The intracellular redox stress caused by hexavalent chromium is selective for proteins that have key roles in cell survival and thiol redox control

Abstract

Hexavalent chromium [Cr(VI)] compounds (e.g. chromates) are strong oxidants that readily enter cells where they are reduced to reactive Cr intermediates that can directly oxidize some cell components and can promote the generation of reactive oxygen and nitrogen species. Inhalation is a major route of exposure which directly exposes the bronchial epithelium. Previous studies with non-cancerous human bronchial epithelial cells (BEAS-2B) demonstrated that Cr(VI) treatment results in the irreversible inhibition of thioredoxin reductase (TrxR) and the oxidation of thioredoxins (Trx) and peroxiredoxins (Prx). The mitochondrial Trx/Prx system is somewhat more sensitive to Cr(VI) than the cytosolic Trx/Prx system, and other redox-sensitive mitochondrial functions are subsequently affected including electron transport complexes I and II. Studies reported here show that Cr(VI) does not cause indiscriminant thiol oxidation, and that the Trx/Prx system is among the most sensitive of cellular protein thiols. Trx/Prx oxidation is not unique to BEAS-2B cells, as it was also observed in primary human bronchial epithelial cells. Increasing the intracellular levels of ascorbate, an endogenous Cr(VI) reductant, did not alter the effects on TrxR, Trx, or Prx. The peroxynitrite scavenger MnTBAP did not protect TrxR, Trx, Prx, or the electron transport chain from the effects of Cr(VI), implying that peroxynitrite is not required for these effects. Nitration of tyrosine residues of TrxR was not observed following Cr(VI) treatment, further ruling out peroxynitrite as a significant contributor to the irreversible inhibition of TrxR. Cr(VI) treatments that disrupt the TrxR/Trx/Prx system did not cause detectable mitochondrial DNA damage. Overall, the redox stress that results from Cr(VI) exposure shows selectivity for key proteins which are known to be important for redox signaling, antioxidant defense, and cell survival.

Fig. 1

Representative 2D electrophoresis of oxidized protein thiols in untreated (left) vs. Cr(VI)-treated (25 µM, 90 min) (right) BEAS-2B cells. M = marker lane at left of each gel. An expanded view of the regions containing Trx and Prx are shown at the bottom. Increased fluorescence following Cr(VI) treatment of the cells indicates greater oxidation of that protein (the 6 circled spots). After image capture, the gels were transferred to nitrocellulose and sequentially probed with different antibodies. Spots 4, 5, and 6 exactly correspond to the positions of Prx3, Trx1, and Trx2, respectively. Spots 1–3 have are have not been identified.

Fig. 2

GAPDH activity (mean ± S.D., n = 3) in BEAS-2B cells treated with Cr(VI) for 90 min. One-way ANOVA indicated that the values are not significantly different (p > 0.05). There was no change in GAPDH protein level by western blot (not shown).

Fig. 3

Representative redox western blots examining the Cr(VI)-mediated oxidation of Trx1, Trx2, and Prx3 in NHBE cells. A, redox status of cytosolic Trx1 after 6hr Cr(VI) treatment, and of mitochondrial Trx2 after 3 and 6 hr of treatment. For Trx2, the lanes marked DTT are isolated mitochondria that had been pre-treated with DTT which reduces Trx2 (Myers et al. 2008); these serve as a reference for the migration of reduced Trx2. B, redox status of mitochondrial Prx3 after 3 hr and 6 hr of treatment.

Fig. 4

The effects of pre-loading BEAS-2B cells with 1 mM DHA for 90 min vs. vehicle control. analyzed for TrxR specific activity (A), or by redox western blots for Trx2 (B) or Prx3 (C). Data are the mean ± S.D. for independent experiments (n = 5 for TrxR activity, n = 4 for Trx2 and Prx3). For all assays, two-way ANOVA indicates that there were no significant differences between the presence and absence of DHA at each Cr(VI) concentration (p > 0.05).

Fig. 5

MnTBAP does not protect the TrxR/Trx system from Cr(VI) exposure. BEAS-2B cells were pre-treated with 0.2 mM MnTBAP or vehicle for 2 hr and then Cr(VI) was added at the indicated concentrations for 3 hr. The cells were analyzed for TrxR specific activity (A), or by redox western blots for Trx1 (B) Prx1 (C), or Trx2. Data are the mean ± S.D. for independent experiments (n = 4 for TrxR activity except n = 3 for 25 µM; n = 5 for Trx1; n = 3 for Prx1 and Trx2). For all assays, two-way ANOVA indicates that there were no significant differences between the presence and absence of MnTBAP at each Cr(VI) concentration (p > 0.05).

Fig. 6

Representative EPR spectra of BEAS-2B cells showing that MnTBAP does not protect mitochondrial electron transport centers from Cr(VI) treatment. Cells were pre-treated with 0.2 mM MnTBAP (c, d) or vehicle (a, b) for 2 hr, and then 25 µM Cr(VI) was added for 3 hr, after which the cells were washed and harvested as described in the Methods. The final cell suspension (ca. 8 × 10⁶ cells in 0.3 ml HBSS in a 4-mm quartz EPR tube) was immediately frozen in liquid nitrogen. Each sample was analyzed at liquid helium temperature (10 K), and each spectrum was corrected for background. The samples were analyzed using a microwave power of 20 mW (a, c), or 5 mW (b, d). Other instrument settings were: 5 G modulation amplitude, 60 dB receiver gain, 82 msec time constant, 9.633 GHz microwave frequency, modulation frequency = 100 kHz, scan time = 83.9 sec; number of scans, 9.

Fig. 7

Representative western blot to determine if TrxR nitration occurs as a result of Cr(VI) treatment. BEAS-2B cells were treated for 3 hr with the indicated concentrations of Cr(VI). The cells (ca. 1 ×10⁷) were washed, harvested, and lysed and TrxR was immunoprecipitated from the lysates using anti-TrxR and protein A/G agarose as described in the methods. The immunoprecipitates were run on SDS-PAGE. The lanes that were probed with anti-nitrotyrosine (top) were loaded with 10-fold more of the immunoprecipitate than those that were probed with anti-TrxR (bottom). The top gel also includes nitrated BSA (1 µg) as a positive control, and BSA as a negative control.

Fig. 8

Cr(VI) treatment does not cause detectable DNA damage in the mitochondria or nucleus of BEAS-2B cells. Representative PCR of total DNA using primers that amplify 16.2 kb of mitochondrial DNA (A), or a 13.5-kb region of nuclear DNA encoding the β-globin gene (B). Total DNA was isolated from cells that had been treated with the indicated concentrations of Cr(VI) for 3 hr. The relative amount of DNA template that was used in each PCR reaction is indicated. The leftmost lane in each panel shows a decreased template control with 50% less (A) or 75% less (B) DNA template from control cells used for the PCR reaction.

Fig. 9

Representative western blots of whole cell lysates showing Txnip levels in NHBE or BEAS-2B cells treated for 3 or 6 hr with 0, 25, or 50 µM Cr(VI) as indicated. There was no change in GAPDH protein level in these cells as shown by western blot (not shown).