Viscum album L. Extracts Protects HeLa Cells against Nuclear and Mitochondrial DNA Damage

Viscum album L. is a semiparasitic plant grown on trees and widely used for the treatment of many diseases in traditional and complementary therapy. It is well known that some activities of Viscum album extracts are varied depending on the host trees, such as antioxidant, apoptosis-inducing, anticancer activities of the plant. The aim of the present study is to examine the comparative effects of methanolic extracts of V. album grown on three different host trees (locust tree, lime tree, and hedge maple tree) on H2O2-induced DNA damage in HeLa cells. Oxidative damage in mitochondrial DNA and two nuclear regions was assessed by QPCR assay. The cells were pretreated with methanolic extracts (10 μg/mL) for 48 h, followed by the treatment with 750 μM H2O2 for 1 hour. DNA damage was significantly induced by H2O2 while it was inhibited by V. album extracts. All extracts completely protected against nuclear DNA damage. While the extract from lime tree or white locust tree entirely inhibited mitochondrial DNA damage, that from hedge maple tree inhibited by only 50%. These results suggest that methanolic extracts of V. album can prevent oxidative DNA damage, and the activity is dependent on the host tree.


Introduction
Reactive oxygen species (ROS) which originate from a range of cellular processes, external factors, and/or various diseases can damage cellular components [1]. The enzymatic and nonenzymatic antioxidant defense systems are natural protectors against oxidative stress caused by ROS. However, these mechanisms cannot completely protect DNA against damage [2]. Although oxidative DNA damage can be repaired, unrepaired damage can accumulate in the cell. The most dramatic results of this accumulation are mutations and cell death [3]. Hence, oxidative DNA damage is an important factor for the aging process and age-related diseases, such as cancer [1,4,5].
Viscum album L. (mistletoe) is a semiparasitic perennial plant that grows on different host trees [6]. Different Viscum album extracts have been used in traditional medicine for the treatment of various diseases such as stroke, atherosclerosis, hypertension, and diabetes [7]. This plant has many biological activities such as anticancer, antiviral, antioxidant, apoptosis-inducing and immunomodulatory properties [8][9][10][11][12][13][14][15]. Although methanolic extract of Viscum album leaves is able to reduce the malondialdehyde (MDA) and reduce glutathione (GSH) levels on kidney and heart of streptozotocininduced diabetic rats and has strong antioxidant activity [11][12][13], the effect on oxidative DNA damage has not been examined in HeLa cells. The main purpose of the present study is to investigate whether the methanolic extract of

Materials and Methods
2.1. Plant Materials. Viscum album L. plants grown on three different host trees (lime tree (Tilia argentea Desf. Ex DC, Tv), hedge maple tree (Acer campestre L. ssp. campestre, Av), and locust tree (Robinia pseudoacacia L., Rv)) were harvested from the Northern part of Istanbul in September 2006. The voucher specimen was identified and deposited in the Herbarium of Biology Department, Istanbul University, Istanbul, Turkey (Tv, ISTF 37486; Av, ISTF 37487; Rv, ISTF 37488). Fresh leaves of each sampling were picked and washed by tap water, followed by distilled water. After drying they were cut into small pieces, weighed, and used immediately or stored separately at −20 • C until use.

Preparation of Extracts.
The extracts were prepared as described earlier [12]. Fresh leaves (20 g) were macerated in methanol (160 mL) in an incubatory shaker (150 rev/min, 25 • C) for 24 hours. After removing the plant residues by filtration, each filtrate was evaporated to dryness under vacuum, and the dried material was weighed. Crude extracts were dissolved in DMSO at a concentration of 40 mg/mL and stored at −20 • C until use. They were designated according to the trees where the plants were collected as Tv (Tilia viscum), Av (Acer viscum), and Rv (Robinia viscum).

Cell Culture.
Human HeLa cervical carcinoma cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and antibiotic-antimycotic mixture (penicillin (100 U/mL), streptomycin (100 μg/mL), amphotericin B (0.25 μg/mL)). Cells were seeded at a concentration of 10 5 cells/mL and maintained at 37 • C in an atmosphere with 5% CO 2 . V. album extract was added to the growth medium, after dissolving in DMSO at a final concentration not exceeding 0.5% (v/v), since DMSO is able to inhibit cell growth above this concentration (data not shown).

Cytotoxicity Test.
The cytotoxic activity of the extracts was tested on HeLa cells by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay based on the reduction of MTT to a colored formazan product by mitochondrial dehydrogenase, which is active only in living cells [16]. The stock solutions of the extracts were diluted with EMEM. Cells (10 5 cells/mL) were seeded into each well of a 96-well plate, containing 200 μL EMEM. After reaching confluence (24 h later), the cells were treated with increasing concentrations (1-500 μg/mL) of V. album methanol extract diluted with EMEM for 48 h. To determine cytotoxic activity of H 2 O 2 the cells were treated with different concentrations (0.05-5 mM) of H 2 O 2 diluted with Hank's balanced salt solution (HBBS) for 1 h at the end of 72 h. In the next step, upper layers were discarded. After washing the adherent cells with phosphate buffer saline (PBS) to minimize the interference of upper layer residue, 10 μL of MTT stock solution (5 mg/mL) was added to each well, and the plates were further incubated for 4 hours at 37 • C. After formation of water insoluble purple formazan crystals, they were dissolved in 200 μL of DMSO, and the resulting optical density was measured by a microplate reader (μQuant, BioTek Instruments Inc., Winooski, VT, USA) at 570 nm and 690 nm (reference) wavelengths. The cell viability was calculated as percentage of viable cells in experimental group versus untreated control group using the following formula: The EC 50 doses on HeLa cells were calculated from a graph of cell viability versus methanolic extract of V. album and nontoxic dose of the plant extract (10 μg/mL) was used for further experiments.  [17]. Depletion of H 2 O 2 in culture media was measured at 10, 30 and 60 min as indicated in Figure 2 by the colorimetric method of Pick and Keisari [18]. In this assay H 2 O 2 is reduced while phenol red is oxidized by the action of HRP. The final purple product exhibits maximum absorption at 612 nm. Briefly, the cells (10 5 cells/mL) were seeded into each well, containing 200 μL EMEM in a 96-well plate. Cell-free (negative) controls were run in parallel with the experimental groups. The cell culture medium (100 μL) containing HBSS plus 50 μM and 100 μM H 2 O 2 was thoroughly mixed with 200 μL phenol red/horseradish peroxidase solution. The mixture was incubated at 37 • C for 5 min, and the reaction was terminated by adding 2 μL of 1 N NaOH. H 2 O 2 concentrations were calculated using a standard curve of H 2 O 2 versus absorbance (610 nm). The standard curve showed a linear relationship between absorbance at 610 nm and H 2 O 2 concentration in the 12.5-100 μM range.

Intracellular ROS Level.
Intracellular ROS level was estimated by using a fluorescent probe, 2 ,7dichlorofluorescein diacetate (DCFH-DA) [19]. After incubation and exposure to oxidative stress, the culture medium was immediately removed, and the cells were washed with PBS followed by incubation for 15 min in 10 μM Evidence-Based Complementary and Alternative Medicine 3 DCFH-DA (100 μL) at 37 • C in an atmosphere with 5% CO 2 . The fluorescence of hydrolyzed 2,7-dichlorofluorescein (DCF) was measured at 10 min intervals for 1 h in a microplate fluorometer with 485 nm excitation/530 nm emission wavelengths (FLx800, BioTek Instruments Inc., Winooski, VT, USA). The relative percentage of ROS production was calculated according to the following equation: where F 0 is fluorescence intensity of untreated control group and F 1 is fluorescence intensity of experimental group.  unpaired t-test. The probability values of P < 0.05 were considered as significant.

Results
All extracts and H 2 O 2 decreased the viability of HeLa cells in a dose-dependent manner ( Figure 1). As illustrated in Figure 1, the half maximal inhibitory concentration (IC 50 ) (Table 1). V. album methanolic extracts have no effect on the steady-state level of intracellular ROS. Av had the highest ROS inhibiting activity compared to 200 μM H 2 O 2 -treated cells. Also, other two extracts (Tv and Rv) were found to have significant inhibitory effects on ROS generation following the oxidative stress induction (Table 1) by using gallic acid (GA) as reference substance.
The gene-specific QPCR assay was performed on nuclear regions (APEX1 and β-globin) and mitochondrial DNA for the detection of oxidative damage caused by H 2 O 2 . The assay is based on the fact that many DNA lesions can block the Taq polymerase, and as a result, the product amplification is decreased [24]. When the different H 2 O 2 concentrations (300 and 750 μM) were applied to determine lesion frequencies in HeLa cells, it was found that in APEX1 and mtDNA regions were similarly induced by 300 μM H 2 O 2 , while in β-globin region no significant induction was  seen relative to control (unpublished data). Therefore, to determine the protective effect of the plant extract on cells, only 750 μM H 2 O 2 has been applied. In this study, lesion numbers per 10 kb on both nuclear and mitochondrial DNA in HeLa cells incubated with or without Viscum extracts were very low under nonstressed conditions (Figure 3). A negative value obtained for Tv in the β-globin region indicated that lesion frequency was much lower than in the others (P < 0.05). However considerable damage in all regions was detected, after the control cells were treated by 750 μM H 2 O 2 . Lesion frequency of mtDNA was higher 2 times than nDNA. As expected, mtDNA was the most sensitive

Discussion
Oxidative stress is an imbalance between formation of ROS and antioxidant defense, resulting in potential cellular damage [3]. Aging and several diseases such as cancers, diabetes, and degenerative disorders have been linked to oxidative stress caused by ROS [1,25]. In living cells ROS are the primary source of oxidative DNA damage under both physiological and increased oxidative stress conditions [25]. Eukaryotic cells protect themselves by antioxidant defence mechanisms such as enzymes, radical scavengers, hydrogen donors, electron donors, peroxide decomposers, singlet oxygen quenchers, enzyme inhibitors, synergists, and metal-chelating agents [26]. Antioxidants may also repair oxidative damage or enhance antioxidant defense either by induction of phase II enzymes or by stimulating mitochondrial biogenesis [27]. Hence, DNA, proteins, and lipid constituents of the cells are protected by antioxidants against oxidative damage. Plants contain various antioxidant constituents that act in different ways and are used in traditional medicine. Depending on the bioactivity, plant extracts as well as many compounds isolated from them are extensively used in pharmaceutical industry as an ingredient drugs or cosmetics; some of these are able to protect against molecular damage [28]. For example, various phenolics compounds and antioxidants reduce oxidative DNA damage in different cell types [20,29,30]. The purpose of this study was to evaluate the protective effect of Viscum album methanolic extracts against nuclear and mitochondrial DNA damage in HeLa cells. In vitro antioxidant activity of this extract has been reported earlier [12,13]. In our previous study composition and activity of Viscum album methanolic extract are found to be dependent on the host tree as well as time of harvesting [12]. In this present study we first showed that Viscum album methanolic extracts are able to inhibit ROS formation induced by H 2 O 2 in HeLa cells. Similar results were obtained in an animal study that reported protective effect of Viscum album against oxidative stress on kidney and heart of diabetic rats [11]. Hence, it is suggested that V. album may protect living cells by inhibition of ROS formation. Surprisingly, the experimental dose of V. album (10 μg/mL) used in the assay did not alter the level of basal damage of both nDNA and mtDNA when there was no induction of stress ( Figure 3). This result may show that undetectable damage has occurred under physiological conditions. We also found that the mitochondrial genome is significantly (P < 0.001) more sensitive to H 2 O 2 -induced oxidative stress than both nuclear loci ( Figure 3). Previous studies demonstrated that mtDNA is more susceptible to oxidative agents than nDNA [23,31]. The susceptibility of mtDNA to oxidative damage is due to chain-propagation reactions or electron leakage from the respiratory chain [32]. mtDNA does not have introns with a high transcription rate, providing a high probability of oxidative modification of the expressed region [33]. Also the presence of localized metal ions in mitochondria may function as catalysts for the generation of ROS and the stimulation of secondary ROS reactions due to damage to the ETC and/or through lipid peroxidation [23].
Furthermore, we demonstrate that Viscum album methanolic extracts are able to prevent DNA damage induced by H 2 O 2 . It is predicted that several thousand DNA lesions per cell occur each day in humans by different stress factors 6 Evidence-Based Complementary and Alternative Medicine [34]. If the cells do not have effective DNA repair and/or antioxidant defense mechanisms, this damage accumulates in DNA (in especially mtDNA), and this may result in agerelated disorders. DNA damage in tumor suppressor genes and oncogenes may also be a likely cause of cancer [3][4][5]. Therefore it is especially significant that Viscum album can protect mtDNA, which has an important role in the aging and APEX1 gene (actively transcribed for a DNA repair enzyme) against this accumulation via inhibition of oxidative DNA damage. Nevertheless, no significant correlation between prevention of DNA damage and the inhibition of ROS was obtained, suggesting that the antioxidant potential of Viscum album extract is generated by a complex synergy and different mechanisms of active molecules, not only by ROS inhibition.
Antioxidant activity of plants is associated with their bioactive compounds, mainly antioxidant phenolics, because of their ability to scavenge free radicals. In V. album methanolic extracts, the total quercetin content was measured as 4.92 μg/g crude extract of Tv, 3.92 μg/g crude extract of Av, 7.97 μg/g crude extract of Rv, respectively (unpublished data). Thus, the high quercetin content in V. album extract may be responsible for the protection of DNA damage in HeLa cells.
In conclusion, taken together these results suggest that methanolic extract of Viscum album can protect against DNA damage via direct inhibition of ROS formation and/or indirectly by other mechanisms, through induction of oxidative damage repair and phase II enzymes. Therefore, dietary intake of Viscum album extract may lower the risk of oxidative stress-mediated diseases such as some cancers, diabetic complications, degenerative and gastrointestinal diseases, or atherosclerosis via reduction of oxidative DNA damage and intracellular levels of ROS. Further studies are needed in order to define the possible beneficial outcomes of its dietary use and to identify mechanisms of action.