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. 2013 Jul 31;221(1):64-72.
doi: 10.1016/j.toxlet.2013.05.641. Epub 2013 Jun 5.

Mechanism study of goldenseal-associated DNA damage

Affiliations

Mechanism study of goldenseal-associated DNA damage

Si Chen et al. Toxicol Lett. .

Abstract

Goldenseal has been used for the treatment of a wide variety of ailments including gastrointestinal disturbances, urinary tract disorders, and inflammation. The five major alkaloid constituents in goldenseal are berberine, palmatine, hydrastine, hydrastinine, and canadine. When goldenseal was evaluated by the National Toxicology Program (NTP) in the standard 2-year bioassay, goldenseal induced an increase in liver tumors in rats and mice; however, the mechanism of goldenseal-associated liver carcinogenicity remains unknown. In this study, the toxicity of the five goldenseal alkaloid constituents was characterized, and their toxic potencies were compared. As measured by the Comet assay and the expression of γ-H2A.X, berberine, followed by palmatine, appeared to be the most potent DNA damage inducer in human hepatoma HepG2 cells. Berberine and palmatine suppressed the activities of both topoisomerase (Topo) I and II. In berberine-treated cells, DNA damage was shown to be directly associated with the inhibitory effect of Topo II, but not Topo I by silencing gene of Topo I or Topo II. In addition, DNA damage was also observed when cells were treated with commercially available goldenseal extracts and the extent of DNA damage was positively correlated to the berberine content. Our findings suggest that the Topo II inhibitory effect may contribute to berberine- and goldenseal-induced genotoxicity and tumorigenicity.

Keywords: Berberine; Comet assay; DNA damage; Goldenseal; Topoisomerase; γ-H2A.X.

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Conflict of interest statement

Conflicts of interest

None.

Figures

Fig. 1.
Fig. 1.
Structures of five goldenseal constituents.
Fig. 2.
Fig. 2.
Berberine induces DNA damage in HepG2 cells. HepG2 cells were exposed to increased concentrations (0, 3.125, 6.25, 12.5, 25, 50, or 100 μM) of berberine for 2, 6, or 24 h. (A) Quantification of the percentages of cells with Comet tails at different concentrations and time points. Data are the means ± S.D. (n = 100 cells in each of three trials; * p < 0.05, ***p < 0.001). (B) Total cellular protein was extracted at the indicated times, and levels of phosphorylated (γ-H2A.X) and total histones H2A.X were detected by Western blotting. β-actin was used as a loading control. Similar results were obtained from three independent experiments.
Fig. 3.
Fig. 3.
Commercial goldenseal extracts induce DNA damage and the extent of DNA damage correlates with berberine content in the extracts. (A) The bar graph represents the berberine concentrations of eight commercially available goldenseal products as measured by HPLC. (B) HepG2 cells were treated with 20 μl of commercial goldenseal extracts for 6 h in 10 ml culture medium and analyzed for γ-H2A.X by Western blotting. β-actin was used as a loading control. (C) Statistical analysis of the relationship between the expression levels of γ-H2A.X and the concentration of berberine in eight commercial goldenseal extracts. Plot represents quantitative expression levels, shown in (B), of γ-H2A.X normalized by β-actin. The band intensities in the Western blots were determined by Image J software. Data were expressed as means ± S.D. of three separate experiments.
Fig. 4.
Fig. 4.
Effects of berberine on cell cycle of HepG2 cells. (A) Flow cytometric analysis for cell cycle distribution. Histograms shown are DNA content analyses for HepG2 cells treated with the indicated concentrations of berberine for 24 h. Treated cells were stained with propidium iodide (PI) and processed for cell cycle analysis. (B) The bar graph depicts the mean percentage of each cell cycle phase ± S.D. from four independent experiments. * p < 0.05, ** p < 0.005, and ***p < 0.001 versus the respective G1, S, or G2/M phase of DMSO-treated cells. (C) Expression of cell cycle checkpoint-related proteins. HepG2 cells were treated with the indicated concentrations of berberine for 6 h. Treated cells were lysed and subjected to Western blotting analyses with antibodies against phospho-Chk1, phosphor-Chk2, and p21Waf1/Clp1. β-actin was used as a loading control.
Fig. 5.
Fig. 5.
Inhibitory effects of goldenseal constituents on topoisomerase activities. Supercoiled DNA relaxation assay for Topo I (A, B) and kDNA decatenation assay for Topo II (D, E). Supercoiled pBR322 plasmid DNA or kDNA was incubated at 37 °C for 30 min with or without Topo I or Topo II enzyme in the presence of the indicated goldenseal constituents (A, D) or increasing concentrations of berberine and palmatine (B, E); CPT (camptothecin) was used as a Topo I positive control inhibitor. DNA samples were separated by electrophoresis on 1% agarose gel, stained with ethidium bromide, and visualized by UV light. RLX, relaxed DNA; SC, supercoiled DNA. Bar graphs C and F represent the densitometric analyses of the results from B and E. Values are means ± S.D. of three separate experiments. * p < 0.05, ** p < 0.005 versus the DMSO control.
Fig. 6.
Fig. 6.
Effect of down-regulation of Topo I or Topo II on berberine induced DNA damage. (A, B) Establish HepG2 cells stably express doxycycline (DOX) inducible sh1-Topo I, sh2-Topo I, sh1-Topo II, sh2-Topo II, or a scrambled (SC) control. These five cell lines were incubated with DOX for 3 days followed by continued culture for another 6 h without DOX; then the decrease in Topo I or Topo II mRNA was assessed by real-time PCR. The results indicated that the mRNA expression levels of Topo I (A) or Topo II (B) were significantly decreased by DOX induction of the shRNAs. Values were means ± S.D. of three separate experiments. *** p < 0.001 versus the SC controls. (C) sh1-Topo I, sh2-Topo I, (D) sh1-Topo II, sh2-Topo II, and SC cells were incubated with DOX for 3 days and then treated with berberine at 50 μM or 100 μM for another 6 h without DOX. Treated cells were then lysed and subjected to Western blot analyses with an antibody against γ-H2A.X. β-actin was used as a loading control. Similar results were obtained from three repeated experiments.

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