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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

Inhibitory effects of the constituents of Ginkgo biloba leaf extract on topoisomerase activities.Using the kDNA decatenation assay (A,C,D), kDNA was incubated at 37 °C for 30 min with or without Topo II enzyme in the presence of the indicated Ginkgo biloba constituents ((A), 500 μM); increasing concentrations of quercetin, kaempferol, and isorhamnetin ((C), 0.78–100 μM); or Ginkgo biloba leaf extract ((D), 0.078–10 μg/ml). Using the supercoiled DNA relaxation assay (B), supercoiled pBR322 plasmid DNA was incubated at 37 °C for 30 min with or without Topo I enzyme in the presence of the indicated Ginkgo biloba constituents (1 mM); and Camptothecin (100 μM) was used as a Topo I positive control inhibitor.
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f4: Inhibitory effects of the constituents of Ginkgo biloba leaf extract on topoisomerase activities.Using the kDNA decatenation assay (A,C,D), kDNA was incubated at 37 °C for 30 min with or without Topo II enzyme in the presence of the indicated Ginkgo biloba constituents ((A), 500 μM); increasing concentrations of quercetin, kaempferol, and isorhamnetin ((C), 0.78–100 μM); or Ginkgo biloba leaf extract ((D), 0.078–10 μg/ml). Using the supercoiled DNA relaxation assay (B), supercoiled pBR322 plasmid DNA was incubated at 37 °C for 30 min with or without Topo I enzyme in the presence of the indicated Ginkgo biloba constituents (1 mM); and Camptothecin (100 μM) was used as a Topo I positive control inhibitor.

Mentions: To validate experimentally the results predicted by the in silico studies, the effects of Ginkgo biloba constituents on Topo II enzymatic activity were investigated in a cell-free system. Topo II activity was measured by detecting human Topo IIα’s ability to relax double-stranded catenated kinetoplast DNA (kDNA) into decatenated relaxed products. Among the seven constituents tested, quercetin, kaempferol, and isorhamnetin showed inhibitory effects on Topo II (i.e., reducing the amount of decatenated DNA and increasing the amount of catenated DNA), whereas ginkgolide A, ginkgolide B, and ginkgolide C, and bilobalide did not show Topo II inhibitory effects up to a concentration of 500 μM when compared to DMSO (Fig. 4A).


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)

Inhibitory effects of the constituents of Ginkgo biloba leaf extract on topoisomerase activities.Using the kDNA decatenation assay (A,C,D), kDNA was incubated at 37 °C for 30 min with or without Topo II enzyme in the presence of the indicated Ginkgo biloba constituents ((A), 500 μM); increasing concentrations of quercetin, kaempferol, and isorhamnetin ((C), 0.78–100 μM); or Ginkgo biloba leaf extract ((D), 0.078–10 μg/ml). Using the supercoiled DNA relaxation assay (B), supercoiled pBR322 plasmid DNA was incubated at 37 °C for 30 min with or without Topo I enzyme in the presence of the indicated Ginkgo biloba constituents (1 mM); and Camptothecin (100 μM) was used as a Topo I positive control inhibitor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Inhibitory effects of the constituents of Ginkgo biloba leaf extract on topoisomerase activities.Using the kDNA decatenation assay (A,C,D), kDNA was incubated at 37 °C for 30 min with or without Topo II enzyme in the presence of the indicated Ginkgo biloba constituents ((A), 500 μM); increasing concentrations of quercetin, kaempferol, and isorhamnetin ((C), 0.78–100 μM); or Ginkgo biloba leaf extract ((D), 0.078–10 μg/ml). Using the supercoiled DNA relaxation assay (B), supercoiled pBR322 plasmid DNA was incubated at 37 °C for 30 min with or without Topo I enzyme in the presence of the indicated Ginkgo biloba constituents (1 mM); and Camptothecin (100 μM) was used as a Topo I positive control inhibitor.
Mentions: To validate experimentally the results predicted by the in silico studies, the effects of Ginkgo biloba constituents on Topo II enzymatic activity were investigated in a cell-free system. Topo II activity was measured by detecting human Topo IIα’s ability to relax double-stranded catenated kinetoplast DNA (kDNA) into decatenated relaxed products. Among the seven constituents tested, quercetin, kaempferol, and isorhamnetin showed inhibitory effects on Topo II (i.e., reducing the amount of decatenated DNA and increasing the amount of catenated DNA), whereas ginkgolide A, ginkgolide B, and ginkgolide C, and bilobalide did not show Topo II inhibitory effects up to a concentration of 500 μM when compared to DMSO (Fig. 4A).

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