without cells are shown at right. For each, the total reaction volume was 0.25 ml. Instrument settings were the same as for Fig. 1.

Fig. 6

Representative Cr(V) signals from BEAS-2B cells exposed to 0.4 mM Na₂CrO₄ for 5 min at 37°C in LHC-9 medium under room air. Cells were grown in DMEM-HEPES medium with 10% FBS, harvested by scraping, washed in pre-warmed LHC-9 medium, and resuspended in a small volume of LHC-9 for incubation with Na₂CrO₄. Reactions contained 3 × 10⁶ cells (A, B, C), or 5 × 10⁶ cells (D). Prior to harvesting and washing in LHC-9, the cells in B were pretreated with 0.25 mM diamide for 30 min, and those in D with 0.1 mM PMS. For each, the total reaction volume was 0.25 ml and instrument settings were the same as in Fig. 1 except that the field set was 3480 G.

Fig. 7

Relative levels of GSH in cells exposed to various chemicals. Cells were either untreated or pre-exposed as indicated to: diamide (0.25 mM for 30 min), or Cr(VI) as Na₂CrO₄ (100 µM for 30 min, 5 µM for 24 h, or 400 µM for 10 min). **, P <0.01 vs. the other treatments.

Fig. 8

Hyperfine structure of the g = 1.979 Cr(V) ESR signal. The "Cells" spectrum was obtained following 5 min incubation of 10⁷ cells at 37°C in 0.25 ml of LHC-medium containing 800 µM Na₂CrO₄. The "NADPH" spectrum was obtained following 30 min incubation of 5 mM NADPH plus 5 mM Na₂CrO₄ at 37°C in 0.3 ml 43.8 mM potassium phosphate buffer (pH 7.4). Instrument settings were: 0.1 G modulation amplitude, 50 mW microwave power, 6.32 × 10⁴ receiver gain, 327.68 msec time constant, 9.764 GHz microwave frequency, sweep width = 20 G, field set = 3516 G, modulation frequency = 100 kHz, scan time = 83.89 sec; conversion time = 81.92 msec. The number of scans was 5 and 50 for the "NADPH" and "Cells" samples, respectively. Note the different y-axis intensity scales for the two samples. The signals intensities were divided by 1000 to obtain the values on the graphs.

Fig. 9

ESR spectra of samples that were incubated and then frozen and analyzed by ESR at 6.3 K. "Cells" (top): BEAS-2B cells were incubated with 400 µM Na₂CrO₄ for 5 min at 37°C prior to freezing. "His" (middle): 10 mM histidine plus 1.25 mM Cr(III) (as CrCl₃) were incubated in 0.5 M HEPES buffer (pH 7.4) at room temperature for 96 h prior to freezing. "GSH" (bottom): 100 mM GSH plus 1.25 mM Cr(III) were incubated in 0.5 M HEPES buffer (pH 7.4) at room temperature for 72 h prior to freezing. Instrument settings were: temperature = 6.3 K, 5 G modulation amplitude, 0.63 mW microwave power, 60 dB receiver gain, 82 msec time constant, 9.633 GHz microwave frequency, scan time = 83 sec; number of scans, 9.

Fig. 10

ESR spectra of BEAS-2B cells incubated with Na₂CrO₄ (A, D), ZnCrO₄ (B, E), or PbCrO₄ (C, F) for 5 min (A, B, C) or 1 h (D, E, F) at 37°C in LHC-9 medium under room air. For each, the initial chromate concentration was 0.4 mM, and the cell density was 5 × 10⁶ cells in a total reaction volume of 0.25 ml. Instrument settings were as for Fig. 1 except that the field set was 3480 G.

Fig. 11

Relative survival/viability of BEAS-2B cells exposed for 24 h in LHC-9 medium to different concentrations of Na₂CrO₄ (A), ZnCrO₄ (B), or PbCrO₄ (C), determined by a clonogenic assay and trypan blue exclusion. Results are the means ± SD, n = 3 experiments.

Fig. 12

Immunofluorescent detection of the proteins P450 reductase (P450red), cytochrome b₅ (b₅), and b₅ reductase (B₅R) using antibodies specific for each protein, and a fluorescein conjugated secondary antibody. The control was treated identically except that only a secondary antibody was applied (no primary antibody).

Fig. 13

Representative Cr(V) signals following incubation of 400 µM Na₂CrO₄ with 1 mM GSH for 5 min in 10 mM potassium phosphate pH 7.4 under room air. The reaction volume was 0.3 ml and the instrument settings were the same as those in Fig. 1.

Fig. 14

General scheme for the formation of the Cr species responsible for the ESR signals observed in BEAS-2B cells. The species associated with each signal are in boldface type. The dashed arrows indicate possible pathways to the formation of certain species, but the exact pathways are not known. The diagram is not meant to be inclusive of all possible pathways or Cr species, but rather is intended to relate the various species observed in these cells.