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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

DNA damage induced by Ginkgo biloba leaf extract in HepG2 cells.(A) HepG2 cells were exposed to increased concentrations (0.2–1.2 mg/ml) of Ginkgo biloba leaf extract for 4 h. The data points represent the means ± S.D. for three independent experiments and asterisk indicates p < 0.05 when the treatment group was compared with the concurrent control. (B) HepG2 cells were exposed to Ginkgo biloba leaf extract for 2, 4, 6, and 24 h. Total cellular protein was extracted and levels of γ-H2A.X, p-Chk1, and p-Chk2 were detected by Western blot analysis. GAPDH was used as a loading control. Similar results were obtained from three independent experiments.
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f1: DNA damage induced by Ginkgo biloba leaf extract in HepG2 cells.(A) HepG2 cells were exposed to increased concentrations (0.2–1.2 mg/ml) of Ginkgo biloba leaf extract for 4 h. The data points represent the means ± S.D. for three independent experiments and asterisk indicates p < 0.05 when the treatment group was compared with the concurrent control. (B) HepG2 cells were exposed to Ginkgo biloba leaf extract for 2, 4, 6, and 24 h. Total cellular protein was extracted and levels of γ-H2A.X, p-Chk1, and p-Chk2 were detected by Western blot analysis. GAPDH was used as a loading control. Similar results were obtained from three independent experiments.

Mentions: We then determined if Ginkgo biloba leaf extract induces DNA damage in human hepatic cells, because Ginkgo biloba leaf extract has been shown to increase an incidence in liver tumors in mice in an NTP 2-year bioassay5. DNA damage was measured by the Comet assay and by the induction of phosphorylation of histone H2A.X (γ-H2A.X) using HepG2 cells. In the standard Comet assay, Ginkgo biloba leaf extract resulted in the induction of DNA damage in a concentration-dependent manner, with a significant difference being observed at concentrations above 400 μg/ml (Fig. 1A). The induction of γ-H2A.X, which occurs after DNA double strand breaks and is generally considered as a hallmark of DNA damage23, was investigated by measuring γ-H2A.X at Ser139 by Western blot analysis. In addition, DNA damage-responsive cell cycle checkpoint-related proteins, phosphorylated-Chk1 (p-Chk1) and phosphorylated-Chk2 (p-Chk2), were also examined using Western blots. Ginkgo biloba leaf extract induced DNA damage in HepG2 cells as demonstrated by a concentration- and time-dependent increase in γ-H2A.X, accompanied by cell cycle perturbation that was evidenced by enhanced expression of both p-Chk1 and p-Chk2 (Fig. 1B).


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)

DNA damage induced by Ginkgo biloba leaf extract in HepG2 cells.(A) HepG2 cells were exposed to increased concentrations (0.2–1.2 mg/ml) of Ginkgo biloba leaf extract for 4 h. The data points represent the means ± S.D. for three independent experiments and asterisk indicates p < 0.05 when the treatment group was compared with the concurrent control. (B) HepG2 cells were exposed to Ginkgo biloba leaf extract for 2, 4, 6, and 24 h. Total cellular protein was extracted and levels of γ-H2A.X, p-Chk1, and p-Chk2 were detected by Western blot analysis. GAPDH was used as a loading control. Similar results were obtained from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: DNA damage induced by Ginkgo biloba leaf extract in HepG2 cells.(A) HepG2 cells were exposed to increased concentrations (0.2–1.2 mg/ml) of Ginkgo biloba leaf extract for 4 h. The data points represent the means ± S.D. for three independent experiments and asterisk indicates p < 0.05 when the treatment group was compared with the concurrent control. (B) HepG2 cells were exposed to Ginkgo biloba leaf extract for 2, 4, 6, and 24 h. Total cellular protein was extracted and levels of γ-H2A.X, p-Chk1, and p-Chk2 were detected by Western blot analysis. GAPDH was used as a loading control. Similar results were obtained from three independent experiments.
Mentions: We then determined if Ginkgo biloba leaf extract induces DNA damage in human hepatic cells, because Ginkgo biloba leaf extract has been shown to increase an incidence in liver tumors in mice in an NTP 2-year bioassay5. DNA damage was measured by the Comet assay and by the induction of phosphorylation of histone H2A.X (γ-H2A.X) using HepG2 cells. In the standard Comet assay, Ginkgo biloba leaf extract resulted in the induction of DNA damage in a concentration-dependent manner, with a significant difference being observed at concentrations above 400 μg/ml (Fig. 1A). The induction of γ-H2A.X, which occurs after DNA double strand breaks and is generally considered as a hallmark of DNA damage23, was investigated by measuring γ-H2A.X at Ser139 by Western blot analysis. In addition, DNA damage-responsive cell cycle checkpoint-related proteins, phosphorylated-Chk1 (p-Chk1) and phosphorylated-Chk2 (p-Chk2), were also examined using Western blots. Ginkgo biloba leaf extract induced DNA damage in HepG2 cells as demonstrated by a concentration- and time-dependent increase in γ-H2A.X, accompanied by cell cycle perturbation that was evidenced by enhanced expression of both p-Chk1 and p-Chk2 (Fig. 1B).

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