Retinal pigment epithelium (RPE) is an essential vision component of the human eye due to its nutritional and functional support to photoreceptors. Age-related macular degeneration (AMD) is a progressive, degenerative retinal disease characterized by RPE loss. Ultraviolet (UV) light exposure induces injurious effects on human eyes by generating excess reactive oxygen species (ROS) and is responsible for photoaging. Bilberry (Vaccinium myrtillus L.) and its extract (VME) are known for their potent antioxidant properties. In the present study, we examined the effect of VME on UVA-induced RPE injury. Using an in vitro system with human RPE (ARPE-19) cells, pretreatment with VME suppressed cell death, mitochondrial superoxide production, and activation of the stress response that occurs following UVA irradiation. Furthermore, VME attenuated rotenone-induced mitochondrial ROS and concomitant reduction in cell viability. Our findings suggest that VME has a protective effect against UVA-induced RPE cell damage.

Ultraviolet (UV) radiation is one of the hazardous environmental factors affecting human health. UV radiation is divided by wavelength into three classifications: UVA (320–400 nm), UVB (290–320 nm), and UVC (100–290 nm), primarily due to the difference among the biological activity of each UV subcategory (Sliney, 2002). All UVC and a substantial proportion of UVB is absorbed by the ozone layer; hence, UVA accounts for most of the UV radiation that reaches the surface of the Earth. UVA and UVB affect living organisms, including humans; UVB is responsible for sunburn and some ocular diseases, such as cataract and photokeratitis on the outer layer of the cornea (Holick, 2016; Yam and Kwok, 2014). UVB is primarily absorbed by the cornea and lens, whereas UVA is less attenuated and can reach the retina and retinal pigment epithelium (RPE) (Young, 2006). UVA-induced DNA breakdown, as well as the production of reactive oxygen species (ROS) and the activation of signal transduction pathways such as the extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) pathways, play a key role in UVA-induced cellular apoptosis (McCubrey et al., 2006). Some compounds found in food, such as carotenoids, are known for their antioxidative activities. Food enriched with antioxidants have been attracting attention for their potential role in protecting human vision against the oxidative stress induced by exposure to excess light, including UV.

RPE, which is found at the back of the retina, absorbs the light that passes through the outer segment of the photoreceptors; RPE is also indirectly exposed to light stress through phagocytosis of light-oxidized photoreceptor outer segments and digesting such toxic substrates (Kim et al., 2013). Due to its high metabolic demands, RPE contains abundant mitochondria (Toms et al., 2019). Mitochondria are intracellular organelles with a long and branched bilayer membrane structure and are responsible for ATP production through oxidative phosphorylation via the citric acid cycle and the electron transfer system. Mitochondria are particularly sensitive to excessive ROS generated by UV irradiation, and the dysfunction of mitochondria has been linked to photoaging (Naidoo et al., 2018). It has been shown that RPE from patients with age-related macular degeneration (AMD) possesses features of mitochondrial dysfunction, such as fewer mitochondria, loss of mitochondrial cristae, and mitochondrial DNA mutation (Feher et al., 2006; Karunadharma et al., 2010). Therapeutic approaches through the restoration of mitochondrial function have been attempted, and preclinical experiments and clinical studies are in progress (Tong et al., 2022). It is also thought that the administration of natural antioxidant compounds could be effective for preventing the life-long deterioration of mitochondria in RPE.

Bilberry (Vaccinium myrtillus L.), a plant belonging to the Ericaceae family, grows abundantly in wild, montane areas in northern and central Europe. Bilberry is one of the plants that forms an important part of the local diet in Nordic countries (Vaneková and Rollinger, 2022), while extracts from the fruits and leaves have been used as a medical herb to treat various physical conditions since ancient times. Bilberry is intensely colored because both peel and pulp contain large quantities of anthocyanins (Vaneková and Rollinger, 2022), which have been shown to possess substantial antioxidant capacity (Ogawa et al., 2008) as well as stimulating antioxidative defenses by the induction of antioxidant enzymes such as glutathione S-transferase (GST), glutathione peroxidase (GSH-Px) (Juadjur et al., 2015), and heme oxygenase-1 (HO-1) (Milbury et al., 2007). In animal studies, bilberry extract has been reported to improve visual function, contributing to the prevention of retinal inflammation and cataract (Fursova et al., 2005; Miyake et al., 2012). Our previous studies have shown that bilberry extract has a neuroprotective effect against retinal damage induced by N-methyl-D-aspartic acid and an inhibitory effect against angiogenesis in oxygen-induced retinopathy in mice (Matsunaga et al., 2009, 2010). Furthermore, in vitro studies showed that bilberry extract had protective effects against UVA-induced photoreceptor damage and blue light-emitting diode (LED) light-induced damage (Ogawa et al., 2013, 2014). However, the effect of bilberry extract on RPE damage induced by UVA has yet to be determined.

Here, we investigated the effect of bilberry extract (Vaccinium myrtillus L. extract, VME) on UVA-induced cellular damage in an RPE cell line, ARPE-19, as an in vitro system. Cell death, cell viability, mitochondrial superoxide production, and oxygen consumption were analyzed following UVA exposure in ARPE-19 cells pretreated with or without VME. We also used immunoblotting to analyze the phosphorylation of stress response proteins following exposure to UVA light. Finally, the effects of VME on rotenone-induced cellular damage were examined.

Reagents Vaccinium myrtillus L. extract (VME) was purchased from Beijing Ginko Group Japan Co., Ltd. (Tokyo, Japan). The constituents of VME were analyzed previously using high-performance liquid chromatography (HPLC) and UV-visible absorption spectroscopy using each standard preparation; 14.1 % delphinidin, 9.1 % cyanidin, and 6.1 % malvidin (Ogawa et al., 2013). Hoechst 33342, propidium iodide (PI), and MitoSOX Mitochondrial Superoxide Indicators were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). Primary antibodies against phosphorylated-p38 mitogen-activated protein kinase (p-p38 MAPK; Thr180/Tyr182, rabbit, Cat# 9211), p38 MAPK (rabbit, Cat# 9212), phosphorylated p44/42 MAPK, extracellular signal-regulated kinases 1/2 (p-ERK1/2; Thr202/Tyr204, rabbit, Cat# 4377), and ERK1/2 (rabbit, Cat# 9102) were purchased from Cell Signaling Technology (Beverly, MA, USA). Rotenone was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture and UV-A irradiation ARPE-19 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained as previously reported (Yako et al., 2022). In brief, ARPE-19 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 (FUJIFILM-Wako, Osaka, Japan) supplemented with 10 % fetal bovine serum (FBS; VALEANT, Costa Mesa, CA, USA) at 37 °C in a humidified atmosphere of 5 % CO₂. UVA light exposure was performed according to a previously reported method with minor modifications (Otsu et al., 2022). ARPE-19 cells were seeded into 96-well plates (Corning, Corning, NY, USA) at 10 000 cells per well and incubated at 37 °C for 24 h. The cells were pretreated with VME at the indicated concentration in DMEM/F-12 supplemented with 1 % FBS for 1 h, then exposed to UVA light (365 nm) using CL-1000L UV crosslinkers (Ultraviolet Products Ltd., Cambridge, UK) until the UVA irradiation reached 5 J/cm². The cells were incubated at 37 °C in a humidified atmosphere of 5 % CO₂, then subjected to the subsequent experiments.

Cell death and viability assay After 24 h of UVA light exposure and incubation with Hoechst 33342 and PI (Thermo Fisher Scientific) added to the culture medium based on a previous report (Ogawa et al., 2013), images were collected using a Lionheart FX Automated Microscope (Bio Tek Instruments, Winooski, VT, USA), and the percentage of PI-positive cells was automatically calculated as a cell death ratio by the Gen5 software (Bio Tek Instruments). For the cell viability assay, CCK-8 reagent was added to each well 24 h after exposure to UVA light and evaluated according to the same methods as in the previous report (Ogawa et al., 2013).

Mitochondrial ROS production assay Mitochondrial superoxide production was determined using MitoSOX and according to the manufacturer's instructions (Thermo Fisher Scientific). In brief, at 24 h following UVA irradiation, Hoechst 33342 and MitoSOX reagent were added to the culture medium at a final concentration of 8.1 and 5 |iM, respectively, followed by incubation at 37 °C for 15 min. The fluorescence intensity of MitoSOX was measured using a Lionheart FX Automated Microscope (Bio Tek Instruments) and normalized by the cell numbers obtained by Hoechst 33342 staining. The relative mitochondrial ROS production represents fold changes compared with the control.

Mitochondrial respiration measurement A Cell Mito Stress Test (Agilent Technologies, Santa Clara, CA, USA) was performed according to the manufacturer's instructions. Briefly, ARPE-19 cells were plated at 32 000 cells per well into Seahorse XF HS 8-well plates (Agilent Technologies), 24 h prior to the assay. The medium was changed to fresh DMEM/F-12 medium (FUJIFILM-Wako) containing 1 % FBS with or without 30 μg/mL VME and incubated for 1 h; then, the entire plates were exposed to UVA radiation. Following the irradiation, the medium was replaced with Seahorse XF DMEM Medium (Agilent Technologies) supplemented with 10 mM glucose, 1 mM pyruvate, and 2 mM L-Glutamine, then incubated at 37 °C for 1 h in a non-CO₂ incubator. The plates were transferred to a Seahorse XF HS Mini Analyzer (Agilent Technologies), and the Cell Mito Stress Test was carried out with inhibitors at the following concentrations: 1.5 μM oligomycin A, 1.0 μM FCCP, and a mixture of 0.5 μM rotenone and 0.5 μM antimycin A (Agilent Technologies). The wells of the control group were covered with aluminum foil to avoid exposure to UVA. Data analyses were conducted using Agilent Seahorse Analytics (Agilent Technologies).

Western blotting analysis Following UVA exposure, ARPE-19 cells were harvested and Western blotting analysis was performed as previously reported, respectively (Ogawa et al., 2013, 2014).

Statistics All data are presented as means ± the standard error of the mean (SEM). Statistical analyses were performed using a paired Student's t-test, Dunnett's test, or Tukey's test, using the Statistical Package for the Social Sciences 15.0 J for Windows software (SPSS Japan Inc., Tokyo, Japan). A value of P < 0.05 was considered to indicate statistical significance.

Protective effects of VME against cellular damage in ARPE-19 cells following exposure to UVA light To examine the effect of VME on UVA-induced RPE cell damage, cells of the human RPE cell line, ARPE-19, were treated with 1, 3, 10, or 30 μg/mL of VME and exposed to UVA at 5 J/cm² (Fig. 1A). At 24 h after exposure to UVA light, the cell death rate had increased to 13.0 ± 1.5 %; this was significantly reduced to 7.11 ± 1.0 % following pretreatment with VME at 30 μg/mL (Fig. 1B, C). Moreover, pretreatment with 10 or 30 μg/mL of VME prevented reductions in cell viability in ARPE-19 cells following UVA irradiation (Fig. 1D).

(A) A schematic diagram of the experiment. Either Hoechst 33342 and propidium iodide (PI) staining or a CCK-8 assay was performed in ARPE-19 cells without or with 1, 3, 10, or 30 μg/mL VME at 24 h following exposure to UVA radiation at 5 J/cm². (B) Representative images of ARPE-19 cells stained with Hoechst 33342 (blue) and PI (red). Arrowheads indicate PI-positive cells. Bar = 100 μm. (C) The graph shows the ratio of PI-positive cells as the cell death rate. (D) Quantitation of cell viability by the CCK-8 assay. Data are shown as the percentage of the control. n = 5–6, means ± SEM; **p < 0.005, ***p < 0.001, two-tailed Student's t-test vs control (Cont); ^†††p < 0.001, Dunnett's test vs vehicle (Veh).

Fig. 1.

Effect of VME on UVA-induced cell damage in ARPE-19 cells.

Preventive effects of VME against UVA-induced mitochondrial damage in ARPE-19 cells Mitochondria are known to be a major site of intracellular ROS production. To assess the effect of VME on mitochondrial ROS production following UVA irradiation, staining with the mitochondrial superoxide indicator MitoSOX was performed. Exposure to UVA light increased mitochondrial ROS levels, as indicated by the fluorescence intensity of MitoSOX in ARPE-19 cells, which was suppressed in the presence of VME at concentrations of 10 and 30 μg/mL (Fig. 2A, B). Notably, VME reduced the MitoSOX signal intensity even in ARPE-19 cells not treated with UVA (Fig. 2B), suggesting that VME can suppress mitochondrial ROS production not only under stress conditions but also under steady-state conditions. To evaluate the effect of VME on mitochondrial function, the oxygen consumption rate (OCR) in ARPE-19 cells was measured following UVA irradiation (Fig. 3A). Exposure to UVA light at 5 J/cm² decreased the maximal OCR, while the UVA-induced decrease in maximal OCR was restored in ARPE-19 cells treated with 30 μg/mL of VME (Fig. 3B, C), suggesting that mitochondrial ATP production ability was preserved by the presence of VME under oxidative stress induced by UVA irradiation.

(A) Representative images of ARPE-19 cells stained with Hoechst 33342 (blue) and mitochondrial ROS indicator (MitoSOX, red) at 24 h following exposure to UVA light. Bar = 100 μm. (B) The graph shows relative MitoSOX fluorescence intensity as mitochondrial ROS production. n = 6, means ± SEM; ^*p < 0.05 two-tailed Student's t-test vs control (Cont); ^†p < 0.05, ^††p < 0.005, Dunnett's test vs vehicle (Veh); ^$$$p < 0.001 two-tailed Student's t-test vs Cont.

Fig. 2.

VME suppresses mitochondrial ROS production in ARPE-19 cells following exposure to UVA radiation.

Fig. 3.

VME ameliorates the decrease in maximal oxygen consumption rates of ARPE-19 cells following UVA exposure.

Regulatory effects of VME on the phosphorylation of stress response proteins in ARPE-19 cells following UVA radiation Next, we performed Western blotting analysis to examine the effect of VME on the p38 and ERK1/2 MAPK signaling pathways in ARPE-19 cells. UVA light exposure increased the phosphorylation of p38; this phosphorylation was inhibited following treatment with 30 μg/mL of VME (Fig. 4A, B). Similarly, the phosphorylation of ERK1/2 was also increased by UVA irradiation, and VME significantly inhibited this phosphorylation (Fig. 4C, D). These results suggest that VME pretreatment has regulatory effects on the UVA-induced activation of the stress response in RPE cells.

(A,C) Representative images of immunoblotting for the phosphorylation of p38 (A) or ERK1/2 (C). (B,D) The ratio of phospho- vs total-p38 (B) or -ERK1/2 (D). n = 6, means ± SEM; ***p < 0.001 Tukey's test vs control (Cont); ^###p < 0.001, Tukey's test vs vehicle (Veh).

Fig. 4.

VME inhibits activation of the apoptosis pathway in UVA-irradiated ARPE-19 cells.

Inhibitory effects of VME on rotenone-induced mitochondrial ROS production in ARPE-19 cells Rotenone is known to increase mitochondrial ROS production by inhibiting complex I. To investigate the effect of VME on mitochondria-derived ROS and cellular damage, we treated ARPE-19 cells with rotenone (Fig. 5A). Mitochondrial ROS production was increased in ARPE-19 cells incubated with 10 μM of rotenone, whereas the increase in mitochondrial ROS in cells incubated with rotenone was restrained following pretreatment with 30 μg/mL of VME (Fig. 5B, C). Furthermore, VME attenuated the reduction in cell viability induced by rotenone (Fig. 5D). Our findings indicate that VME has an inhibitory effect against mitochondria-derived ROS and subsequent damage to RPE.

(A) A schematic diagram of this experiment. ARPE-19 cells were incubated with 10 μM of rotenone for 24 h prior to the assays. (B) Representative images of ARPE-19 cells stained with Hoechst 33342 (blue) and MitoSOX (red) following rotenone treatment with or without VME. Bar = 100 μm. (C) Quantitative results for B. (D) Quantitation of cell viability using the CCK-8 assay. Data are shown as the percentage of the control. n = 6, means ± SEM **p < 0.005, ***p < 0.001 two-tailed Student's t-test vs control (Cont), ^†p < 0.05, Dunnett's test vs vehicle (Veh), ^$p < 0.001 two-tailed Student's t-test vs Cont.

Fig. 5.

Effect of VME on rotenone-induced mitochondrial injury in ARPE-19 cells.

In this study, we focused on the preventive effects of VME against RPE damage following exposure to UVA light. Recently, it was reported that ARPE-19 cells are particularly sensitive to UVA radiation between the wavelengths of 350 and 380 nm (Anderson et al., 2022). Excessive exposure to light has been shown to be a cause of increased cellular ROS production, while the accumulation of chronic oxidative stress accelerates tissue aging, suggesting that either a reduction in ROS itself or the activation of the cellular capacity to process ROS is key to anti-photoaging processes. Pretreatment with 10 or 30 μg/mL VME prevented the loss of cell viability following UVA irradiation at 5 J/cm² (Fig. 1). VME pretreatment suppressed UVA light-induced mitochondrial ROS production at a similar concentration (Fig. 2). Exposure to UVA resulted in mitochondrial dysfunction and the activation of the stress response, which could be remediated by pretreatment with 30 μg/mL VME (Figs. 3, 4). VME also attenuated both mitochondrial ROS production and the decrease in cell viability induced by rotenone (Fig. 5). We conclude that VME has a protective effect against cellular damage and mitochondrial dysfunction caused by UVA-induced oxidative stress.

Most of the phenolic compounds present in bilberry are anthocyanins (Heinonen, 2007; Kähkönen et al., 2003). In a previous study, we determined the constituents of VME; it contains 15 different anthocyanins, comprising 5 anthocyanidins (delphinidin, cyanidin, malvidin, petunidin, and peonidin) and 3 sugars (glucose, galactose, and arabinose) (Ogawa et al., 2013). Our results showed that VME was effective in protecting ARPE-19 cells against UVA irradiation at 10 μg/mL, which contains 1.41 μg/mL (4.65 μM) delphinidin, 0.91 μg/mL (3.17 μM) cyanidin, and 0.61 μg/mL (1.84 μM) malvidin (Ogawa et al., 2013). It has been demonstrated that the main constituent anthocyanidins of VME have inhibitory effects against UVA-induced ROS production and cellular damage in the murine retinal cell line 661W (Ogawa et al., 2013). In the previous report, we also demonstrated that treatment with delphinidin, cyanidin, or malvidin at concentrations ranging from 1 to 30 mM reduced UVA-induced intracellular ROS production and cell death in 661W cells. Additionally, cyanidin treatment suppressed p38 MAPK phosphorylation induced by UVA irradiation. It is known that UV irradiation generates superoxide anion radical and hydrogen peroxide in cellular mitochondria, and hydrogen peroxide is changed to hydroxyl radical by the catalytic action of iron or copper ions. Hydroxyl radical causes lipid peroxidation, protein denaturation, mitochondrial dysfunction, and DNA damage in cells, then it induces cellular damage and apoptosis. In contrast, delphinidin, cyanidin, and malvidin in VME have been demonstrated to have direct radical scavenging activity for hydroxyl radical and/or superoxide anion radical (Ogawa et al., 2011), which induce the phosphorylation of MAPKs. In addition, another previous report also suggested that a single treatment of delphinidin or cyanidin suppressed H₂O₂-induced intracellular ROS increase and the phosphorylation of p38 MAPK and ERK1/2 in fibroblast cells (Nam et al., 2016). Furthermore, delphinidin has been known to inhibit the phosphorylation of MAPK activation by binding to MKK4 in an ATP-competitive manner (Kwon et al., 2009). For these reasons, anthocyanins included in VME might suppress ROS generation, ameliorate mitochondrial dysfunction, and inhibit the phosphorylation of p38 MAPK and ERK1/2 by UVA irradiation in ARPE-19 cells (Figs. 2, 3, and 4). It is important to prevent the phosphorylations of p38 MAPK and ERK1/2 by UVA, because they participate in cellular responses to UV-induced apoptosis (Bode and Dong, 2003).

It has been reported that cyanidin- and delphinidin-3-glucosides act as electron acceptors at complex I and subsequently increased ATP synthesis in ischemia-damaged mitochondria of rat hearts (Skemiene et al., 2015). We demonstrated the inhibitory effect of VME against rotenone-induced mitochondrial ROS production and cell damage, indicating that the protective effect of VME could be achieved by the activation of complex I. The inhibitory effect of VME on rotenone-induced mitochondrial ROS production was observed at a concentration of 30 μg/mL (Fig. 5), which was higher than the effective concentration of the UVA-induced model. Since UVA irradiation triggers ROS generation in mitochondria as well as in the cytoplasm and cell membranes, the radical scavenging activity of VME and its inhibition of cellular ROS generation would contribute to the protection against UVA-induced cell damage to some extent. Further studies are needed to reveal the molecular mechanisms of VME and anthocyanins against mitochondrial dysfunction in RPE exposed to UVA.

The plasma concentration of anthocyanins ranged between 0.56 and 4.46 nmol/L following the consumption of 480 mL cranberry juice, containing 94.47 mg of anthocyanins (Milbury et al., 2010), suggesting that the plasma concentration of anthocyanins following oral administration may be lower than the effective concentration we obtained in our experiments. Although the absorption of anthocyanins from food has been shown to be limited, the accumulation of anthocyanins in ocular tissues has been detected in some animal studies (Kalt et al., 2008; Matsumoto et al., 2006). Further studies are necessary to determine the metabolism of VME in the eye and to investigate its protective effects against UVA-induced RPE damage in vivo. Additionally, in nature anthocyanins mainly occur in glycosylated forms, therefore the effect of glycosidic anthocyanins on UVA-induced RPE damage should be investigated.

In conclusion, anthocyanin-rich extract derived from bilberry, Vaccinium myrtillus L., improves mitochondrial function and protects RPE injury induced by UVA by its antioxidative action. Our findings suggest that the daily intake of bilberry could be beneficial for RPE health via the attenuation of oxidative damage caused by ROS production.

Conflict of interest There are no conflicts of interest to declare.

Author Contributions W.O., K.O., and H.H. designed the study. W.O. and K.O. performed the experiments, analyzed the data, and drafted the manuscript. H.H. supervised the execution of the study. All the authors contributed to the preparation of the manuscript and approved the final version.

age-related macular degeneration

light-emitting diode

mitogen-activated protein kinase

oxygen consumption rate

propidium iodide

retinal pigment epithelium

reactive oxygen species

Vaccinium myrtillus L. extract

ultraviolet-A

•  Anderson,  G.,  McLeod,  A.,  Bagnaninchi,  P., and  Dhillon,  B. (2022). Quantitative action spectroscopy reveals ARPE-19 sensitivity to ultraviolet radiation at 350 nm and 380 nm. Scientific Reports, 12, 14223.
•  Bode,  A. M., and  Dong,  Z. (2003). Mitogen-activated protein kinase activation in UV-induced signal transduction. Science's STKE: Signal Transduction Knowledge Environment, 2003, RE2.
•  Feher,  J.,  Kovacs,  I.,  Artico,  M.,  Cavallotti,  C.,  Papale,  A., and  Balacco Gabrieli,  C. (2006). Mitochondrial alterations of retinal pigment epithelium in age-related macular degeneration. Neurobiology of Aging, 27, 983–993.
•  Fursova,  A. Z.,  Gesarevich,  O. G.,  Gonchar,  A. M.,  Trofimova,  N. A., and  Kolosova,  N. G. (2005). Dietary supplementation with bilberry extract prevents macular degeneration and cataracts in senesce-accelerated OXYS rats. Advances in gerontology = Uspekhi gerontologii / Rossiiskaia akademiia nauk, Gerontologicheskoe obshchestvo, 16, 76–79.
•  Heinonen,  M. (2007). Antioxidant activity and antimicrobial effect of berry phenolics-a Finnish perspective. Molecular Nutrition and Food Research, 51, 684–691.
•  Holick,  M. F. (2016). Biological Effects of Sunlight, Ultraviolet Radiation, Visible Light, Infrared Radiation and Vitamin D for Health. Anticancer Research, 36, 1345–1356.
•  Juadjur,  A.,  Mohn,  C.,  Schantz,  M.,  Baum,  M.,  Winterhalter,  P., and  Richling,  E. (2015). Fractionation of an anthocyanin-rich bilberry extract and in vitro antioxidative activity testing. Food Chemistry, 167, 418–424.
•  Kähkönen,  M. P.,  Heinämäki,  J.,  Ollilainen,  V., and  Heinonen,  M. (2003). Berry anthocyanins: isolation, identification and antioxidant activities. Journal of the Science of Food and Agriculture, 83, 1403–1411.
•  Kalt,  W.,  Blumberg,  J. B.,  McDonald,  J. E.,  Vinqvist-Tymchuk,  M. R.,  Fillmore,  S. A. E.,  Graf,  B. A.,  O'Leary,  J. M., and  Milbury,  P. E. (2008). Identification of anthocyanins in the liver, eye, and brain of blueberry-fed pigs. Journal of Agricultural and Food Chemistry, 56, 705–712.
•  Karunadharma,  P. P.,  Nordgaard,  C. L.,  Olsen,  T. W., and  Ferrington,  D. A. (2010). Mitochondrial DNA damage as a potential mechanism for age-related macular degeneration. Investigative Ophthalmology and Visual Science, 51, 5470–5479.
•  Kim,  J.-Y.,  Zhao,  H.,  Martinez,  J.,  Doggett,  T. A.,  Kolesnikov,  A. V.,  Tang,  P. H.,  Ablonczy,  Z.,  Chan,  C.-C.,  Zhou,  Z.,  Green,  D. R., and  Ferguson,  T. A. (2013). Noncanonical autophagy promotes the visual cycle. Cell, 154, 365–376.
•  Kwon,  J. Y.,  Lee,  K. W.,  Kim,  J.-E.,  Jung,  S. K.,  Kang,  N. J.,  Hwang,  M. K.,  Heo,  Y.-S.,  Bode,  A. M.,  Dong,  Z., and  Lee,  H. J. (2009). Delphinidin suppresses ultraviolet B-induced cyclooxygenases-2 expression through inhibition of MAPKK4 and PI-3 kinase. Carcinogenesis, 30, 1932–1940.
•  Matsumoto,  H.,  Nakamura,  Y.,  Iida,  H.,  Ito,  K., and  Ohguro,  H. (2006). Comparative assessment of distribution of blackcurrant anthocyanins in rabbit and rat ocular tissues. Experimental Eye Research, 83, 348–356.
•  Matsunaga,  N.,  Chikaraishi,  Y.,  Shimazawa,  M.,  Yokota,  S., and  Hara,  H. (2010). Vaccinium myrtillus(Bilberry) Extracts Reduce Angiogenesis In Vitro and In Vivo. In Evidence-Based Complementary and Alternative Medicine, 7, 47–56.
•  Matsunaga,  N.,  Imai,  S.,  Inokuchi,  Y.,  Shimazawa,  M.,  Yokota,  S.,  Araki,  Y., and  Hara,  H. (2009). Bilberry and its main constituents have neuroprotective effects against retinal neuronal damage in vitro and in vivo. Molecular Nutrition and Food Research, 53, 869–877.
•  McCubrey,  J. A.,  Lahair,  M. M., and  Franklin,  R. A. (2006). Reactive oxygen species-induced activation of the MAP kinase signaling pathways. Antioxidants and Redox Signaling, 8, 1775–1789.
•  Milbury,  P. E.,  Graf,  B.,  Curran-Celentano,  J. M., and  Blumberg,  J. B. (2007). Bilberry (Vaccinium myrtillus) anthocyanins modulate heme oxygenase-1 and glutathione S-transferase-pi expression in ARPE-19 cells. Investigative Ophthalmology and Visual Science, 48, 2343–2349.
•  Milbury,  P. E.,  Vita,  J. A., and  Blumberg,  J. B. (2010). Anthocyanins are bioavailable in humans following an acute dose of cranberry juice. The Journal of Nutrition, 140, 1099–1104.
•  Miyake,  S.,  Takahashi,  N.,  Sasaki,  M.,  Kobayashi,  S.,  Tsubota,  K., and  Ozawa,  Y. (2012). Vision preservation during retinal inflammation by anthocyanin-rich bilberry extract: cellular and molecular mechanism. Laboratory Investigation; a Journal of Technical Methods and Pathology, 92, 102–109.
•  Naidoo,  K.,  Hanna,  R., and  Birch-Machin,  M. A. (2018). What is the role of mitochondrial dysfunction in skin photoaging? Experimental Dermatology, 27, 124–128.
•  Nam,  D.C.,  Hah,  Y.S.,  Nam,  J.B.,  Kim,  R.J., and  Park,  H.B. (2016). Cytoprotective mechanism of cyanidin and delphinidin against oxidative stress-induced tenofibroblast death. Biomolecules and therapeutics, 24, 426–432.
•  Ogawa,  K.,  Sakakibara,  H.,  Iwata,  R.,  Ishii,  T.,  Sato,  T.,  Goda,  T.,  Shimoi,  K., and  Kumazawa,  S. (2008). Anthocyanin composition and antioxidant activity of the Crowberry (Empetrum nigrum) and other berries. Journal of Agricultural and Food Chemistry, 56, 4457–4462.
•  Ogawa,  K.,  Oyagi,  A.,  Tanaka,  J.,  Kobayashi,  S., and  Hara,  H. (2011). The protective effect and action mechanism of vaccinium myrtillus L. on gastric ulcer in mice. Phytotherapy Research, 25, 1160–1165.
•  Ogawa,  K.,  Tsuruma,  K.,  Tanaka,  J.,  Kakino,  M.,  Kobayashi,  S.,  Shimazawa,  M., and  Hara,  H. (2013). The protective effects of bilberry and lingonberry extracts against UV light-induced retinal photoreceptor cell damage in vitro. Journal of Agricultural and Food Chemistry, 61, 10345–10353.
•  Ogawa,  K.,  Kuse,  Y.,  Tsuruma,  K.,  Kobayashi,  S.,  Shimazawa,  M., and  Hara,  H. (2014). Protective effects of bilberry and lingonberry extracts against blue light-emitting diode light-induced retinal photoreceptor cell damage in vitro. BMC Complementary and Alternative Medicine, 14, 120.
•  Otsu,  W.,  Yako,  T.,  Sugisawa,  E.,  Nakamura,  S.,  Tsusaki,  H.,  Umigai,  N.,  Shimazawa,  M., and  Hara,  H. (2022). Crocetin protects against mitochondrial damage induced by UV-A irradiation in corneal epithelial cell line HCE-T cells. Journal of Pharmacological Sciences, 150, 279–288.
•  Skemiene,  K.,  Liobikas,  J., and  Borutaite,  V. (2015). Anthocyanins as substrates for mitochondrial complex I -protective effect against heart ischemic injury. The FEBS journal, 282, 963–971.
•  Sliney,  D. H. (2002). How light reaches the eye and its components. International Journal of Toxicology, 21, 501–509.
•  Toms,  M.,  Burgoyne,  T.,  Tracey-White,  D.,  Richardson,  R.,  Dubis,  A. M.,  Webster,  A. R.,  Futter,  C., and  Moosajee,  M. (2019). Phagosomal and mitochondrial alterations in RPE may contribute to KCNJ13 retinopathy. Scientific Reports, 9, 3793.
•  Tong,  Y.,  Zhang,  Z., and  Wang,  S. (2022). Role of Mitochondria in Retinal Pigment Epithelial Aging and Degeneration. Frontiers in Aging, 3, 926627.
•  Vaneková,  Z., and  Rollinger,  J. M. (2022). Bilberries: curative and miraculous - A review on bioactive constituents and clinical research. Frontiers in Pharmacology, 13, 909914.
•  Yam,  J. C. S., and  Kwok,  A. K. H. (2014). Ultraviolet light and ocular diseases. International Ophthalmology, 34, 383–400.
•  Young,  A. R. (2006). Acute effects of UVR on human eyes and skin. Progress in Biophysics and Molecular Biology, 92, 80–85.