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Mutagenesis Advance Access originally published online on September 28, 2007
Mutagenesis 2007 22(6):403-408; doi:10.1093/mutage/gem035
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© The Author 2007. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Arsenic salt-induced DNA damage and expression of mutant p53 and COX-2 proteins in SV-40 immortalized human uroepithelial cells

Chee-Yin Chai1,2,3,*, Ya-Chun Huang1,4, Wen-Chun Hung3,5, Wan-Yi Kang1,2 and Wan-Tzu Chen2

1Department of Pathology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan 2Department of Pathology, Kaohsiung Medical University Chung-Ho Memorial Hospital, Kaohsiung 807, Taiwan 3National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, Kaohsiung 804, Taiwan 4Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan 5Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan

Arsenic is widely distributed in the environment, and is a proven toxic and carcinogenic agent that is associated with various human malignancies, including bladder cancer. However, the mechanisms of its carcinogenic action are still not well understood. In addition, over-expression of mutant p53 and cyclooxygenase-2 (COX-2) frequently occurs in a variety of human malignancies. It is therefore of interest to study the genotoxicity of arsenic salts on human uroepithelial cells and the expression of oncoproteins p53 and COX-2. In this study, the relative genotoxicity of sodium arsenite was evaluated in SV-40 immortalized human uroepithelial cells (SV-HUC-1) using the alkaline comet assay. The expression of mutant p53 and COX-2 was also evaluated by immunocytochemistry and western blotting. Our results revealed that sodium arsenite was able to induce DNA damage, and that its genotoxicity is correlated with its concentration. In addition, the expression of mutant p53 increased in parallel with comet scores, and the maximal expression of mutant p53 was observed at 4 µM arsenite. Similarly, sodium arsenite stimulated a concentration-dependent increase in COX-2 expression. In conclusion, this study demonstrated that sodium arsenite is genotoxic to uroepithelial cells in vitro, and that it will induce expression of mutant p53 and COX-2 proteins, indicating a possible key event in carcinogenesis. This study provides us with knowledge of the relationship between p53 and COX-2 over-expression in arsenite-treated urothelial cells and suggests a potential therapeutic role of COX-2 inhibitors in human urothelial malignancies.


   Introduction
Top
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Arsenic is widely distributed in the earth's crust, and is a proven toxic and carcinogenic agent (1Go,2Go). The genotoxic and co-genotoxic effects of inorganic arsenicals are well documented in mammalian systems, both in vitro (3Go) and in vivo (4Go). A number of potential mechanisms have been formulated to explain the genotoxicity of these compounds (5Go,6Go). Paradoxically, arsenic has also been used as an effective chemotherapeutic agent in the treatment of certain human cancers, for example, human leukemia (7Go–10Go). However, the mechanisms by which arsenic induces proliferation or the death of cancer cells are not well understood.

Arsenite has been shown to induce DNA strand breakage and DNA–protein cross-links in a variety of cell lines (11Go–13Go); DNA strand breaks usually trigger the accumulation of wild-type p53 protein, a short half-life protein denoted as the guardian of the genome. The wild-type p53 protein plays a pivotal role in maintaining genome integrity by inducing growth arrest for the purposes of repairing DNA damage (14Go,15Go). On the other hand, the TP53 gene is thought to be the most frequently mutated gene in a variety of human malignancies. In addition, the half-life of mutated p53 protein is increased compared to wild type and accumulates within cells. Over-expression of mutated p53 protein and the mutation of p53 have also been shown in a high percentage of transitional cell carcinomas of the urinary bladder (16Go,17Go). This phenomenon is also noticed in the urothelial carcinoma in the endemic region of ‘black foot disease’ on the southwest coast of Taiwan where high arsenic levels were found in artesian well water and where an elevated risk of bladder cancer has been reported (18Go).

Cyclooxygenase-2 (COX-2) has been known to act as a survival factor in a variety of cellular stress conditions and to protect normal human cells such as neurons, cardiomyocytes, renal cells, mammary epithelial cells, fibroblasts and endothelial cells from apoptosis induced by various stresses including nerve growth factor withdrawal, ischemia, hypertonicity or DNA-damaging agents (19Go–22Go). However, COX-2 is also considered a pro-carcinogen as demonstrated by experiments where tumorigenesis was inhibited in COX-2 knockout mice and by cancer chemoprevention studies, which used non-steroidal anti-inflammatory drugs (23Go). Elevated eicosanoid (e.g. prostaglandin E2) levels also occur in both basal and squamous cell carcinoma of the skin (24Go) and are associated with increased tumor progression and metastasis (25Go). Over-expression of COX-2 in mouse skin transforms the epidermis into an ‘auto-promoted’ state, and this sensitizes the tissue to DNA damage by genotoxic carcinogens (26Go). COX-2 also contributes to proliferation in keratinocytes (27Go). Therefore, the mechanism by which inorganic trivalent arsenic modulates inflammatory and proliferative events in the skin may also involve aberrant COX-2 expression and activity. Furthermore, a previous report revealed that arsenite elevated COX-2 mRNA in normal human epidermal keratinocytes (NHEKs) at concentrations relevant to those seen in arsenite-exposed populations (28Go). Thus, we reasoned that COX-2 might play an important role in arsenic-induced bladder cancer. However, a literature review revealed that only a few papers included SV-HUC-1 cells and until now only one report used arsenic-treated SV-HUC-1 cells (29Go). To the best of our knowledge, until recently, there are still no reports on the analysis of DNA damage using the comet assay and the expression of mutant p53 and COX-2 by immunocytochemistry (ICC) of arsenite-treated SV-HUC-1 cells, concurrently.

The aim of this study was to evaluate the degree of damage induced by arsenic salt in SV-HUC-1 cells using the comet assay. We also used ICC and western blotting to detect the expression of mutant p53 and COX-2 in order to address a possible mechanism of arsenic-induced carcinogenesis.


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Chemicals and monoclonal antibodies
Sodium arsenite (NaAsO2), ethylenediaminetetraacetic acid (EDTA), EDTA disodium salt (Na2EDTA), Triton X-100, 2-(4-(2-hydroxyethyl)-1-piperazinl) ethanesulfonic acid sodium salt (HEPES-Na), sodium dodecyl sulfate (SDS), laurosylsarcosinate, dimethylsulfoxide (DMSO) and all other chemicals and stains of the highest purity were purchased from Sigma (St Louis, MO, USA). Growth medium (i.e. Ham's F-12 medium), fetal bovine serum (FBS), phosphate-buffered saline (PBS), antibiotics and trypsin were obtained from HyClone (Logan, UT, USA). The DAKO Universal LSAB kit (biotinylated second antibody and peroxidase-conjugated streptavidin) and 3,3-diaminobenzidine (DAB) were obtained from DAKO (Glostrup, Denmark). The mouse monoclonal primary antibody used was p53 PAb240 (NCL-p53-240) from Novocastra (Newcastle, UK). The rabbit polyclonal antibody COX-2 (CP222C) was obtained from BioCare Medical (Concord, Canada).

Cell line
The SV-40 immortalized human uroepithelial cell line (SV-HUC-1) was obtained from the (American Type Culture Collection, Wiltshire, USA), was grown in 25-cm2 flasks (Nunc, Roskilde, Denmark) (initial density 1 x 105 cells per ml) and was used for all experiments. Cells were cultured in Ham's F-12 medium supplemented with 10% FBS, 100 units per ml penicillin and 100 µg/ml streptomycin at 37°C and 5% CO2. SV-HUC-1 was established by transformation of normal uroepithelium. Although SV-HUC-1 cells are transformed cells, the cell line has been repeatedly tested by the African Green Monkey cell plaque assay for the production of infectious SV40, and results were negative. The cell line has neither a mutation nor a deletion in chromosome 17, where p53 is located (30Go).

Treatment of cells
Subcultures for experiments were prepared the day before treatment. Approximately 2 x 105 cells at logarithmic growth phase were treated with NaAsO2. Final concentrations of NaAsO2 were 1, 2, 4, 8 and 10 µM. Cells were incubated at 37°C in a 5% CO2 incubator for 48 h. All experiments were done three times to assess reproducibility.

Cell viability
Logarithmically growing cells were incubated with varying concentrations of NaAsO2 (1, 2, 4, 8 and 10 µM) for 48 h. Cultures were harvested and cell viability was assessed by the trypan blue exclusion assay. Briefly, cultures were washed with PBS, trypsinized for 3 min and neutralized by the addition of supplemented medium. After centrifuging (1000 rpm, 5 min), cells were suspended in 1 ml of PBS and a 50-µl aliquot was taken and suspended in 400 µl of PBS and 50 µl of trypan blue. Viable cells were counted using a Neubauer chamber.

Alkaline comet assay
Cells in growth phase were washed twice with PBS, and 5 x 105 cells were subjected to the alkaline comet assay to assess NaAsO2-induced DNA damage. The comet assay was performed using the protocol of Singh and Wrenn (31Go) with some modifications, as described previously (32Go–34Go). Briefly, the cell samples were carefully re-suspended in 75 µl of 0.5% low melting agarose (LMA), layered onto microscope slides pre-coated with 150 µl of 0.5% normal melting agarose (NMA) (dried for 10 min at 65°C) and spread with a coverslip. After solidification (for 10 min at 4°C) and removal of the coverslip, 75 µl of 0.5% LMA was again added to the slides. The slides were covered again and kept for 15–20 min at 4°C. The coverslips were then removed and the slides were immersed in cold fresh lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100 and 1% laurosylsarcosinate; pH 10) for 1 h at 4°C in a dark chamber. To avoid the occurrence of additional DNA damage, the following steps were performed under dim light. The slides were placed for 40 min in a horizontal gel electrophoresis tank filled with cold electrophoretic buffer (1 mM Na2EDTA and 300 mM NaOH, pH 13.5) to allow DNA unwinding. Electrophoresis was performed in the same buffer for 20 min at 0.73 V/cm and 300 mA. Unwinding and electrophoresis were carried out in an ice bath. After electrophoresis, the slides were neutralized twice for 5 min with 0.4 M Tris (pH 7.5) and fixed with 3 ml absolute ethanol for 3 min. The slides were stained with silver stain for analysis. An intensity score [from class 0 (undamaged) to class 4 (severely damaged)] was assigned to each cell based on the procedures described by Visvardis et al. (35Go). The positive control for comet assay is 1 µM H2O2 at 37°C in a 5% CO2 incubator for 10 min, as H2O2 caused DNA damage via oxidative stress to the cells (36Go).

Image analysis
Observations were made at 400x magnification using a NIKON E600 light microscope equipped with CCD-IRS color video camera (Tokyo, Japan). The image for each individual cell was acquired immediately to the computer monitor, linking to an image analysis system Image Pro Plus 4.0 program (Media Cybernetics, Silver Spring, USA). Pictures of 50 randomly selected comets per slide were analysed, and then a repeated analysis of 50 randomly selected comets per slide was carried out. The mean score was recorded.

ICC of SV-HUC-1
Cells were cytospun down to a silane-coated glass slide, and fixed in ethanol for 30 min. Antigen retrieval was performed by incubating slides in 0.1 M citrate buffer (pH 6.0) in an autoclave at 121°C for 10 min. Endogenous peroxidase activity was blocked by incubation in 0.5% hydrogen peroxide for 10 min followed by washing with Tris buffer solution, pH 7.0. Nonspecific binding of antibody was prevented by blocking for 20 min in 5% bovine serum albumin. The mouse monoclonal primary antibody used was p53 PAb240, which recognizes mutant p53 protein (37Go,38Go). This antibody was added at a dilution of 1:50 for 60 min at room temperature. The rabbit polyclonal antibody used was COX-2 at a dilution of 1:50 for 4 h at room temperature. Biotinylated secondary antibody and peroxidase-conjugated streptavidin from the DAKO Universal LSAB kit were applied for 20 min each. Finally, cells were incubated in DAB for 5 min and hematoxylin was used as a nuclear counterstain. The labeling index (LI), expressed as the percentage of immunostained cells, was determined by counting 1000 cells in the areas of densest immunostaining using light microscope at 400x magnification. A colon cancer is used as positive control for mutant p53 and COX-2 protein.

Western blotting
p53 protein is a well-documented marker of DNA damage. The effects of arsenite on p53 protein levels were examined using immunoblotting (39Go). SV-HUC-1 cells were grown to approximately 80–90% confluency and were treated with NaAsO2 at concentrations of 0, 1, 2, 4, 8 and 10 µM for 48 h. Cells were harvested with lysis buffer (500 mM NaCl, 20% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 20 mM HEPES-Na) for 1 h. Protein concentrations were determined by a Bradford assay (Bio-Rad, Hercules, CA), and samples were loaded onto a 10% SDS gel. After protein separation, the samples were transferred to a polyvinylidenedifluoride membrane; the mouse monoclonal primary antibody used was p53 PAb240 and the rabbit polyclonal antibody used was COX-2. Secondary antibody was conjugated with horseradish peroxidase. p53 and COX-2 were then visualized by enhanced chemiluminescence (Amersham, Buckinghamshire, England). In separate experiments, SV-HUC-1 cells were exposed to varying concentrations of NaAsO2 for 48 h to determine the concentration-dependent effect on p53 and COX-2 expression.

Statistical analysis
All experiments were repeated at least three times. The data obtained were analysed using the SPSS 8.0 statistical software program (Chicago, IL, USA). Cell viability and western blotting were analysed by Student's t-test. The effect of chemical treatment on the frequency of tailed cells, the grade of damaged cells and migration were analysed using Student's t-test, Wilcoxon signed ranks test and the one-way ANOVA.


   Results
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 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Effects of NaAsO2 on SV-HUC-1 cell viability
We determined cell viability after incubation with 0, 1, 2, 4, 8 and 10 µM of NaAsO2 for 48 h. Sodium arsenite treatment reduced cell viability in a concentration-dependent manner. Concentrations of 1, 2, 4, 8 and 10 µM NaAsO2 reduced cell viability to 75.10, 71.96, 59.64, 44.34 and 33.99%, respectively (Table I).


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  		Table I. Effects of sodium arsenite on the cell viability, mutant p53 and COX-2 LI

 
Effects of NaAsO2 treatment
Figure 1 illustrates the scoring of comet images of SV-HUC-1 cells. Table II shows the concentration-dependent increase in DNA damage induced by NaAsO2 in the comet assay. When cells with tails were considered, the Wilcoxon signed ranks test demonstrated that there were significant differences between the control group and the experimental groups (P < 0.05). The mean DNA migration values of the 50 nuclei in the control and experimental groups were compared using a one-way ANOVA (P < 0.001).


Figure 1
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  		Fig. 1. Comet images of SV-HUC-1 cells, illustrating the visual scoring classification. (A) class 0, (B) class 1, (C) class 2, (D) class 3, (E) and (F) class 4.

 


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  		Table II. Effects of sodium arsenite on the tail length and the percentage of cells with tails

 
Student's t-test was used to compare the migration values for the different treatments. Mean DNA tail length increased with increasing concentration of NaAsO2 (P < 0.001). There were significant differences between the control group and the experimental groups (Table II).

Immunocytochemistry
Figure 2 shows the expression levels of mutant p53 and COX-2 in the SV-HUC-1 cell line as analysed by ICC after treatment with NaAsO2. p53 staining was observed in nuclei of cells. COX-2 expression was seen as brown staining of the cytoplasm of cells.


Figure 2
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  		Fig. 2. Immunocytochemical analysis of the effect of sodium arsenite on mutant p53 (left) and COX-2 (right) expressed at 48 h in SV-HUC-1 cells (200x). The maximal induction of mutant p53 and COX-2 was observed at 4 µM arsenite. (Scale bar = 150 m.)

 
The LI of mutant p53, for concentrations of 1, 2, 4, 8 and 10 µM NaAsO2, was 19.38, 28.14, 45.66, 32.69 and 13.75%, respectively, and the LI of COX-2 was 20.50, 30.71, 66.08, 33.68, and 17.77%, respectively. Sodium arsenite treatment increased the LI in a concentration-dependent manner at 1–4 µM NaAsO2 (Table I).

In SV-HUC-1 cells, a major induction in the level of p53 protein and LI with 1 µM arsenite for 48 h was observed. However, the p53 and COX-2 LI decreased with higher concentrations of arsenite.

Western blotting analysis
Treatment of SV-HUC-1 cells with arsenite concentrations ranging from 1 to 4 µM for 48 h dramatically increased p53 levels, but these levels decreased at 8 and 10 µM of arsenite (Figure 3A). As measured by a densimeter, the relative densities of p53 expression in arsenite-treated cells with concentrations of 1, 2, 4, 8 and 10 µM are 4.07, 4.30, 5.01, 4.49 and 2.95, respectively (Figure 3B).


Figure 3
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  		Fig. 3. Effects of sodium arsenite on cellular levels of mutant p53 and COX-2 at various concentrations in SV-HUC-1 cells. (A) Increase of mutant p53 and COX-2 expressed as dose respective to control (B) and (C). *, P<0.05.

 
Expression of COX-2 protein was measured by western blot analysis using a polyclonal COX-2 antibody and chemiluminescence, as shown in Figure 3A. COX-2 protein (~72 kDa) was increased at 1, 2 and 4 µM arsenite. However, the decreased COX-2 expression was noted at 8 and 10 µM arsenite. The relative densities of COX-2 expression in arsenite-treated cells with concentrations of 1, 2, 4, 8 and 10 µM were 1.92, 2.22, 4.99, 2.38 and 1.33, respectively (Figure 3C).


   Discussion
Top
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Inorganic arsenic is an epidemiologically proven human carcinogen; evaluation of its carcinogenicity in animal experiments has been problematical (2Go). In our study, sodium arsenite was clearly genotoxic in the comet assay, which confirms results previously obtained by other authors using the same assay system (40Go–43Go). The range of time of arsenite exposure that we used was similar to previously published papers (44Go–47Go). And in the previously published papers, we also found that comet assay was performed when exposure time was >6 h (48Go–51Go) and when cell viability was <70% (52Go–54Go). Although it has not been proven that inorganic arsenic reacts directly with DNA, it has been suggested that arsenite induces oxidative damage by causing single- and double-strand breaks (55Go). Arsenite treatment can also modify cellular functions by changing the phosphorylation profiles of cellular proteins (56Go) and inhibiting DNA repair enzymes (57Go,58Go). These two mechanisms could be related. Concomitantly with DNA strand breaks, p53 protein was also found to accumulate in HFW cells (human fibroblast cells) (59Go,60Go). Arsenite obviously follows different kinetics in terms of DNA strand break induction and mutant p53 accumulation. Our results show that changes in the expression of mutant p53 accumulation occurred in parallel with changes in comet scores. These results indicate that the level of mutant p53 accumulation occurred in parallel with the incidence of DNA strand breaks after 48 h of arsenite treatment. Our ICC results show that accumulation of mutant p53 in the cell nuclei is similar to a previous report (61Go). Although, it has been reported that the antibody PAb240 recognizes all forms of p53 (including wild-type p53) when they are denatured (38Go). In our study, we performed ICC under non-denaturing condition and western blotting under denaturing condition to detect mutant p53 protein. And expression of p53 was noticed in both methods.

Our western blotting and ICC results revealed that, in SV-HUC-1 cells treated with 1 µM arsenic for 48 h, over-expression of mutant p53 protein occurred. Maximal induction of mutant p53 was observed at 4 µM arsenite. The immunoblot signal and LI decreased with higher concentrations of arsenite (i.e. at 8 and 10 µM arsenite), which could be due to cell death at those concentrations; arsenite causes cellular transformation/carcinogenesis at low concentrations and induces cell death at higher concentrations (62Go–64Go). This is supported by the viability data. In Jurkat cells, a major induction in the level of mutant p53 protein with 1 µM arsenic for 24 h was observed; the immunoblot signal also decreased with higher concentrations of arsenite (65Go).

It has been shown that expression of high levels of wild-type p53 results in cell cycle arrest and apoptosis. Furthermore, it could serve as a transcriptional activator of genes containing the p53-binding sites as well as inhibiting gene transcription (66Go). Therefore, mutations in the core domain of p53 may alter its binding ability and affect its role in transcription regulation. One of the ways by which p53 inhibits gene expression is via interaction with the TATA-binding proteins (TBPs) and by interfering with the assembly of a functional transcription initiation complex (67Go,68Go). A recent in vitro study also demonstrated that wild-type p53 inhibits the formation of the complex between TBP and human COX-2 promoters in a cell-free system (69Go). Accordingly, a p53 mutation would result in a loss of the inhibitory effect of COX-2 expression in human cancer. In addition, Hsu et al. (70Go) revealed that the p53 gene mutation rate in arsenic-related skin cancer from the black foot disease endemic area of Taiwan is high, and that the mutation types are different from those in UV-induced skin cancer. Shibata et al. (71Go) also showed that 62% of urothelial carcinomas showed p53 mutations. Their results showed a high frequency of p53 mutations and frequent double mutations in urothelial cancers from the endemic area of the black foot disease in Taiwan. Therefore, it is worth investigating the role of mutant p53 and COX-2 in arsenic carcinogenesis as well as the mechanisms involved in the regulation of several functions in urothelial carcinomas, including inflammation, proliferation and apoptosis. Furthermore, our data revealed the possibility of a close regulation of COX-2 activity by p53.

Elevated expression and activity of COX-2 occurs in numerous carcinomas including those in the bladder and skin, and studies in rodent and human tissues have indicated that over-expression of COX-2 contributes to skin carcinogenesis (72Go). A previous study has shown that arsenic increases expression of the pro-carcinogenic enzyme COX-2 in NHEKs and that this occurs via specific mitogen and stress signaling pathways (73Go). In SV-HUC-1 cells, sodium arsenite stimulated concentration-dependent changes in COX-2 expression. Our data are consistent with a recent report by Tsai et al. (74Go) that shows the induction of COX-2 protein in human umbilical vein endothelial cells by arsenite. Our data indicate that arsenite modulates COX-2 expression in SV-HUC-1 at concentrations within the range of those found in contaminated drinking water and in the urine of humans consuming contaminated drinking water (75Go). By reviewing the literature, we found a report on hepatocyte growth factor inhibiting anoikis by induction of COX-2 in human head and neck cancer UMSCC1 cells, the mechanism of which remains to be explained (76Go). COX-2 also inhibits anoikis by activation of the PI-3K/Akt pathway in human bladder cancer EJ cells, which provides one of the mechanisms for the anti-anoikis effect of COX-2 (77Go). In addition, COX-2 has been known to inhibit apoptosis in human cancer cells through the regulation of Bcl-2 family protein expression (78Go–80Go). Accumulating evidence indicates that COX-2 plays an important role in tumor development and progression. In several epidemiologic studies, regular administration of COX-2 promotes colon cancer development (81Go,82Go). However, how and why the COX-2 gene is constitutively and highly expressed in tumor cells has not yet been clarified, so the mechanism by which arsenite treatment causes an increase in COX-2 levels in culture cells still warrants further investigation.

In conclusion, this study provides us with knowledge regarding the relationship between mutant p53 and COX-2 over-expression in arsenite-treated urothelial cells. The information supports not only the role of COX-2 in arsenite carcinogenesis but also a potential therapeutic role of COX-2 inhibitors in human urothelial malignancies.


   Funding
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 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, Kaohsiung, Taiwan.


   Notes
 
* To whom correspondence should be addressed. Department of Pathology, Kaohsiung Medical University Chung-Ho Memorial Hospital, No. 100, Tzyou 1st Road, Kaohsiung 807, Taiwan. Tel: +886 7 3208233; Fax: +886 7 3136681; Email: cychai{at}kmu.edu.tw


   References
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 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received on June 13, 2007; revised on July 13, 2007; accepted on August 13, 2007.


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