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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

Interaction of Topo II with quercetin and kaempferol.(A) The superimposition of quercetin (purple) and kaempferol (yellow) in the binding site of Topo II. The Topo II protein backbone is displayed. (B) The H-bond interactions between quercetin and the binding site of Topo II. (C) The H-bond interactions between kaemferol and the binding site of Topo II.
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f3: Interaction of Topo II with quercetin and kaempferol.(A) The superimposition of quercetin (purple) and kaempferol (yellow) in the binding site of Topo II. The Topo II protein backbone is displayed. (B) The H-bond interactions between quercetin and the binding site of Topo II. (C) The H-bond interactions between kaemferol and the binding site of Topo II.

Mentions: Previously, we evaluated the genotoxicity of Ginkgo biloba leaf extract and eight of its constituents in mouse L5178Y cells and found that Ginkgo biloba leaf extract and two flavonoid constituents, quercetin and kaempferol, are mutagenic due to the induction of DNA double-strand breaks6. Considering that some flavonoids target DNA topoisomerases and interrupt the process of DNA replication and that Topo II introduces transient breakage in double strands of DNA, we determined the binding potentials for Ginkgo biloba’s constituents to Topo II using molecular docking. Seven constituents of Ginkgo biloba (quercetin, kaempferol, isorhamnetin, ginkgolide A, ginkgolide B, ginkgolide C, and bilobalide; chemical structures are shown in Supplementary Fig. 2) were docked into the binding pocket of Topo II (Fig. 3) and the docking scores were calculated (Table 1). Three chemicals had Surflex-dock scores >6.0 with quercetin being the highest (7.39), followed by kaempferol (6.69) and isorhamnetin (6.18). For the other 4 chemicals, the Surflex-dock scores range from 3.74 to 4.82. This result suggests that quercetin, kaempferol, and isorhamnetin have relatively higher potentials to bind to the Topo II. For a consensus evaluation, a C-score (0–5) was also calculated and the larger value of C-score represents the better consensus. In our calculation, quercetin and isorhamnetin reached 4 (Table 1). The high C-score further indicates that quercetin has the highest potential to interact with Topo II. As shown in Fig. 3B, a total of 7H-bonds are formed between quercetin and amino acids Asp94, Ser149, Arg98, Asn120, Lys123 (2 H-bonds), and Thr215. Seven H-bonds are formed between kaempferol and Asn91 (2 H-bonds), Lys168, Ile141, Asn120, Lys123, and Thr215 (Fig. 3C). It is worth noting that Asn120, Lys123, and Thr215 are important for determining the orientations of both quercetin and kaempferol. In addition, hydrophobic interaction is also an important factor affecting the binding activity. The binding pocket of Topo II contains hydrophobic bottom, which are suited for the binding of quercetin and kaempferol with hydrophobic aromatic rings.


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)

Interaction of Topo II with quercetin and kaempferol.(A) The superimposition of quercetin (purple) and kaempferol (yellow) in the binding site of Topo II. The Topo II protein backbone is displayed. (B) The H-bond interactions between quercetin and the binding site of Topo II. (C) The H-bond interactions between kaemferol and the binding site of Topo II.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Interaction of Topo II with quercetin and kaempferol.(A) The superimposition of quercetin (purple) and kaempferol (yellow) in the binding site of Topo II. The Topo II protein backbone is displayed. (B) The H-bond interactions between quercetin and the binding site of Topo II. (C) The H-bond interactions between kaemferol and the binding site of Topo II.
Mentions: Previously, we evaluated the genotoxicity of Ginkgo biloba leaf extract and eight of its constituents in mouse L5178Y cells and found that Ginkgo biloba leaf extract and two flavonoid constituents, quercetin and kaempferol, are mutagenic due to the induction of DNA double-strand breaks6. Considering that some flavonoids target DNA topoisomerases and interrupt the process of DNA replication and that Topo II introduces transient breakage in double strands of DNA, we determined the binding potentials for Ginkgo biloba’s constituents to Topo II using molecular docking. Seven constituents of Ginkgo biloba (quercetin, kaempferol, isorhamnetin, ginkgolide A, ginkgolide B, ginkgolide C, and bilobalide; chemical structures are shown in Supplementary Fig. 2) were docked into the binding pocket of Topo II (Fig. 3) and the docking scores were calculated (Table 1). Three chemicals had Surflex-dock scores >6.0 with quercetin being the highest (7.39), followed by kaempferol (6.69) and isorhamnetin (6.18). For the other 4 chemicals, the Surflex-dock scores range from 3.74 to 4.82. This result suggests that quercetin, kaempferol, and isorhamnetin have relatively higher potentials to bind to the Topo II. For a consensus evaluation, a C-score (0–5) was also calculated and the larger value of C-score represents the better consensus. In our calculation, quercetin and isorhamnetin reached 4 (Table 1). The high C-score further indicates that quercetin has the highest potential to interact with Topo II. As shown in Fig. 3B, a total of 7H-bonds are formed between quercetin and amino acids Asp94, Ser149, Arg98, Asn120, Lys123 (2 H-bonds), and Thr215. Seven H-bonds are formed between kaempferol and Asn91 (2 H-bonds), Lys168, Ile141, Asn120, Lys123, and Thr215 (Fig. 3C). It is worth noting that Asn120, Lys123, and Thr215 are important for determining the orientations of both quercetin and kaempferol. In addition, hydrophobic interaction is also an important factor affecting the binding activity. The binding pocket of Topo II contains hydrophobic bottom, which are suited for the binding of quercetin and kaempferol with hydrophobic aromatic rings.

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