Persistence of DNA damage following exposure of human bladder cells to chronic monomethylarsonous acid

Abstract

Malignant transformation was demonstrated in UROtsa cells following 52-weeks of exposure to 50 nM monomethylarsonous acid (MMA(III)); the result was the malignantly transformed cell line, URO-MSC. URO-MSC cells were used to study the induction of DNA damage and the alteration of DNA repair enzymes in both the presence of MMA(III) [URO-MSC(+)] and after subsequent removal of MMA(III) [URO-MSC(-)] following chronic, low-level exposure. In the presence of MMA(III), URO-MSC(+) cells demonstrated a sustained increase in DNA damage following 12-weeks of exposure; in particular, a significant increase in DNA single-strand breaks at 12-weeks of exposure consistently elevated through 52 weeks. The persistence of DNA damage in URO-MSC cells was assessed after a 2-week removal of MMA(III). URO-MSC(-) cells demonstrated a decrease in DNA damage compared to URO-MSC(+); however, DNA damage in URO-MSC(-) remained significantly elevated when compared to untreated UROtsa and increased in a time-dependent manner. Reactive oxygen species (ROS) were demonstrated to be a critical component in the generation of DNA damage determined through the incubation of ROS scavengers with URO-MSC cells. Poly (ADP-ribose) polymerase (PARP) is a key repair enzyme in DNA single-strand break repair. URO-MSC(+) resulted in a slight increase in PARP activity after 36-weeks of MMA(III) exposure, suggesting the presence of MMA(III) is inhibiting the increase in PARP activity. In support, PARP activity in URO-MSC(-) increased significantly, coinciding with a subsequent decrease in DNA damage demonstrated in URO-MSC(-) compared to URO-MSC(+). These data demonstrate that chronic, low-level exposure of UROtsa cells to 50 nM MMA(III) results in: the induction of DNA damage that remains elevated upon removal of MMA(III); increased levels of ROS that play a role in MMA(III) induced-DNA damage; and decreased PARP activity in the presence of MMA(III).

Figure 1

Summary of experimental design. Non-tumorigenic UROtsa cells were chronically exposed to 50 nM MMA^III for 52 wk resulting in the malignantly transformed URO-MSC cell line. Cells were exposed to MMA^III for 12, 24, 36 and 52 wk to examine the progressive changes following MMA^III exposure. For experimentation purposes, cells were analyzed in the presence of MMA^III [URO-MSC12(+), URO-MSC24(+), URO-MSC36(+), URO-MSC52(+)], or URO-MSC cells were grown in the absence of MMA^III for 2 wk following previous chronic exposure [URO-MSC12(-), URO-MSC24(-), URO-MSC36(-), URO-MSC52(-)] to analyze the biological alterations in UROtsa cells in the presence and absence of the MMA^III.

Figure 2

Comparison of the average comet moment in URO-MSC cells in the presence/absence of MMA^III following chronic exposure. Graph depicts changes in comet moment between MMA^III exposed URO-MSC cells and untreated UROtsa control (n=4), each n value ≥ 75 cells. (*) Marks statistically significant difference (p ≤ 0.05) between URO-MSC(+) and URO-MSC(-) determined using Student's t-test. (†) Marks statistically significant difference in DNA comet moment of URO-MSC(+)/URO-MSC(-) cells with UROtsa control identified with ANOVA followed by Bonferroni's multiple comparison test with p ≤ 0.05 considered statistically significant. Non-parametric test for trend demonstrates a time-dependent increase in comet moment of URO-MSC(-) cells, (#) marks statistically significant (p ≤ 0.001) upward trend. Error bars within each column represent the standard error of the mean (± SEM).

Figure 3

Detection of ROS generation following chronic MMA^III exposure in URO-MSC(+) and URO-MSC(-) cells. Spectrofluorometric analysis of ROS in URO-MSC12(+), URO-MSC24(+), and URO-MSC52(+) demonstrate a constitutive increase in ROS levels beginning at 12 wk exposure through 52 wk. URO-MSC12(-), URO-MSC24(-), and URO-MSC52(-) cells demonstrate a general time-dependent increase in ROS following previous chronic exposure to MMA^III. (*) Marks statistically significant difference (p ≤ 0.05) between URO-MSC(+) and URO-MSC(-) determined using Student's t-test. (†) Marks statistically significant changes in ROS levels (n=3) of treated URO-MSC(+)/URO-MSC(-) samples compared to untreated UROtsa control identified with ANOVA followed by Bonferroni's multiple comparison test; p ≤ 0.05 was considered statistically significant. Error bars within each column represent the standard error of the mean (± SEM).

Figure 4

a.) Decrease in DNA damage in URO-MSC12(-) cells incubated with ROS scavengers following chronic, low-level exposure to MMA^III. b.) Decrease in DNA damage in URO-MSC52(-) cells incubated with ROS scavengers following chronic, low-level exposure to MMA^III. Graphs depict average comet moment of URO-MSC12(-)/URO-MSC52(-) cells after treatment with ROS scavengers: SOD, CAT, and KI. Average comet moment is decreased in URO-MSC12(-)/URO-MSC52(-) cells incubated with SOD (100 units/ml), CAT (200 units/ml), or KI (5 mM) for 2 wk. Statistically significant differences in DNA comet moment were identified with ANOVA followed by Bonferroni's multiple comparison test; p ≤ 0.01 was considered statistically significant and marked by asterisk (*). Error bars within each column represent the standard error of the mean (± SEM).

Figure 5

Comparison of ssDNA damage in the presence/absence of MMA^III following chronic, low-level exposure to MMA^III in URO-MSC cells. (*) Marks statistically significant difference (p ≤ 0.05) between URO-MSC(+) and URO-MSC(-) determined using Student's t-test. (†) Marks statistically significant difference in DNA single strand breaks of URO-MSC(+)/URO-MSC(-) cells compared to UROtsa control identified with ANOVA followed by Bonferroni's multiple comparison test with p ≤ 0.05 considered statistically significant. Non-parametric test for trend demonstrates a time-dependent increase in ssDNA breaks of URO-MSC(-) cells, (#) marks statistically significant (p ≤ 0.001) upward trend. Error bars within each column represent the standard error of the mean (± SEM).

Figure 6

Comparison of relative PARP activity in the presence/absence of MMA^III following chronic, low-level exposure to MMA^III in URO-MSC cells. Graph depicts changes in PARP activity between MMA^III exposed URO-MSC cells and untreated UROtsa control. (*) Marks statistically significant difference (p ≤ 0.05) between URO-MSC(+) and URO-MSC(-) determined using Student's t-test. (†) Marks statistically significant difference in PARP activity of URO-MSC(+)/URO-MSC(-) cells compared to UROtsa control identified with ANOVA followed by Bonferroni's multiple comparison test with p ≤ 0.05 considered statistically significant. Error bars within each column represent the standard error of the mean (± SEM).