Aqueous Extracts of the Edible Gracilaria tenuistipitata are Protective Against H2O2-Induced DNA Damage, Growth Inhibition, and Cell Cycle Arrest

Potential antioxidant properties of an aqueous extract of the edible red seaweed Gracilaria tenuistipitata (AEGT) against oxidative DNA damage were evaluated. The AEGT revealed several antioxidant molecules, including phenolics, flavonoids and ascorbic acid. In a cell-free assay, the extract exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity that significantly reduced H2O2-induced plasmid DNA breaks in a dose-response manner (P < 0.001). The AEGT also suppressed H2O2-induced oxidative DNA damage in H1299 cells by reducing the percentage of damaged DNA in a dose-response manner (P < 0.001) as measured by a modified alkaline comet-nuclear extract (comet-NE) assay. The MTT assay results showed that AEGT confers significant protection against H2O2-induced cytotoxicity and that AEGT itself is not cytotoxic (P < 0.001). Moreover, H2O2-induced cell cycle G2/M arrest was significantly released when cells were co-treated with different concentrations of AEGT (P < 0.001). Taken together, these findings suggest that edible red algae Gracilaria water extract can prevent H2O2-induced oxidative DNA damage and its related cellular responses.


AEGT Modulates H 2 O 2 -Induced Plasmid DNA Strand Breaks
This study assessed the protective effect of AEGT against pBR322 plasmid DNA cleavage induced by H 2 O 2 treatment. In the absence of H 2 O 2 , plasmid DNA appeared mainly in supercoiled form (S) (Figure 2a

AEGT Promotes Cell Survival under H 2 O 2 Treatment
We further tested whether the modulating effect of AEGT also promotes H1299 cell survival under H 2 O 2 treatment. The H1299 cells were treated with or without H 2 O 2 in the presence of 0.5, 1, 2, and 4 mg/mL of AEGT for 24 h. The cell viability was then determined by MTT assay. Treating H1299 with 0.5, 1, and 4 mg/mL AEGT alone showed no significant adverse effects on cell viability (P > 0.05 versus control) (Figure 4a). In the presence of H 2 O 2 -induced oxidative DNA damage, AEGT increased cell viability in a dose-dependent manner. From a baseline cell viability of 55.7 ± 3.8, treatment with 0.5, 1, 2 and 4 mg/mK AEGT significantly increased the cell viability of H 2 O 2 -treated cells to 63.3 ± 8.3, 76.5 ± 4.7, 89.1 ± 3.2 and 100.3 ± 16.0, respectively (P < 0.05 to 0.005, Figure 4b). Notably, the viability of H1299 cells treated with both H 2 O 2 and 4 mg/mL AEGT did not significantly differ from that in control cells (P > 0.05, Figure 4b).  Data are means ± S.D. (n = 3). Levels not connected by same big letter significantly differed. Except (b) A vs. BC (P < 0.05), comparisons between other different letters in (a) and (b) showed significant differences (P < 0.005).

AEGT Prevents Cell Cycle Arrest by H 2 O 2
After 24 h treatment with H 2 O 2 in the presence of 0.5, 1, 2, and 4 mg/mL AEGT, the cell cycle distributions of H1299 cells were further analyzed by flow cytometry. The control H1299 displayed a major G1 peak and a minor G2/M peak comprising 62.0% and 18.0% of the cell population, respectively ( Figure

Discussion
This study investigated the antioxidant activity of AEGT in terms of its biochemical characteristics, DNA damage protection, cell growth, and cell cycle recovery after treatment with H 2 O 2 alone or with H 2 O 2 and AEGT. The selected damaging agent in this study was H 2 O 2 because it exhibits either direct (oxidation of its target) or indirect (involving peroxiredoxins) signaling [18] and induction of oxidative stress [20][21][22]. The AEGT effectively suppressed ROS-induced DNA strand break in cell-free assay ( Figure 2). Sensitive comet-NE assay of cellular DNA damage ( Figure 3) and cell cycle profiling ( Figure 5) further showed that AEGT increases H1299 cell survival by modulating oxidative stress ( Figure 4). In accordance with many other reports of H 2 O 2 treatment in various cell lines [23][24][25], H 2 O 2 -treated H1299 also showed prominent G2/M arrest, and AEGT conferred effective recovery from H 2 O 2 -induced G2/M arrest although the sub-G1 population in H 2 O 2 /4 mg/mL AEGT is mildly increased ( Figure 5). Earlier reports that ROS correlate with immunity [26], anti-hepatoma activity [27], HCV-related activity [28], and Alzheimer's disease [29] suggest that AEGT has potential applications in testing recovery from ROS-related cellular responses.
Typical phenolic compounds known to exhibit antioxidant activity include phenolic acids and flavonoids [30,31]. In addition to phenolic compounds, tannins [32], ascorbic acid [33], and pigments [34] are other potential antioxidant compounds contained in seaweeds. In the current study, polyphenols, flavonoids, and ascorbic acid were also detected in AEGT. Algal polyphenols are known to confer the major antioxidant activity of seaweed extracts [35]. Analysis of DPPH radical scavenging is largely performed to assess the free radical scavenging effect of specific compounds or extracts and can quickly indicate antioxidant activity [36,37]. The DPPH radical scavenging and phenolic content reportedly have a strong correlation in plant-based foods such as herbs [38] and seaweeds [39]. Moreover, flavonoids [40] and ascorbic acid [36] also exhibit free radical scavenging activities.
Oxidative DNA damage can result from ROS that are generated by cellular metabolism or environmental stress and it is believed to contribute to aging, carcinogenesis and other diseases [41]. For In vitro assays, the conversion of the supercoiled form of plasmid DNA into either open-circular or linear form has been used as a standard indicator of DNA damage [42].
The literature shows that boiled G. tenuistipitata extract exhibits radical scavenging activities with an IC 50 of 24.22 mg/mL in DPPH radical scavenging assay [10]. This study shows that AEGT exhibits an even more potent antioxidant activity. A 60% DPPH scavenging activity was observed at only 3 mg/mL. Considering the rapid growth of seaweed and the extensive use of seaweed extracts in the food and cosmetics industries, AEGT represents an attractive multifunctional alternative for such applications. Future studies to identify the bioactive fraction of AEGT would further enhance the economic value of red algal extract. The potential protective role of AEGT to other types of cancer cells warrants further investigation. Moreover, many algal extracts reportedly exhibit other interesting biological effects, such as the protective effects of enzymatic extracts from microalgae against H 2 O 2 -induced DNA damage [43] and the protective effects of Phaeodactylum tricornutum lipid-rich algae extract against proteasome activity [44]. Similarly, we found that AEGT protects against H 2 O 2 -induced plasmid and cellular DNA damage, cytotoxicity, and cell cycle arrest without affecting cell viability as observed in AEGT alone.

Raw Materials
Specimens of G. tenuistipitata collected during spring 2009 from a culture farm at Kouhu beach, Yunlin County, Taiwan, were delivered to the laboratory at 0 °C. In the laboratory, the seaweeds were washed with running tap-water to remove epiphytes and encrusting material, immersed twice in distilled water, and then lyophilized. After pulverizing the dried sample and passing it through a 60-mesh sieve, the lyophilized sample was ground to fine powder and stored at −40 °C.

Extraction and Isolation of Seaweed G. tenuistipitata
After the addition of 1,000 mL deionized water, the dried samples (50 g) were agitated in a mechanical shaker at room temperature for 24 h. The extract was then filtered with Whatman No. 1 filter paper. The filtrate solution was evaporated to dryness at 40 ± 2 °C in a rotary evaporator (Buchi Laboratoriums-Technik, Buchs, Switzerland) and then lyophilized. The lyophilized extract was stored in a sealed container at −40 °C until use.

Determination of Total Phenolics, Flavonoid, and Ascorbic Acid of AEGT
The total phenolic compounds in AEGT extracts were determined with Folin-Ciocalteu reagent as described by Singleton and Rossi [45] using gallic acid as standard. The total phenolic content was expressed as gallic acid equivalent in μg per mg of dry sample. Ascorbic acid was quantitatively determined using the 2,6-dichloroindophenol-Na dye method as described by Jones and Hurghes [46]. Results were presented on a dry matter basis (μg ascorbic acid per mg of dry sample). Flavonoid content was determined by the colorimetric method described by Woisky and Salatino [47]. Total flavonoid content was calculated in quercetin equivalents based on a calibration curve and expressed as μg quercetin equivalents per mg of dry sample.

Free Radical Scavenging Activity
The ability of AEGT to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was determined as described earlier [48]. Briefly, a 1 mM methanolic solution of DPPH (1 mL) was mixed with solution of each extract (1, 2, and 4 mg/mL, 3 mL). After vigorous vortexing, the mixture was kept in darkness for 30 min at room temperature. Absorbance was measured at 517 nm, and activity was expressed as percentage of DPPH scavenging compared to control. The percentage of scavenging activity was calculated as [(A c − A s )/A c ] × 100 where A s is the absorbance measured with the extract sample in the assay and A c is the absorbance of control (without extract sample). Butylated hydroxyanisole (BHA) and ascorbic acid (Vit. C) were used as positive controls.

Plasmid DNA Cleavage Assay
Conversion of the supercoiled (S) form of plasmid DNA to the open-circular (OC) and/or further linear (L) forms was analyzed as an indicator of DNA strand breaks [49]. Reaction mixtures (10 μL) containing 150 ng of pBR322 plasmid DNA, 0.1 mM FeSO 4 , 0.05% H 2 O 2 and AEGT at concentrations of 0, 1, 2, and 4 mg/mL were incubated at 37 °C for 30 min. After stopping the reaction by adding 2 μL of 6 × gel loading dye (0.05% bromophenol blue, 40 mM EDTA and 50% glycerol (v/v), electrophoresis was performed on 0.8% agarose gel in 0.5 × TAE buffer at 50 V for 1-2 h. The DNA in the gel was stained with ethidium bromide (final concentration 0.8 g/mL) and then visualized and photographed under ultraviolet light. The formula used for calculating the percentage of supercoiled DNA was as follows: S% = (band density of S / band density of (S + L + OC), where the band density was determined by the Gel-pro Analyzer 4.0 (Media Cybernetics, Bethesda, MD, USA). Relative S% = S% of test sample/S% of control.

Comet-NE Assay
The comet-NE is more sensitive than the traditional comet assay [50,51]. The comet-NE assay using nuclear extracts (NEs) prepared from NB4 cells (human acute promyelocytic leukemia) cell line [52][53][54] was performed using a protocol described previously [52][53][54]. Aliquots (100 L of H1299 cell suspensions (1 × 10 6 cells/mL in PBS) were mixed with equal volumes of 1.2% low-melting agarose (in PBS, pH 7.4) and immediately pipetted onto a glass slide precoated with 1% regular agarose (in distilled water). The slides were then immersed in freshly prepared ice-cold cell lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris pH 10, 1% N-laurylsarcosine, 1% Triton X-100 and 10% dimethylsulfoxide or DMSO) incubated at 4 °C for 2 h and then rinsed three times with deionized water. A 20 L excision mixture containing 0.6 g NE, 50 mM Hepes-KOH (pH 7.9), 70 mM KCl, 5 mM MgCl 2 , 0.4 mM EDTA, 2 mM ATP, 40 mM phosphocreatine and 2.5 mM creatine phosphokinase was then added to each slide. After applying the cover slips, the slides were incubated at 37 °C for 2 h in a humidified space for NE digestion. The slides were denatured in 0.3 N NaOH, 1 mM EDTA for 20 min and then electrophoresed at 20 V, 300 mA for 25 min. After washing with deionized water, the slides were neutralized in 0.4 M Tris-HCl, pH 7.5 and stained with 40 L propidium iodide (PI, 50 g/mL). Under a fluorescence microscope (TE2000-U; Nikon, Tokyo, Japan), the migration of DNA from the nucleus of each cell was measured with the CometScore [55] software program. The % tail DNA parameter [56,57] was calculated as the percentage of DNA in the comet tail (sum of intensities of pixels in the tail). The formula used for calculating the % tail DNA was as follows: Tail % DNA = 100 − Head % DNA, where Head % DNA = (Head Optic Intensity / (Head Optic Intensity + Tail Optic Intensity)) × 100.

Cell Viability Assay
The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed as described previously [58]. Briefly, fresh medium (100 μL) containing 0.5 mg/mL MTT was added into each well of a 96-well plate containing 5 × 10 3 cells/well and incubated for 2 h at 37 °C. After removing MTT-containing medium, 100 μL of DMSO was added into each well to dissolve the purple formazan crystal. The plates were then shaken gently for 20 min in darkness and then read at 595 nm on a microtiter plate reader.

Cell Cycle Histogram Obtained by Propidium Iodide Staining in Flow Cytometry
The cell cycle histogram was determined as described previously [59]. Briefly, cells with 5 × 10 5 cells/100-mm Petri-dish were plated. After recovery, cells were treated for 24 h with 0, 1, 2, and 4 mg/mL AEGT with or without 0.003% H 2 O 2 . After treatment, cells were collected, washed twice with PBS, and fixed in 70% ethanol overnight. The cells were then centrifuged at 700 rpm for 5 min at 4 °C and then resuspended in PBS buffer containing 10 μg/mL PI (Sigma, St Louis, MO, USA) and 10 μg/mL RNase A. After 15 min incubation in darkness at room temperature, the cells were analyzed with a FACScan flow cytometer (Becton-Dickinson, Mansfield, MA, USA) in cell counts of 10,000 with gated setting (forward light scatter versus side light scatter such that only single cells were assayed) [60], and the gated data were analyzed by Cell-Quest and Modfit softwares (Becton-Dickinson, Mansfield, MA, USA).

Statistical Analysis
All data were presented as means ± SEM. Experimental groups were compared by one-way ANOVA with Tukey HSD Post Hoc Test using JMP® 9 software [61]. Levels not connected by the same big letter significantly differed.

Conclusions
Taken together, the experimental results in this study confirmed the hypothesis that edible red algae Gracilaria extract prevents ROS-induced DNA damage and its related cellular responses.