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. Author manuscript; available in PMC: 2015 May 13.
Published in final edited form as: Toxicol Appl Pharmacol. 2012 Apr 19;262(1):11–21. doi: 10.1016/j.taap.2012.04.014

Figure 7.

Figure 7

ROS generation induced by carcinogenic heavy metal mediated β-catenin activation. (A) ROS generation induced by carcinogenic metals in CRL-1807 cells was determined by DCF assay. CRL-1807 cells were treated with As(III) or Cr(VI), alone or in combination, for 24 h; the ratio of carboxy-H2DCFDA (oxidation sentive) to carboxy-DCFDA (Oxidation insensitive) was compared. Each data point was the mean ± SEM of three independent experiments. *p < 0.05, statistically significant difference from untreated control cells. As2+Cr1, As2+Cr2, As2+Cr4: 2 µM of As(III) combination with 1, 2, 4 µM of Cr(VI), respectively. (B) Addition of catalase inhibited carcinogenic metals-induced ROS generation. CRL-1807 cells were treated with Cr(VI) (0 or 4 µM), As(III) (0 or 10 µM), As(III) combination with Cr(VI) [As: 2 µM; Cr(VI): 2 µM], or pre-treated with catalase (1000 Unit/ml) for 2 h followed by carcinogenic metals exposure for 24 h. After exposure, cells were labeled with H2DCFDA (10 µM) as described under the Material and Method. (C) Addition of PEG-SOD or catalase inhibited the β-catenin expression level induced by carcinogenic metals in CRL-1807 cells. CRL-1807 cells were treated with Cr(VI) (0 or 4 µM), As(III) (0 or 10 µM), As(III) combination with Cr(VI) [As: 2 µM, Cr(VI): 2 µM], or pre-treated with SOD (1000 Unit/ml) or catalase (1000 Unit/ml) for 2 h followed by carcinogenic metals exposure for 24 h. The expression of β-catenin was determined with immunoblotting. The expression of actin served as an internal control. (D) Addition of PEG-SOD or catalase inhibited the β-catenin activity induced by carcinogenic metals in CRL-1807 cells. The cells were transfected with super TOP flash luciferase reporter and renilla reporter followed by treatment with carcinogenic metals treatment. The cells were harvested for the measurement of luciferase activity after 24 h. Values are expressed as relative units after internal normalization with renilla luciferase. *p < 0.05, statistically significant difference from control cells. The data are expressed as the mean ± S.E. of three independent experiments. #p < 0.05, statistically significant difference from carcinogenic metals-treated cells.