**1. Introduction**

Diabetic retinopathy (DR) is a microvascular consequence of diabetes mellitus and remains the leading cause of blindness among the working-age population [1]. DR is defined as the progressive, irreversible deterioration of retinal microvasculature as a result of chronic hyperglycemia [2]. Studies have found the relationship between oxidative stress and DR that oxidative stress plays a role in pathogenesis of DR and DR can increase the reactive oxygen species (ROS) level. Hyperglycemia is thought to be one of the main causes of the disease and higher level of oxidative stress can accelerate the process by blocking the downstream flow of glycolysis [3,4]. DR also increases oxidative stress because high glucose level and retinal vascularization by diabetic induction elevate arginase activity which later increases oxidative stress [5].

As the relationship between ROS and various retinal pathogenesis have been studied, defense mechanisms against ROS have been also studied [6–11]. Organisms have defense mechanisms against oxygen metabolites and the mechanism includes removal of free radicals by enzymes, proteins, and pro-oxidant metal reactions, and reduction of free radicals by antioxidants (vitamin C, vitamin E, glutathione) [6]. The cellular antioxidant response element is essentially important for the amelioration of oxidative stress. It responds to hyperglycemia and can be used to evaluate the complications of diabetes. In a previous study, Busik et al. [12] suggested that diabetes-related endothelial injury in the retina may be due to glucose-induced cytokine release by other retinal cells, such as retinal pigment epithelium (RPE) and Müller cells, and not a direct e ffect of high glucose. Therefore, it is important to investigate the oxidative stress as well as e ffects of antioxidants on RPE cells in order to determine the pathogenesis regarding oxidative stress in DR.

Studies have found that with aging, endogenous antioxidants level [13] and antioxidant enzyme activity along with its gene expression and protein level decrease [14]. This alteration in the antioxidative defense system worsens the imbalance between ROS production and its removal. As a consequence, oxidatively damaged macromolecules including lipids, deoxyribonucleic acid (DNA), and proteins accumulate accelerating the aging process with oxidative-stress-induced aging [15].

For this reason, it becomes more important to maintain the antioxidant defense system and one way is to supplement antioxidants from an outer source. Supplements actively studied for their antioxidative e ffect are ascorbic acid (vitamin C), glutathione, alpha-tocopherol (vitamin E), and other carotenoids (i.e., astaxanthin, lutein, β-carotene) [16–18]. One frequently used way to evaluate their antioxidant activity is by studying their reactivity with free radicals and metal ions (DPPH, ABTS, FRAP, CUPRAC, ORAC, HORAC, TRAP) [19–22]. However, giving them enough credence for their antioxidant capacity assumption is often controversial since one same antioxidant can have a di fferent relative capacity to other antioxidants when measured with di fferent methods [23–28].

For this reason, it is necessary to study potential antioxidants' capacities and properties based on a solid oxidative stress model. A solid oxidative stress model portrays the biological environment well so that a more accurate assumption is possible, and the result is reproducible. Hydrogen peroxide (H2O2) [29–31] and ultraviolet B (UVB) irradiation [32–34] have been studied to establish an oxidative stress model within cells. H2O2 represents endogenous ROS production and UVB represents an outer source of oxidative stress to retinal cells. In this study, both a H2O2-induced oxidative stress model and UVB-induced oxidative stress model will be used to evaluate the antioxidative potential of ascorbic acid and astaxanthin on ARPE-19 cells.

#### **2. Materials and Methods**

#### *2.1. ARPE-19 Cell Culture*

ARPE-19 cells (american type culture collection, Manassas, VA, USA) were cultured and maintained as a monolayer in 1:1 mixture of Dulbecco's modified eagle's medium and nutrient mixture F-12 (DMEM/F-12) (Invitrogen, Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 1% penicillin-streptomycin (Invitrogen). Cells were incubated at 37 ◦C in a humidified 5% CO2 incubator in the complete medium with a 2–3-times-a-week change until they reached 80% confluency. Cells used for this study were in a passage between 25 and 30.

#### *2.2. Hydrogen Peroxide Exposure Procedure*

Cells were seeded in a 96-well plate with a density of 2.5 × 10<sup>4</sup> cells/well and allowed to attach to the bottom of the well and to become confluent overnight. The next day, the medium was changed to a serum-free medium and cells were maintained in it up to 7 days until the day of the procedure. 30% ( *w*/*w*) H2O2 in H2O-containing stabilizer (Sigma Aldrich, St. Louis, MO, USA) was used to make medium with intended H2O2 concentration. H2O2 solution was diluted fresh each time. For the exposure, the used medium of the cells was changed to serum-free DMEM/F-12 without phenol red (Invitrogen) with the desired concentration of H2O2. The viability was checked by MTT assay after 24 h of exposure to H2O2.

#### *2.3. Ultraviolet B Irradiation Procedure*

Cells were seeded in a 96-well plate with a density of 2.5 × 10<sup>4</sup> cells/well and allowed to attach to the bottom of the well and to become confluent overnight. The next day, the medium was changed to serum-free medium and cells were maintained in it up to 7 days until the day of the procedure. At UVB irradiation, the medium was changed to DMEM/F-12 without phenol red without serum. As a UVB source, Sankyo Denki lamps (G15T8E, Tokyo, Japan) was used. Its irradiation intensity was 0.2 mW/cm<sup>2</sup> when measured 20 cm below the lamp where the plates were put. The intensity was measured with a UVB meter (UVX Digital Radiometer, UVP, Upland, CA, USA). Cells were irradiated with intended doses of UVB and for the control group and di fferential dose of UVB irradiation, remaining wells in the same plate were thoroughly masked.

#### *2.4. DPPH Scavenging Assay*

Total antioxidative capacities of ascorbic acid and astaxanthin were estimated using DPPH (2,2-diphenyl-1-picrylhydrazyl) ROS scavenging assay. DPPH solution was made by dissolving DPPH in methanol to 0.16 mM. Ascorbic acid and astaxanthin were dissolved to various concentrations in dimethyl sulfoxide (DMSO) (Sigma Aldrich). Ascorbic acid (20 μL) or astaxanthin (20 μL) solution was mixed with 100 μL DPPH solution for 30 min with vigorous shaking at room temperature. After the reaction absorbance at 517 nm was measured and the relative amount of scavenged DPPH was calculated using the following equation.

$$\text{Scavenged DPPH } f \text{ration } (\%) = \frac{Ab\_{\text{Control}} - Ab\_{AO}}{Ab\_{\text{Control}}} \times 100\tag{1}$$

*AbControl* is the absorbance of the groups with only DPPH and *AbAO* is the absorbance of the groups of the mixture of DPPH and various concentrations of antioxidants.

## *2.5. Antioxidant Treatment*

Cells were treated with either ascorbic acid (Sigma Aldrich) or astaxanthin (Sigma Aldrich) in DMEM/F-12 without phenol red to study their antioxidative e ffect on ARPE-19 cells. Ascorbic-acid-containing medium was made from ascorbic acid stock (0.5 M in PBS) and astaxanthin-containing medium was made from astaxanthin stock (1 mg/mL in DMSO). Cells were pretreated with ascorbic acid or astaxanthin for 6 h and then they were irradiated by UVB or exposed to H2O2. For UVB irradiation group, after pretreatment, used medium was changed to the fresh medium containing the same concentrations of compounds and followed the UVB irradiation (20 mJ/cm<sup>2</sup> or 100 mJ/cm2) procedure. For the H2O2 exposure group, after pretreatment, the used medium was changed to the fresh medium containing the same concentrations of the compounds with a sublethal or lethal dose of H2O2 (0.2 mM or 0.4 mM).

## *2.6. MTT Assay*

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma Aldrich) was used to determine cell viability. MTT is enzymatically turned into purple formazan crystals by mitochondrial respiration activity. The procedure was done following the manufacturer's instructions. Briefly, after antioxidants, UVB, or H2O2 treatment to the cells, the medium was removed and MTT (0.5 mg/mL) was added diluted in serum-free medium. After 3 h of incubation at 37 ◦C in a humidified 5% CO2 incubator, MTT-containing medium was carefully aspirated from the well and DMSO was added to each well to solubilize formazan crystals. Absorbance at 570 nm was measured using a microplate reader (EPOCH 2, BioTek Instruments Inc. Winoosky, VT, USA) with a reference wavelength of 630 nm. Cells untreated or treated with the only vehicle were set to be 100% cell viability for the normalization of the absorbance and experiments had more than three replicates for each condition.

#### *2.7. Crystal Violet Assay*

The relative number of cells attached to the bottom of the well was measured by crystal violet uptake assay. The procedure was done as previously described [35]. Briefly, after UVB, or H2O2 treatment to the cells, the medium was removed, and cells were fixed with 4% paraformaldehyde in 4 ◦C. After they were washed 3 times and 0.1% crystal violet (Sigma Aldrich) in 10% ethanol was added to each well for 5 min. After washing 3 times, the remaining stain was dissolved in 10% acetic acid and absorbance at 540 nm was measured.

#### *2.8. DCFH-DA Intracellular ROS Level Assay*

Intracellular ROS level was measured by 2-,7--dichlorodihydrofluorescein diacetate (DCFH-DA) assay. DCFH-DA is cell-permeable and is not fluorescent which enters cells to be de-esterified to 2-,7--dichlorodihydrofluorescein (DCFH), and become impermeable to the cell membrane. It then reacts with ROS to be highly fluorescent 2-,7--dichlorofluorescein (DCF). Before UVB irradiation or H2O2 exposure, cells were cultured with 10 μM DCFH-DA (Sigma Aldrich) in DMEM/F-12 without phenol red for 30 min at 37 ◦C in a humidified 5% CO2 incubator. After incubation, they were washed 2 times in phosphate-buffered saline (PBS) and antioxidant treatment, UVB irradiation or H2O2 exposure was done following measurement of fluorescence of DCF at excitation and emission wavelength of 495 nm and 529 nm, respectively, with a microplate reader (Synergy Mix, BioTek Instruments Inc. Winoosky, VT, USA). Cells untreated or treated with the only vehicle were set to be 100% intracellular ROS level for the normalization of the fluorescence intensity and experiments had more than three replicates for each condition.
