Ginkgo biloba L. Leaf Extract Protects HepG2 Cells Against Paraquat-Induced Oxidative DNA Damage

Ginkgo biloba L. leaf extracts and herbal infusions are used worldwide due to the health benefits that are attributed to its use, including anti-neoplastic, anti-aging, neuro-protection, antioxidant and others. The aim of this study was to evaluate the effect of an aqueous Ginkgo biloba extract on HepG2 cell viability, genotoxicity and DNA protection against paraquat-induced oxidative damage. Exposure to paraquat (PQ), over 24 h incubation at 1.0 and 1.5 µM, did not significantly reduce cell viability but induced concentration and time-dependent oxidative DNA damage. Ginkgo biloba leaf extract produced dose-dependent cytotoxicity (IC50 = 540.8 ± 40.5 µg/mL at 24 h exposure), and short incubations (1 h) produced basal and oxidative DNA damage (>750 and 1500 µg/mL, respectively). However, lower concentrations (e.g., 75 µg/mL) of Ginkgo biloba leaf extract were not cytotoxic and reduced basal DNA damage, indicating a protective effect at incubations up to 4 h. On the other hand, longer incubations (24 h) induced oxidative DNA damage. Co-incubation of HepG2 cells for 4 h, with G. biloba leaf extract (75 µg/mL) and PQ (1.0 or 1.5 µM) significantly reduced PQ-induced oxidative DNA damage. In conclusion, the consumption of Ginkgo biloba leaf extract for long periods at high doses/concentrations is potentially toxic; however, low doses protect the cells against basal oxidative damage and against environmentally derived toxicants that induce oxidative DNA damage.


Introduction
Plants and herbs have been used since ancient times and today many people still rely on medicinal plants for their primary health care, others use them as a natural source of bioactive molecules. Ginkgo Concerning the effect of G. biloba extract on cell viability, cells were exposed for 24 h ( Figure 1B) to concentrations up to 1500 µg/mL (please see Sections 4.1 and 4.2 for experimental details). As observed, cell viability was reduced with the increasing concentration of the extract. In this experiment, we observed an IC 50 of 540.8 ± 40.5 µg/mL after exposure for 24 h, corroborating previous works that reported the cell toxicity effect of G. biloba extract on HepG2 cells [30]. Figure 1B shows that concentrations up to 100 µg/mL were totally safe. Thus, a concentration lower than 100 µg/mL was chosen for the next experiments.

Paraquat Induces DNA Damage in HepG2 Cells
We aimed to study the effect of PQ as an inducer of DNA damage in HepG2 cells, and without compromising cell viability, we evaluated the % of DNA in the tail of cells that were first exposed to PQ and then subjected to comet assay (please see Methods). Figure 2 shows the results obtained for PQ-induced DNA damage in HepG2 cells after 30 and 60 min (Figure 2A,B, respectively) exposure to different concentrations (1.0, 1.5, 2.0 and 5.0 µM PQ). As observed, 30 min ( Figure 2A) incubation with 1.5 µM PQ increased DNA damage, strand breaks (light blue bar; 5.79 ± 1.30% of DNA in the tail), and oxidative damage (dark blue bar; 5.78 ± 1.63%). Similar oxidative damage was also observed at 1.0 µM (dark blue bars; 5.98 ± 0.58%). Higher concentrations of PQ did not result in a significantly higher amount of DNA damage (Figure 2A). Longer exposure to PQ, for 1 h incubation ( Figure 2B), showed an increase in oxidative damage (dark blue bars) for 1.5 and 5.0 µM PQ, compared to the respective control. However, strand breaks (light blue bars) were not different from the control, which may be a result of internal cell repair mechanisms. The asterisk (*) means significant differences compared to the respective control. The # means significant differences between treatment in the presence and absence of FPG. P-value < 0.05.
In a parallel study, two PQ concentrations, 1.0 µM ( Figure 2C) and 1.5 µM ( Figure 2D) were assayed on DNA damage along time, namely, at 0.5, 1, 4 and 24 h incubation. Over time, 1.0 µM PQ did not induce significant strand damage, in comparison to control (P > 0.05). But, although the level of oxidized purines remained similar to control (P > 0.05) at 1 h treatment, it peaked at 4 h of treatment (dark blue bar; 14.92 ± 0.96% of DNA in tail), with this value being 4.5-fold higher on average, than control (P < 0.05). The % DNA in the tail at 24 h (dark blue bar; 16.53 ± 0.37) is slightly higher than at 4 h, but the differences are statistically significant (P < 0.05; Figure 2C). For 1.5 µM PQ ( Figure 2D), the results show a similar pattern, that is, the maximum oxidative damage at 24 h incubation (dark blue bar; 25.28 ± 4.60%) is~7.6-fold higher than control.
Paraquat is an oxidizing molecule that has been shown to induce DNA damage in some studies (e.g., mice hepatocytes [31] alveolar macrophages [29], Caco-2 cells [32]). The hypothesized mechanism involves PQ reduction by NADPH-oxidase, decreasing the levels of NADPH, which is oxidized to NADP. Reduced PQ is regenerated into oxidizing-PQ with the production of a superoxide radical (O 2 •− ), which may be converted into hydrogen peroxide (H 2 O 2 ) by superoxide dismutase (SOD) and then eliminated by catalase (CAT). The activity of these enzymes determines the production of oxygen reactive species (ROS) that contribute to DNA damage [31,[33][34][35]. In the present study, PQ concentration and exposure time were optimized for HepG2 cells. Results show that damage is concentration and time-dependent, which could be attributed to PQ inducing lipid peroxidation and peroxyl radicals, which then contribute to DNA damage, as described previously [33,[36][37][38]. Also, PQ can increase oxidative stress and mitochondrial damage [38]. The values of DNA damage for 1 µM PQ were identical from 4 to 24 h, which may suggest that the produced radicals did not injure the repair enzymes that attempt to restore the initial sequence of DNA.

Effect of Ginkgo biloba Extract on DNA Damage
Concerning the effect of Ginkgo biloba extract by itself, on DNA damage, was evaluated using the following concentrations: 75, 750 and 1500 µg/mL and exposure periods: 5 min and 30 min, and 1 h, 4 h and 24 h. Figure 3A presents the effect of 1 h exposure to various concentrations of G. biloba extract, and shows the dose-dependent DNA damage for higher concentrations, which is more evident when measured as oxidative damage (with FPG). Concentrations of 750 and 1500 µg/mL significantly increased strand breaks (light grey bars) compared to control, and oxidized purines (green bars) attain a higher percentage for 1500 µg/mL of extract (~2.4-fold higher than control, P < 0.05). However, at 75 µg/mL the extract did not induce DNA damage for 1 h exposure ( Figure 3A), it even showed a reduction in oxidative damage, thus this concentration was used to study the damage over time ( Figure 3B). As can be seen in Figure 3B, 75 µg/mL G. biloba extract over time did not significantly affect DNA damage, which was evaluated by strand breaks (P > 0.05), and its effect on oxidative damage (with FPG) only shows a significant increase in oxidized purines at 24 h incubation (~2-fold higher than control). These results are in line with the results of cell viability ( Figure 1A), and corroborate the results reported by Grollino et al., in which no DNA damage was detected by conventional comet assay (strand breaks analysis) in HepG2 cells exposed to Ginkgo biloba extract (IDN5933, Ginkgoselect®Plus) at concentrations up to 2 mg/mL. These authors, pointed out that cell toxicity is a result of oxidative stress [39], and here we report that Ginkgo biloba aqueous extract at higher concentrations induces oxidative DNA damage ( Figure 3B). Thus, high concentrations of Ginkgo biloba aqueous extract induced DNA damage, suggesting the direct and nonspecific harmful effects of G. biloba leaf extracts in DNA when used at high concentrations. Babich and co-workers [17] reported that polyphenols can injure DNA depending on their concentration. G. biloba compounds that may account for this genotoxicity include ginkgetin [40], quercetin [41] and ginkgolic acids [18,19,42], which contribute as individual compounds depending on their concentration in the extract [43].

Effect of Ginkgo biloba Extract on DNA Damage
Concerning the effect of Ginkgo biloba extract by itself, on DNA damage, was evaluated using the following concentrations: 75, 750 and 1500 µg/mL and exposure periods: 5 min and 30 min, and 1 h, 4 h and 24 h. Figure 3A presents the effect of 1 h exposure to various concentrations of G. biloba extract, and shows the dose-dependent DNA damage for higher concentrations, which is more evident when measured as oxidative damage (with FPG). Concentrations of 750 and 1500 µg/mL significantly increased strand breaks (light grey bars) compared to control, and oxidized purines (green bars) attain a higher percentage for 1500 µg/mL of extract (∼2.4-fold higher than control, P < 0.05). However, at 75 µg/mL the extract did not induce DNA damage for 1 h exposure ( Figure 3A), it even showed a reduction in oxidative damage, thus this concentration was used to study the damage over time ( Figure 3B). As can be seen in Figure 3B, 75 µg/mL G. biloba extract over time did not significantly affect DNA damage, which was evaluated by strand breaks (P > 0.05), and its effect on oxidative damage (with FPG) only shows a significant increase in oxidized purines at 24 h incubation (∼2-fold higher than control). These results are in line with the results of cell viability ( Figure 1A), and corroborate the results reported by Grollino et al., in which no DNA damage was detected by conventional comet assay (strand breaks analysis) in HepG2 cells exposed to Ginkgo biloba extract (IDN5933, Ginkgoselect®Plus) at concentrations up to 2 mg/mL. These authors, pointed out that cell toxicity is a result of oxidative stress [39], and here we report that Ginkgo biloba aqueous extract at higher concentrations induces oxidative DNA damage ( Figure 3B). Thus, high concentrations of Ginkgo biloba aqueous extract induced DNA damage, suggesting the direct and nonspecific harmful effects of G. biloba leaf extracts in DNA when used at high concentrations. Babich and co-workers [17] reported that polyphenols can injure DNA depending on their concentration. G. biloba compounds that may account for this genotoxicity include ginkgetin [40], quercetin [41] and ginkgolic acids [18,19,42], which contribute as individual compounds depending on their concentration in the extract [43].
Hecker and co-workers [19] reported the release of lactate dehydrogenase (LDH) from human keratinocyte (HaCaT) and in rhesus monkey kidney tubular epithelial cell line (LLC-MK2) incubated with Ginkgo extract (EGb 761), which is indicative of loss of membrane integrity. Other studies have suggested that the pro-oxidant activity of G. biloba extract causes cell toxicity [16,39,43]. Future studies should explore the involvement of oxidative stress enzymatic system to long-term treatment with G. biloba extract.
From the above experiment, we chose to use 75 µg/mL G. biloba extract and 4 h incubation, as this concentration and period of incubation do not cause cell toxicity and DNA damage (basal and oxidative).  Hecker and co-workers [19] reported the release of lactate dehydrogenase (LDH) from human keratinocyte (HaCaT) and in rhesus monkey kidney tubular epithelial cell line (LLC-MK2) incubated with Ginkgo extract (EGb 761), which is indicative of loss of membrane integrity. Other studies have suggested that the pro-oxidant activity of G. biloba extract causes cell toxicity [16,39,43]. Future studies should explore the involvement of oxidative stress enzymatic system to long-term treatment with G. biloba extract.
From the above experiment, we chose to use 75 µg/mL G. biloba extract and 4 h incubation, as this concentration and period of incubation do not cause cell toxicity and DNA damage (basal and oxidative).

Ginkgo biloba Extract Protects HepG2 Cells Against Paraquat-Induced DNA Damage
From the above experiment, we chose to use 75 µg/mL G. biloba extract and 4 h incubation, because this concentration and period of incubation do not cause cell toxicity or DNA damage with significant differences. HepG2 cells were concomitantly exposed to 75 µg/mL G. biloba and to 1.0 or 1.  The asterisk (*) means significant differences compared with the respective control (non-exposed cells). The same letter means significant differences between treatments. P-value < 0.05.
Our findings corroborate the results of other experimental models, such as in the case of simultaneous incubation of Saccharomyces cerevisiae with G. biloba water extract plus hydrogen peroxide, where G. biloba showed a DNA protective effect [14].
The pro-oxidant potential of G. biloba extract at high concentrations, which may induce mutations in the DNA sequence that will join to naturally occurring mutations, is of interest due to concerns about the potential health risks for the general population who are exposed to diet supplements. Over time, cell antioxidant defenses weaken or become inadequate, increasing the risk of developing genetically-based diseases, such as cancer. A study in Swiss albino mice showed that long-term treatment with G. biloba extract can cause aneuploids, decreased fertility and hampers the pre-implantation of embryos due to embriotoxic and neurotoxic effects. This also warrants its careful use as a remedy for male impotence and/or erectile dysfunction [3]. Some recent studies highlight some negative results from relatively large clinical trials on the frequent consumption of G. biloba for long periods of time [43]. We have also observed that high doses of G. biloba extract are cytotoxic ( Figure 1A) and cause oxidative DNA damage (Figure 3).
Transposing this study to the consumer point of view, a tea-bag contains about 1.5 g of dry material which serves to make one cup of infusion (~250 mL). Taking into consideration that most of the compounds in the infusion will suffer pH degradation (in the stomach and intestine) and/or oxidation, and that absorption will not be complete as some compounds will not be absorbed because of the presence of fibers in the gut, we may speculate that the amount of extract reaching the whole body is in the antioxidant range. However, caution must be taken if several G. biloba infusions are consumed per day on a continuous basis. Although, the standardized G. biloba supplements have a known composition and are composed of selected compounds that are beneficial to the human organism, consumers should also pay attention to the manufacturer's recommendations for dosage and not overconsume these supplements.

Conclusions
Paraquat may be used experimentally as oxidative stress inducer. The aqueous extract of Ginkgo biloba leaf at low concentrations (up to 200 µg/mL), did not produce cell toxicity or induce DNA damage for short exposures, but instead resulted in a reduction in oxidative DNA damage, indicating an antioxidant effect. However, at higher concentrations, cell toxicity and oxidative DNA damage were observed in a concentration and time-dependent manner. Most importantly, at low concentrations, i.e., as found in the regular usage of a tea-bag per day, a protective effect against PQ-induced DNA damage was observed. Given these results, an antioxidant effect for low concentrations and oxidant properties of Ginkgo biloba extract at high concentrations were observed, thus care should be taken when using frequent infusions or extracts directly from leaves for long periods and at high dosages. The moderate consumption of G. biloba infusions might contribute flavonoids and other phenolic compounds that act as free radical scavengers to reduce cell oxidative stress. However, high doses, in addition to providing beneficial compounds, also appear to contribute compounds that generate cellular stress, which can be translated into DNA damage. PQ was used as a model for oxidative DNA damage, further studies could examine the protective effect of Ginkgo biloba extract (at low doses) against other environmental stressors, such as food containing contaminants (e.g., pesticides, heavy metals).

Preparation of Ginkgo biloba Aqueous Extract
Ginkgo biloba leaves were collected at Vila Real, Portugal from a tree sample in the University Botanical Garden (voucher ID: HVR4565, UTAD, Quinta de Prados, Vila Real; WGS84 103 (41.2884381; 7.739039). Aqueous extraction was performed as described by Ding and co-workers in [44]. Briefly, the leaves were washed with distilled water, air-dried at room temperature in the shade and then powdered. Then, 30 mL of boiling distilled water was added to 3.6 g of plant material and this mixture was heated in a water bath at 100 • C for 5 min and then centrifuged at 2000 ×g for 15 min. The supernatant was collected, filtrated, the pH adjusted to 6.5 and it was then frozen at −20 • C before its lyophilization. After lyophilization, a yield of 15.3% ((dried extract weight/ dried plant material) × 100%) was obtained.

Cell Viability/Cytotoxicity Assay
HepG2 cells were handled as described in [45,46]. Briefly, HepG2 cells were seeded in 96-well plates, at 5 × 10 4 cells/mL (100 µL/well) for 24 h. After seeding, the culture media was replaced by FBS-free culture media supplemented with PQ (1-8 µM) or with G. biloba extract at the desired concentration (from 50 to 1500 µg/mL). To prepare these solutions, a 20 mg/mL stock solution of G. biloba extract was prepared in FBS-free culture medium, and from this, subsequent dilutions were made in FBS-free culture medium. Cell viability was evaluated with Alamar blue assay (Alfagene, Invitrogen, Portugal). Thus, 24 h after exposure of the cells, FBS-free culture media supplemented with 10% (v/v) of Alamar blue was added (100 µL to each well), and absorbance at 570 nm (reduced form; resorufin) and 620 nm (oxidized form; resazurin) was read~4 h later. Data were analyzed by calculating the percentage of Alamar blue reduction (according to the manufacturer's recommendation) and expressed as a percentage of control (untreated cells), as reported in [47].

Cell Treatment and Experimental Conditions for Comet Assay
HepG2 cells were seeded onto 12-well culture plates at a density of 1 × 10 5 cells/mL (1 mL/well). After seeding for 24 h, the culture medium was discarded and replaced by Ginkgo biloba extract and/or paraquat (at desired concentrations, please see the Results section for details) diluted in FBS-free culture medium. Negative controls were made throughout using FBS-free culture medium.
Treatments were performed in three distinct conditions: (i) For optimization of the binomial concentration-time to maximize PQ-induced oxidative stress, cells were incubated with PQ at final concentrations of 1.0, 1.5, 2.0 and 5.0 µM for 30 min or 1 h, and for lower PQ concentrations (1.0 and 1.5 µM), incubations of 0.5, 1, 4 and 24 h were used. (ii) To study the effect of G. biloba extract on DNA damage, cells were exposed to 75, 750 and 1500 µg/mL of G. biloba extract (stock solution diluted in FBS-free culture media) for 1 h, and for 75 µg/mL of G. biloba extract, comet assay was performed at 1, 4 and 24 h after cell exposure. (iii) Finally, a set of cells were submitted to G. biloba extract (75 µg/mL) incubation for 4 h and simultaneously incubated (co-incubation) with 1.0 or 1.5 µM PQ.

Measurement of Strand Breaks
The two gels, placed on each side of a microscope slide, served as duplicates. Slides with cells were immersed into cold lysis solution (2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris, pH 10 adjusted with NaOH, and 1% Triton X-100, which was added immediately before use) for 1 h at 4 • C. Then, slides were placed in electrophoresis solution (0.3 M NaOH, 1 mM EDTA) for 40 min at 4 • C in the dark (alkaline treatment). Subsequently, gels underwent electrophoresis at 25 V and 300 mA, for 30 min at 4 • C in the dark. After electrophoresis, gels were washed twice with cold (4 • C) distilled water. Strand breaks allow DNA to extend from the nucleoids towards the anode, forming a "comet tail".

Measurement of Oxidative DNA Damage
FPG (a lesion specific enzyme) was used to measure oxidized purines, including 8-oxoguanine. After lysis the slides were washed three times with enzyme reaction buffer (0.1 M KCl, 0.5 mM EDTA, 40 mM HEPES, 0.2% (w/v) bovine serum albumin, pH adjusted to 8 with KOH) at 4 • C. After washing, 50 µL of FPG was dropped onto each gel, covered with glass and incubated at 37 • C for 30 min in a humid chamber. Alkaline treatment and electrophoresis then followed as described above. The additional breaks formed at the oxidized purines lead to an increase in the amount of DNA in the tail.

Comet Evaluation
Comets were visualized by fluorescence microscopy (Nikon Eclipse E400) after staining with ethidium bromide (Sigma Chemical), and computerized image analysis was applied using Comet Assay IV software. The percentage of DNA in the tail (mean value from 50 comets per gel) was taken as a measure of DNA damage.

Data and Statistical Analysis
Data from different experiments are presented as mean ± standard deviation (S.D.) or as mean ± S.E.M. (as mentioned). The value of IC 50 (concentration that inhibits 50% of cell viability/proliferation or half maximal inhibitory concentration ) was calculated as reported by Silva et al [50]. Data analysis was performed using the software Statistical Program for Social Sciences (SPSS), version 20.0, as described in [49]. Statistical significance of differences between mean values was assessed by the ANOVA test (confidence level 95%; P-value < 0.05). The Tukey test was applied for multiple comparisons.