Homologous Recombination and Translesion DNA Synthesis Play Critical Roles on Tolerating DNA Damage Caused by Trace Levels of Hexavalent Chromium

Abstract

Contamination of potentially carcinogenic hexavalent chromium (Cr(VI)) in the drinking water is a major public health concern worldwide. However, little information is available regarding the biological effects of a nanomoler amount of Cr(VI). Here, we investigated the genotoxic effects of Cr(VI) at nanomoler levels and their repair pathways. We found that DNA damage response analyzed based on differential toxicity of isogenic cells deficient in various DNA repair proteins is observed after a three-day incubation with K2CrO4 in REV1-deficient DT40 cells at 19.2 μg/L or higher as well as in TK6 cells deficient in polymerase delta subunit 3 (POLD3) at 9.8 μg/L or higher. The genotoxicity of Cr(VI) decreased ~3000 times when the incubation time was reduced from three days to ten minutes. TK mutation rate also significantly decreased from 6 day to 1 day exposure to Cr(VI). The DNA damage response analysis suggest that DNA repair pathways, including the homologous recombination and REV1- and POLD3-mediated error-prone translesion synthesis pathways, are critical for the cells to tolerate to DNA damage caused by trace amount of Cr(VI).

Fig 1. K₂CrO₄ reduces viability of human cells as well as DT40 cells deficient in certain DNA repair pathways at the maximum contaminant levels (MCL) set by the U.S. EPA.

Various human cells (A) and DT40 parental cells and their mutant cells (B) deficient in REV1, BRCA1, or RAD54 were exposed to K₂CrO₄ for ~3 days to determine their survival rates. Survival data were log-transformed giving approximate normality using Prism 5. Each LC₅₀ value was then calculated. The black arrow indicates the maximum contaminant levels (MCL) set by the U.S. EPA, while the open arrow shows the highest Cr(VI) levels detected in U.S. city water (12.9 μg/L). (C) Point-of-departure analysis showed a significant difference (p<0.0001) in DT40 (log10(BMD) = 1.98 by the Hill model) vs. REV1-deficient cells (log10(BMD) = 1.28 (19.2 μg/L) by the Hill model). (D) DT40 cell-based DNA damage response analysis was performed for K₂CrO₄ using a series of DT40 mutants. Relative LC₅₀ values were normalized according to the LC₅₀ value of the parental (wild type) DT40 cells. Error bars represent standard deviation from at least three independent experiments. Student’s t-test was used to test for significance in mean LC₅₀ values between DT40 parental cells and each mutant. Columns shaded in gray indicate significant differences (p<0.05) between parental DT40 cells and each mutant. Columns shaded in black show the cell lines (REV1, RAD54, POLD3, and BRCA1 mutants) that are markedly sensitive to K₂CrO₄. (p<0.05 for all four cell lines after Bonferroni adjustment for 32 tests).

Fig 2. K₂CrO₄ reduces the viability of human cells deficient in certain DNA repair pathways at Cr(VI) levels detected in U.S. city water.

HeLa cells (A) and TK6 cells (B) transiently or stably knocked down with shRNA against RAD54, BRCA1, or POLD3 were exposed to K₂CrO₄ for ~3 days to determine their survival rate. (C) Point of departure analysis of TK6 indicated significant differences (p<0.0001) for TK6 control (log10(BMD) = 1.53, exponential model) vs. POLD3 shRNA knock down (log10(BMD) = 0.99 (9.8 μg/L), polynomial model). The black arrow indicates the maximum contaminant levels (MCL) set by the U.S. EPA. The open arrow shows the highest Cr(VI) levels detected in US city water.

Fig 3. The genotoxicity of K₂CrO₄ in human and DT40 cells is drastically decreased when K₂CrO₄ incubation time is reduced.

(A) REV1 ko DT40 cells were incubated with K₂CrO₄ for 10 min to 8 hours followed by extensive washing. The cells were further cultivated for 3 days in fresh medium without addition of K₂CrO₄ to determine cell survival. (B) The exposure times (hours) were multiplicatively inversed followed by log transformation (x-axis). LC₅₀ data for each exposure time were log transformed (y-axis). Linear regression analysis was performed to determine the relationship between exposure time and LC₅₀ values (p<0.0001). (C) TK6 POLD3 knock-down cells and TK6 mock shRNA-treated cells were incubated with K₂CrO₄ for 10 min followed by extensive washing. The cells were further cultivated for 3 days in fresh medium without addition of K₂CrO₄ to determine cell survival. The survival curves were compared between 10-min and 3-day exposure groups (p<0.0001). The green arrow indicates the Cr(VI) concentration (172,000 μg/L) that causes an increase in oral cancer, a finding that was previously shown in an NTP rodent study [7].

Fig 4. The mutagenicity of K₂CrO₄ in human TK6 cells is markedly decreased by reducing K₂CrO₄ incubation time.

(A) Cumulative cell growth rates of TK6 cells were monitored during continuous incubation with K₂CrO₄ at different concentrations for up to 6 days. (B) Mutation rates of TK6 cells exposed to K₂CrO₄ for 10 min. After extensive washing, the cells were further cultured for 3 days for the phenotype expression period, followed by a mutant selection process using trifluorothymidine (TFT). The two groups show significantly different mutation rates (p<0.0001). (C) Mutation rates of TK6 cells exposed to K₂CrO₄ for 1 or 6 days. For 1-day exposure experiments, the TK6 cells were treated with K₂CrO₄ for 24 hours followed by washing and 2-day phenotype expression period before TFT treatment (log10(BMD) = 2.01, Hill model). For 6-day exposure experiments, we continuously treated the cells with K₂CrO₄ for 6 days by adding fresh K₂CrO₄ into the culture medium when the cells were subcultured. After the 6-day treatment, the cells were treated with TFT (log10(BMD) = 1.48, Hill model). The number of mutants was counted at 2 and 4 weeks after the first TFT treatment. Error bars represent standard deviation around the mean obtained from at least three independent experiments. The 1-day and 6-day BMD values are significantly different (P = 0.005). (D) Dose-response curves of mutation rates of TK6 cells exposed to trace amounts of K₂CrO₄ were compared between 1-, 6- or 14-day treatment groups. For 1-day and 6-day exposures, the mutation assays were performed as described in Fig 4C. For 14-day exposure experiments, the assays were conducted as described in the 6-day experiment except that the duration of K₂CrO₄ treatment was prolonged. Error bars represent standard deviation around the mean obtained from at least three independent experiments. (E) Since 6- and 14-day dose-response curves overlapped with each other in mutation rates and there was no significant difference between the mutation rates above the second active concentration, the mutation rate data were combined and analyzed as pooled results. (log10(BMD) = 1.02 (10.5 μg/L), Hill model).