Induction of DNA damage in human urothelial cells by the brominated flame retardant 2,2-bis(bromomethyl)-1,3-propanediol: role of oxidative stress

Abstract

2,2-bis(bromomethyl)-1,3-propanediol (BMP) is an extensively used brominated flame retardant found in urethane foams and polyester resins. In a 2-year dietary study conducted by the National Toxicology Program, BMP caused neoplastic lesions at multiple sites including the urinary bladder in both rats and mice. The mechanism of its carcinogenic effect is unknown. In the present study, using SV-40 immortalized human urothelial cells (UROtsa), endpoints associated with BMP induced DNA damage and oxidative stress were investigated. The effects of time (1-24h) and concentration (5-100 μM) on BMP induced DNA strand breaks were assessed via the alkaline comet assay. The results revealed evidence of DNA strand breaks at 1 and 3h following incubation of cells with non-cytotoxic concentrations of BMP. Strand breaks were not present after 6h of incubation. Evidences for BMP associated oxidative stress include: an elevation of intracellular ROS formation as well as induction of Nrf2 and HSP70 protein levels. In addition, DNA strand breaks were attenuated when cells were pre-treated with N-acetyl-l-cysteine (NAC) and oxidative base modifications were revealed when a lesion specific endonuclease, human 8-hydroxyguanine DNA glycosylase 1 (hOGG1) was introduced into the comet assay. In conclusion, these results demonstrate that BMP induces DNA strand breaks and oxidative base damage in UROtsa cells. Oxidative stress is a significant, determinant factor in mediating these DNA lesions. These early genotoxic events may, in part, contribute to BMP-induced carcinogenesis observed in rodents.

Figure 1

Viability of UROtsa cells after 1, 6 and 24h of incubation with vehicle or BMP (10–1500 μM). Data represents the mean ± SEM of 5 determinations.

Figure 2

Effects of time and concentration on DNA strand breaks induced by BMP in UROtsa cells by standard comet assay.(A) Representative comet images after 1h of exposure to BMP (B) DNA strand breaks following BMP (25μM) exposure for 1, 3, 6, 18 and 24h and H₂O₂ (100 μM) for 1h. (C) DNA strand breaks following exposure to various concentrations of BMP (5–100μM) for 1 h (D) The distribution of UROtsa cells with respect to different DNA damage levels (I ~IV) following BMP (5–100 μM) and H₂O₂ (50μM) treatment for 1h. T_%DNA represents % of DNA in the comet tail. Data are shown as mean ± SEM, N=3–7. *P<0.05, **P<0.01 versus vehicle control at each time point.

Figure 2

Effects of time and concentration on DNA strand breaks induced by BMP in UROtsa cells by standard comet assay.(A) Representative comet images after 1h of exposure to BMP (B) DNA strand breaks following BMP (25μM) exposure for 1, 3, 6, 18 and 24h and H₂O₂ (100 μM) for 1h. (C) DNA strand breaks following exposure to various concentrations of BMP (5–100μM) for 1 h (D) The distribution of UROtsa cells with respect to different DNA damage levels (I ~IV) following BMP (5–100 μM) and H₂O₂ (50μM) treatment for 1h. T_%DNA represents % of DNA in the comet tail. Data are shown as mean ± SEM, N=3–7. *P<0.05, **P<0.01 versus vehicle control at each time point.

Figure 2

Effects of time and concentration on DNA strand breaks induced by BMP in UROtsa cells by standard comet assay.(A) Representative comet images after 1h of exposure to BMP (B) DNA strand breaks following BMP (25μM) exposure for 1, 3, 6, 18 and 24h and H₂O₂ (100 μM) for 1h. (C) DNA strand breaks following exposure to various concentrations of BMP (5–100μM) for 1 h (D) The distribution of UROtsa cells with respect to different DNA damage levels (I ~IV) following BMP (5–100 μM) and H₂O₂ (50μM) treatment for 1h. T_%DNA represents % of DNA in the comet tail. Data are shown as mean ± SEM, N=3–7. *P<0.05, **P<0.01 versus vehicle control at each time point.

Figure 2

Effects of time and concentration on DNA strand breaks induced by BMP in UROtsa cells by standard comet assay.(A) Representative comet images after 1h of exposure to BMP (B) DNA strand breaks following BMP (25μM) exposure for 1, 3, 6, 18 and 24h and H₂O₂ (100 μM) for 1h. (C) DNA strand breaks following exposure to various concentrations of BMP (5–100μM) for 1 h (D) The distribution of UROtsa cells with respect to different DNA damage levels (I ~IV) following BMP (5–100 μM) and H₂O₂ (50μM) treatment for 1h. T_%DNA represents % of DNA in the comet tail. Data are shown as mean ± SEM, N=3–7. *P<0.05, **P<0.01 versus vehicle control at each time point.

Figure 3

Generation of intracellular ROS in UROtsa cells exposed to BMP (10–100μM) for up to 1h. H₂DCFDA fluorescence signal was normalized for carboxy-DCFDA fluorescence using the following equation: H₂DCFDA Signal = H₂DCFDA_treatment × (carboxy-DCFDA_control / carboxy-DCFDA_treatment). The results are expressed as the percentage of H2DCFDA signal with respect to vehicle control. Data are expressed as mean ± SEM, N= 6, *P<0.05, **P<0.01 versus vehicle control.

Figure 4

The expression of (A) Nrf2 and (B) HSP70 protein in UROtsa cells following BMP (10μM) exposure for 1, 3, 6, 18 and 24h. Data are shown as mean ± SEM, N= 4. *P<0.05 versus control (0h).

Figure 4

The expression of (A) Nrf2 and (B) HSP70 protein in UROtsa cells following BMP (10μM) exposure for 1, 3, 6, 18 and 24h. Data are shown as mean ± SEM, N= 4. *P<0.05 versus control (0h).

Figure 5

Involvement of oxidative stress in BMP mediated DNA damage. (A) Oxidative base modifications in UROtsa cells following 1h of BMP (25μM), KBrO₃(2mM) and H₂O₂(100μM) exposure measured by hOGG1 modified comet assay. Gray Bars represent the net increase in T_%DNA after incubation with hOGG1. (B) Protective effects of NAC (2mM) on BMP (10–100μM) induced DNA strand breaks in UROtsa cells. Data are shown as mean ± SEM, N=3. *P<0.05 versus vehicle control, ^#P<0.05 versus cells without NAC pre-treatment.

Figure 5

Involvement of oxidative stress in BMP mediated DNA damage. (A) Oxidative base modifications in UROtsa cells following 1h of BMP (25μM), KBrO₃(2mM) and H₂O₂(100μM) exposure measured by hOGG1 modified comet assay. Gray Bars represent the net increase in T_%DNA after incubation with hOGG1. (B) Protective effects of NAC (2mM) on BMP (10–100μM) induced DNA strand breaks in UROtsa cells. Data are shown as mean ± SEM, N=3. *P<0.05 versus vehicle control, ^#P<0.05 versus cells without NAC pre-treatment.