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

Zhuhong Zhang,Xiaoqing Guo,Si Chen,Hu Mei,Nan Mei,Lei Guo,Jiekun Xuan,Vasily N. Dobrovolsky,Letha Couch

doi:10.1038/srep14633

Zhuhong Zhang, Xiaoqing Guo + Show 7 more

Open Access

https://doi.org/10.1038/srep14633

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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.

Highlights

Ginkgo biloba leaves and their extracts have been used to treat a variety of ailments, including memory loss and cognitive disorders, arrhythmias and ischemic heart disease, diabetes, skin infection, cancer, and thromboses[1,2]
In the present study, using a combination of in silico tools and biochemical and molecular approaches, we studied the DNA damaging effect caused by Ginkgo biloba leaf extract and seven of its constituents in human hepatic HepG2 cells, and examined the seven Ginkgo biloba constituents for their potential to interact with topoisomerases
Quercetin showed a stronger inhibitory effect, beginning at as low as 3.13 μ M, whereas kaempferol and isorhamnetin showed relatively low inhibitory effects (Fig. 4C). These results clearly demonstrated that some constituents of Ginkgo biloba inhibited topoisomerase II (Topo II) enzyme activity in a concentration-dependent manner, with inhibition potency in the order of quercetin > kaempferol ≈ isorhamnetin ginkgolide A, ginkgolide B, ginkgolide C, and bilobalide

Summary

Introduction

Ginkgo biloba leaves and their extracts have been used to treat a variety of ailments, including memory loss and cognitive disorders, arrhythmias and ischemic heart disease, diabetes, skin infection, cancer, and thromboses[1,2]. Some of Ginkgo biloba’s ingredients possess mutagenic activities For such reasons, Ginkgo biloba leaf extract was nominated to the National Toxicology Program (NTP) for toxicological evaluation and a 2-year carcinogenicity bioassay. In our previous study using the mouse lymphoma cells, we demonstrated that Ginkgo biloba leaf extract and some of its constituents (including quercetin and keampferol) induced genotoxicity and DNA damage[6]. Inhibition of topoisomerase enzyme activity can generate both single and double strand DNA breaks by blocking the ligation step, resulting in significant genotoxicity and cytotoxicity[11]. In the present study, using a combination of in silico tools and biochemical and molecular approaches, we studied the DNA damaging effect caused by Ginkgo biloba leaf extract and seven of its constituents in human hepatic HepG2 cells, and examined the seven Ginkgo biloba constituents for their potential to interact with topoisomerases. We investigated the involvement of topoisomerases in Ginkgo biloba leaf extract- or quercetin-triggered DNA damage in cells where topoisomerase was silenced

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