Undetectable role of oxidative DNA damage in cell cycle, cytotoxic and clastogenic effects of Cr(VI) in human lung cells with restored ascorbate levels

Abstract

Cultured human cells are invaluable biological models for mechanistic studies of genotoxic chemicals and drugs. Continuing replacement of animals in toxicity testing will further increase the importance of in vitro cell systems, which should accurately reproduce key in vivo characteristics of toxicants such as their profiles of metabolites and DNA lesions. In this work, we examined how a common severe deficiency of cultured cells in ascorbate (Asc) impacts the formation of oxidative DNA damage by hexavalent chromium (chromate). Cr(VI) is reductively activated inside the cells by both Asc and small thiols but with different rates and spectra of intermediates and DNA adducts. We found that Cr(VI) exposure of H460 human lung epithelial cells in standard culture (<0.01 mM cellular Asc) induced biologically significant amounts of oxidative DNA damage. Inhibition of oxidative damage repair in these cells by stable XRCC1 knockdown strongly enhanced cytotoxic effects of Cr(VI) and led to depletion of cells from G(1) and accumulation in S and G(2) phases. However, restoration of physiological levels of Asc (≈ 1 mM) completely eliminated Cr(VI) hypersensitivity of XRCC1 knockdown. The induction of chromosomal breaks assayed by the micronucleus test in Asc-restored H460, primary human lung fibroblasts, and CHO cells was also unaffected by the XRCC1 status. Centromere-negative (clastogenic) micronuclei accounted for 80-90% of all Cr(VI)-induced micronuclei. Consistent with the micronuclei results, Asc-restored cells also showed no increase in the levels of poly(ADP-ribose), which is a biochemical marker of single-stranded breaks. Asc had no effect on cytotoxicity of O(6)-methylguanine, a lesion produced by direct DNA alkylation. Overall, our results indicate that the presence of physiological levels of Asc strongly suppresses pro-oxidant pathways in Cr(VI) metabolism and that the use of standard cell cultures creates a distorted profile of its genotoxic properties.

Fig. 1

Characterisation of Asc-restored H460 cells. (A) Asc concentrations in control and 2 mM DHA-treated H460 cells. (B) GSH levels in control and Asc-restored H460 cells. (C) Clonogenic toxicity of hydrogen peroxide. (D) Clonogenic toxicity of MNNG. Cells were pre-incubated for 1 h with 10 μM O⁶-benzylguanine prior to the addition of MNNG. (E) Cr(VI) uptake by control and Asc-restored H460 cells. Cr(VI) treatments were for 3 h. When not visible, error bars were smaller than the graph symbol. Statistics: *P < 0.05.

Fig. 2

Validation of XRCC1 deficiency in H460 cells. (A) Western blot demonstrating efficiency of XRCC1 knockdowns in three independently infected H460 populations. (B) Increased clonogenic toxicity of hydrogen peroxide in H460 cells with XRCC1 knockdown. Statistics: *P < 0.05, ***P < 0.001.

Fig. 3

Cell cycle effects of Cr(VI) in XRCC1-depleted H460 cells. Cells were treated with Cr(VI) for 3 h and cell cycle distribution was determined 24 h later. Statistics: *P < 0.05, **P < 0.01. (A) Changes in percentage of cells among cell cycle phases in standard H460 cultures. (B) Changes in cell cycle phases in Asc-restored H460 cells.

Fig. 4

Effects of restored Asc levels on cytotoxicity by Cr(VI). (A) Clonogenic toxicity of Cr(VI) in control (shLuc) and XRCC1-depleted (shXRCC1) H460 cells containing low Asc levels (standard tissue culture conditions). Statistics: *P < 0.05, **P < 0.01. (B) Clonogenic toxicity of Cr(VI) in Asc-restored H460 cells. (C) Western blot demonstrating PARP cleavage in Asc-restored H460 cells expressing nonspecific (shLuc) and targeting (shXRCC1) shRNA. Cells were treated with Cr(VI) for 3 h and protein extracts from combined attached and floating cells were prepared 24 h later. (D) Clonogenic toxicity of Cr(VI) in XRCC1-null and XRCC1-complemented CHO-EM9 cells. Both cell lines were pre-incubated with 2 mM DHA prior to Cr(VI) exposure, which resulted in 1.4 mM Asc concentrations inside the cells.

Fig. 5

Formation of micronuclei and PAR by Cr(VI) in Asc-restored cells. Data are from 3 (EM9 cells) or 6 slides (H460, IMR90) with at least 500 cells scored for each slide. Shown is the frequency of cells containing micronuclei. (A) Frequency of centromere-positive (aneugenic) and -negative (clastogenic) micronuclei in human H460 cells expressing control (shLuc) and targeting (shXRCC1) shRNA. (B) Micronuclei in XRCC1-null and XRCC1-expressing CHO-EM9 cells with restored Asc levels to 1.4 mM. (C) Western blot demonstrating XRCC1 knockdown in IMR90 primary human lung fibroblasts. For XRCC1-tageting shRNA, extracts from two independently infected populations are shown. (D) Micronuclei in IMR90 cells expressing nonspecific and XRCC1-targeting shRNA. Asc levels in IMR90 cells were restored to 1.2 mM. (E) Western blot for PAR-modified proteins in Asc-restored H460 cells. Cells were treated for 1 h with Cr(VI) and 30 min with 100 μM H₂O₂. (F) Absence of PAR formation in Asc-restored XRCC1−/− and XRCC1+ CHO cells treated with Cr(VI) for 1 h. PAR, poly(ADP-ribose); NS, nonspecific band.