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Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells.

Zhang Z, Chen S, Mei H, Xuan J, Guo X, Couch L, Dobrovolsky VN, Guo L, Mei N - Sci Rep (2015)

Bottom Line: In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined.In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II.Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA.

ABSTRACT
Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

No MeSH data available.


Related in: MedlinePlus

Effect of silencing of Topo II on Ginkgo biloba extract- or quercetin-induced DNA damage.(A) HepG2 cells stably expressing doxycycline (DOX)-inducible Topo II knockdown and scramble control cell lines were incubated with DOX for 3 days followed by continued culture for another 4 h without DOX; then the Topo II knockdown efficiency was assessed by Western blot. (B,C) Topo II knockdown and scramble control cells were incubated with DOX for 3 days and then treated with Ginkgo biloba leaf extract at 0.8 mg/ml (B) or quercetin at 50 μM (C) for another 4 h without DOX. Treated cells were then lysed and subjected to Western blot analyses with antibodies against γ-H2A.X, p-Chk1, and p-Chk2. Similar results were obtained from three repeated experiments. (D), HepG2 cells were exposed to Ginkgo biloba extract at the concentration of 0.8 mg/ml for 4 h or 24 h. Cells lysis was subjected to Western blot analysis with an antibody against Topo II.
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f8: Effect of silencing of Topo II on Ginkgo biloba extract- or quercetin-induced DNA damage.(A) HepG2 cells stably expressing doxycycline (DOX)-inducible Topo II knockdown and scramble control cell lines were incubated with DOX for 3 days followed by continued culture for another 4 h without DOX; then the Topo II knockdown efficiency was assessed by Western blot. (B,C) Topo II knockdown and scramble control cells were incubated with DOX for 3 days and then treated with Ginkgo biloba leaf extract at 0.8 mg/ml (B) or quercetin at 50 μM (C) for another 4 h without DOX. Treated cells were then lysed and subjected to Western blot analyses with antibodies against γ-H2A.X, p-Chk1, and p-Chk2. Similar results were obtained from three repeated experiments. (D), HepG2 cells were exposed to Ginkgo biloba extract at the concentration of 0.8 mg/ml for 4 h or 24 h. Cells lysis was subjected to Western blot analysis with an antibody against Topo II.

Mentions: Quercetin’s inhibitory potential on Topo II was demonstrated using an in silico approach (Fig. 3) and a cell-free system (Fig. 4A,C), while the inhibitory activity of Ginkgo biloba leaf extract was demonstrated in a cell-free system (Fig. 4D). To investigate whether or not Topo II plays a role in Ginkgo biloba leaf extract- or quercetin-induced DNA damage in cells, a previously generated doxycycline-inducible Topo II knockdown HepG2 cell line27 was used. Topo II was markedly suppressed at the transcriptional level as demonstrated previously27, and at a protein level compared to the scramble control as demonstrated by Western blotting in this study (Fig. 8A). There were no difference between Topo II-silenced HepG2 cells and its scramble control cells by the lactate dehydrogenase activity assay, indicating Topo II-silencing did not affect cell viability (Supplementary Fig. 4).


Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells.

Zhang Z, Chen S, Mei H, Xuan J, Guo X, Couch L, Dobrovolsky VN, Guo L, Mei N - Sci Rep (2015)

Effect of silencing of Topo II on Ginkgo biloba extract- or quercetin-induced DNA damage.(A) HepG2 cells stably expressing doxycycline (DOX)-inducible Topo II knockdown and scramble control cell lines were incubated with DOX for 3 days followed by continued culture for another 4 h without DOX; then the Topo II knockdown efficiency was assessed by Western blot. (B,C) Topo II knockdown and scramble control cells were incubated with DOX for 3 days and then treated with Ginkgo biloba leaf extract at 0.8 mg/ml (B) or quercetin at 50 μM (C) for another 4 h without DOX. Treated cells were then lysed and subjected to Western blot analyses with antibodies against γ-H2A.X, p-Chk1, and p-Chk2. Similar results were obtained from three repeated experiments. (D), HepG2 cells were exposed to Ginkgo biloba extract at the concentration of 0.8 mg/ml for 4 h or 24 h. Cells lysis was subjected to Western blot analysis with an antibody against Topo II.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4588569&req=5

f8: Effect of silencing of Topo II on Ginkgo biloba extract- or quercetin-induced DNA damage.(A) HepG2 cells stably expressing doxycycline (DOX)-inducible Topo II knockdown and scramble control cell lines were incubated with DOX for 3 days followed by continued culture for another 4 h without DOX; then the Topo II knockdown efficiency was assessed by Western blot. (B,C) Topo II knockdown and scramble control cells were incubated with DOX for 3 days and then treated with Ginkgo biloba leaf extract at 0.8 mg/ml (B) or quercetin at 50 μM (C) for another 4 h without DOX. Treated cells were then lysed and subjected to Western blot analyses with antibodies against γ-H2A.X, p-Chk1, and p-Chk2. Similar results were obtained from three repeated experiments. (D), HepG2 cells were exposed to Ginkgo biloba extract at the concentration of 0.8 mg/ml for 4 h or 24 h. Cells lysis was subjected to Western blot analysis with an antibody against Topo II.
Mentions: Quercetin’s inhibitory potential on Topo II was demonstrated using an in silico approach (Fig. 3) and a cell-free system (Fig. 4A,C), while the inhibitory activity of Ginkgo biloba leaf extract was demonstrated in a cell-free system (Fig. 4D). To investigate whether or not Topo II plays a role in Ginkgo biloba leaf extract- or quercetin-induced DNA damage in cells, a previously generated doxycycline-inducible Topo II knockdown HepG2 cell line27 was used. Topo II was markedly suppressed at the transcriptional level as demonstrated previously27, and at a protein level compared to the scramble control as demonstrated by Western blotting in this study (Fig. 8A). There were no difference between Topo II-silenced HepG2 cells and its scramble control cells by the lactate dehydrogenase activity assay, indicating Topo II-silencing did not affect cell viability (Supplementary Fig. 4).

Bottom Line: In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined.In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II.Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA.

ABSTRACT
Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

No MeSH data available.


Related in: MedlinePlus