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 two 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 (1–24 h) 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.

Piperine Causes G1 Phase Cell Cycle Arrest and Apoptosis in Melanoma Cells through Checkpoint Kinase-1 Activation

“…Previous studies have shown the involvement of ROS in inducing DNA damage and cell cycle arrest [14], [17]. Therefore, ROS generation was determined using flow cytometer by measuring the fluorescence of DCF, which is formed due to the oxidation of DCFDA by endogenous peroxides.…”

Comparison of 2,2-bis(bromomethyl)-1,3-propanediol induced genotoxicity in UROtsa cells and primary rat hepatocytes: Relevance of metabolism and oxidative stress

“…This cell line was generated from the normal human bladder epithelium and immortalized with the SV-40 large T antigen (Rossi et al, 2001). Cell culture conditions were as previously described by (Kong et al, 2011; Wnek et al, 2011). …”

“…The assay was performed as previously reported by (Kong et al, 2011). Briefly, UROtsa cells (5 × 10 5 cells/ml) and freshly isolated, suspended hepatocytes (5 × 10 5 cells/ml) were incubated with BMP (10–100 µM) at 37 °C for 1 h. All control and treated cultures received the same final concentration of vehicle (0.5% EtOH).…”

“…They build upon our observation that BMP induces strand breaks, as assessed by the comet assay, in human bladder epithelial cells (UROtsa; considered a target cell population) (Kong et al, 2011). Specifically, the in vitro studies compare the genotoxic potential (induction of DNA strand breaks and covalent binding to DNA) of BMP in UROtsa cells (target cells) and primary rat hepatocytes (non-target cells) and relate these outcomes to the glucuronidation capacity of these two cell types.…”

“…Specifically, the in vitro studies compare the genotoxic potential (induction of DNA strand breaks and covalent binding to DNA) of BMP in UROtsa cells (target cells) and primary rat hepatocytes (non-target cells) and relate these outcomes to the glucuronidation capacity of these two cell types. In addition, as BPM-induced oxidative stress plays a key role in BMP-associated DNA damage (Kong et al, 2011), the basal level of intracellular GSH content and the effect of BMP on GSH levels were assessed in both the target cells and non-target cells.…”