Low level exposure to monomethyl arsonous acid-induced the over-production of inflammation-related cytokines and the activation of cell signals associated with tumor progression in a urothelial cell model.

Human bladder cancer has been associated with chronic exposure to arsenic. Chronic exposure of an immortalized non-tumorigenic urothelial cell line (UROtsa cells) to arsenicals has transformed these cells to a malignant phenotype, but the involved mechanisms are not fully understood. Chronic inflammation has been linked with cancer development mainly because many pro-inflammatory cytokines, growth factors as well as angiogenic chemokines have been found in tumors. In this study the chronology of inflammatory cytokines production was profiled in UROtsa cells chronically exposed to the toxic arsenic metabolite, monomethylarsonous acid [50 nM MMA(III)] to know the role of inflammation in cell transformation. Acute 50 nM MMA(III) exposure induced over-production of many pro-inflammatory cytokines as soon as 12 h after acute exposure. The same cytokines remain over-regulated after chronic exposure to 50 nM MMA(III), especially after 3 mo exposure. At 3 mo exposure the sustained production of cytokines like IL-1, IL-6, IL-8 and TNF is coincident with the appearance of characteristics associated with cell transformation seen in other arsenic-UROtsa studies. The sustained and increased activation of NFkappaB and c-Jun is also present along the transformation process and the phosphorylated proteins p38 MAPK and ERK 1/2 are increased also through the time line. Taken together these results support the notion that chronic inflammation is associated within MMA(III)-induced cell transformation and may act as a promoting factor in UROtsa cell transformation.

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

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).

Reactive oxygen species regulate properties of transformation in UROtsa cells exposed to monomethylarsonous acid by modulating MAPK signaling.

UROtsa cells exposed to 50 nM monomethylarsonous acid [MMA(III)] for 52 wk (MSC52) achieved hyperproliferation, anchorage independent growth, and enhanced tumorgenicity. MMA(III) has been shown to induce reactive oxygen species (ROS), which can lead to activation of signaling cascades causing stress-related proliferation of cells and even cellular transformation. Previous research established the acute activation of MAPK signaling cascade by ROS produced by MMA(III) as well as chronic up regulation of COX-2 and EGFR in MSC52 cells. To determine if ROS played a role in the chronic pathway perturbations by acting as secondary messengers, activation of Ras was determined in UROtsa cells [exposed to MMA(III) for 0-52 wk] and found to be increased through 52 wk most dramatically after 20 wk of exposure. Ras has been shown to cause an increase in O2(-) and be activated by increases in O2(-), making ROS important to study in the transformation process. COX-2 upregulation in MSC52 cells was confirmed by real time RT-PCR. By utilizing both antioxidants or specific COX inhibitors, it was shown that COX-2 upregulation was dependent on ROS, specifically, O2(-). In addition, because previous research established the importance of MAPK activation in phenotypic changes associated with transformation in MSC52 cells, it was hypothesized that ROS play a role in maintaining phenotypic characteristics of the malignant transformation of MSC52 cells. Several studies have demonstrated that cancer cells have lowered superoxide dismutase (MnSOD) activity and protein levels. Increasing levels of MnSOD have been shown to suppress the malignant phenotype of cells. SOD was added to MSC52 cells resulting in slower proliferation rates (doubling time=42h vs. 31h). ROS scavengers of OH also slowed proliferation rates of MSC52 cells. To further substantiate the importance of ROS in these properties of transformation in MSC52 cells, anchorage independent growth was assessed after the addition of antioxidants, both enzymatic and non-enzymatic. Scavengers of OH, and O2(-) blocked the colony formation of MSC52 cells. These data support the role for the involvement of ROS in properties of transformation of UROtsa cells exposed to MMA(III).