Inhibition of Apoptosis in Human Retinal Pigment Epithelial Cells Treated with Benzo(e)Pyrene, a Toxic Component of Cigarette Smoke

Abstract

Purpose.: To study the inhibitory effects of some agents or drugs (inhibitors) on benzo(e)pyrene (B(e)P)-induced cell death and apoptosis on human retinal pigment epithelial (ARPE-19) cells in vitro.

Methods.: ARPE-19 cells were pretreated with varying concentrations of different classes of inhibitors (calpain, benzyl isothiocyanate [BITC], simvastatin, epicatechin, genistein, resveratrol, and memantine) before B(e)P exposure. Cell viability (CV) was determined by a trypan blue dye-exclusion assay. Caspase-3/7 and caspase-9 activities were measured by fluorochrome assays. The production of reactive oxygen/nitrogen species (ROS/RNS) was measured with 2′,7′-dicholorodihydrofluorescein diacetate dye assay.

Results.: At 30-μM concentrations, the genistein, resveratrol, and memantine inhibitors were able to reverse significantly the loss of cell viability, the activation of caspase-3/7 and caspase-9, and the production of ROS/RNS in ARPE-19 cell cultures. Memantine was the most potent and genistein was the least effective inhibitor in blocking the B(e)P-induced effects. Calpain, BITC, simvastatin, and epicatechin did not reverse the loss of cell viability in B(e)P-treated ARPE-19 cells. As a matter of fact, at the concentrations studied (15, 30, 45 μM), the BITC plus B(e)P-treated cultures showed significantly lower cell viability than the B(e)P-treated culture alone, suggesting BITC-related toxicity.

Conclusions.: Genistein, resveratrol, and memantine can reverse the apoptosis and oxidant production generated by B(e)P, a toxic element of smoking. These inhibitors may be beneficial against retinal diseases associated with the loss of RPE cells.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in the United States. The number of people with advanced AMD will increase from 1.75 million today to 3 million by 2020. ¹ Smoking has been identified as a primary risk factor associated with the prevalence and the incidence of AMD. AMD may be found in two forms, neovascular (wet form) or geographic atrophy (dry form). ^2,3 The wet and dry forms of AMD are characterized by abnormal vascular cell proliferation and retinal pigment epithelial (RPE) cell damage, respectively. Data from North America, Europe, and Australia, obtained from the Beaver Dam Eye Study, the Rotterdam Study, and the Blue Mountains Eye Study, suggest that smoking induces early AMD and progression of AMD, in contrast to disease occurrence in nonsmokers. These studies further reported a threefold increased risk for any type of AMD in the smoking population. ⁴  

Retinal pigment epithelium (RPE) appears to be specifically targeted in AMD. Abnormalities in the RPE and Bruch's membrane are present at the early stage of the disease, followed by further RPE degeneration as the disease progresses. ⁵ Studies on postmortem eyes of patients with AMD demonstrated the death of RPE, photoreceptors, and inner nuclear layer cells by apoptosis. ⁶  

Polycyclic aromatic hydrocarbons (PAHs), one of the several thousand toxicants in cigarette smoke, adversely affect DNA more than any of the other toxicants and cause increased cellular proliferation. Several in vitro and in vivo studies using PAH have shown damaging effects of PAHs on different types of cells. ^7–9 Among all PAHs, benzo(a)pyrene (B(a)P) has been studied using various ocular cells and was found to cause damage to bovine RPE cells. ¹⁰ In addition, benzo(e)pyrene (B(e)P), a close derivative of B(a)P, has also been shown to cause cell death and to induce apoptosis by the involvement of caspase-3/7, caspase-8, caspase-9, and caspase-12 in human ARPE-19 cells. ¹¹ In those studies, the minimum concentration of B(e)P causing significant loss of cell viability and apoptosis was 200 μM. Therefore, in the present study, we investigated whether different agents or drugs claiming to have neuroprotective effects were able to reverse the effects of 200 μM B(e)P on ARPE-19 cells. The following agents or drugs were tested for their inhibitory effect: memantine, simvastatin, epicatechin, calpain inhibitors such as ALLN Ac-Leu-Leu-Nle-H (aldehyde) and ALLM Ac-Leu-Leu-Met-H (aldehyde), isothiocyanates such as benzyl isothiocyanate (BITC), and polyphenols such as genistein, and resveratrol. These inhibitors have varied mechanisms of action. Memantine is a noncompetitive antagonist of the glutamatergic N-methyl-d-aspartate receptors and prevents neuronal excitotoxicity. It is used for the treatment of Parkinson's and Alzheimer's diseases. ^12,13 Simvastatin is a powerful lipid-lowering drug that, apart from inhibiting 3-hydroxy-3-methyl-glytaryl coenzyme A reductase, also upregulates Bcl-2, a major cell survival protein, and thus acts as neuroprotective agent. ¹⁴ Epicatechin is one of the naturally found flavonoids that has monoamine oxidase-B (MAO-B) inhibitory properties and can reduce the presence of free radicals responsible for cellular apoptosis and necrosis. Recent reports showed decreased reactive oxygen species (ROS) accumulation in cardiac cells after exposure to epicatechin. ¹⁵ Calpains are intracellular, calcium-dependent cysteine proteases involved with the regulation of key cellular processes, such as differentiation, apoptosis, cell motility, and cell cycle through signal-dependent limited cleavage of substrate proteins. ^16,17 Inappropriate regulation of the calpain proteolytic system is associated with several important pathologic disorders in humans, such as muscular dystrophy, cancer, Alzheimer's disease, neurologic injury, ischemia/reperfusion injury, atherosclerosis, diabetes, and cataract formation. ¹⁸ ALLN and ALLM are widely used as selective cell-permeable calpain I and II inhibitors. ¹⁹ Treatment of human articular chondrocytes (HACs) with calpain inhibitors (ALLN, ALLM) before peroxynitrite (ONOO⁻) treatment markedly reduces cell death, suggesting calpain proteases mediate cell death. ²⁰ BITC, an isothiocyanate, inhibits ROS generation in cultured cells and in animal models. ²¹ Moreover, it is an effective inhibitor of PAH tumorigenesis in mouse lung. ²² Genistein, a polyphenol, is a flavonoid that is widely distributed in fruits, vegetables, and beverages (such as tea, cocoa, and red wine). It has been shown to have preventive and therapeutic effects on cardiovascular diseases, tumor genesis, and ocular diseases, particularly those that involve the loss of nerve cells. The effects include inhibition of angiogenesis, tyrosine kinase phosphorylation, DNA synthesis, and cell cycle arrest. In addition, it is a potent antioxidant and a free-radical scavenger. Recently, it has been reported that genistein may inhibit neovascularization in a surgical model of choriocapillaris atrophy in rabbits ²³ and retinal ischemia in mice. ²⁴ Both flavonoids and resveratrol (hydroxystilbenes) have been found to exert various biological activities, many of which are beneficial for the health of animals and humans. Their effects include tumor suppression, neuroprotection, cardioprotection, and anti-inflammation. ^25,26 Furthermore, resveratrol has been considered to be a caloric restriction mimetic because it increases the lifespan of various species of lower animals. ²⁷ These biological activities of polyphenols are suspected to be related to their anti-oxidative and radical scavenging activities. ²⁸  

In this experiment, ARPE-19 cells were pretreated with the different classes of inhibitors before B(e)P treatment, and the effects on cell viability, caspase activity, and ROS/reactive nitrogen species (RNS) generation were measured. 

Materials and Methods

Cell Culture and Treatments

ARPE-19 cells (ATCC, Manassas, VA) were grown in a 1:1 mixture (vol/vol) of Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12 (Invitrogen-Gibco, Carlsbad, CA), 10 mM 1× nonessential amino acids, 0.37% sodium bicarbonate, 0.058% l-glutamine, 10% fetal bovine serum, and antibiotics (penicillin G 100 U/mL, streptomycin sulfate 0.1 mg/mL, gentamicin 10 μg/mL, amphotericin-B 2.5 μg/mL). Cells were plated in 6- and 24-well plates (Becton Dickinson Labware, Franklin Lakes, NJ) for cell viability (5.0 × 10⁵ cells per well) and caspase (1.2 × 10⁵ cells per well) assays, respectively. Similarly, 120,000 cells/well were plated in 24-well plates for the detection of ROS/RNS. When ARPE-19 cells became confluent, they were incubated for 24 hours in serum-free medium so that they became relatively nonproliferating, which is the state of natural human RPE cells. Passages 12 to 15 were used for the experiments. Keeping in mind that levels of enzyme activity can vary with passage and time, experiments were performed in triplicate, and the entire experiment was repeated three different times. 

Pretreatment with Inhibitors

For each experiment, cells were pretreated for 6 hours with different classes of inhibitors using the following concentrations: memantine: 2 μM, 10 μM, 20 μM, 30 μM, and 40 μM; simvastatin: 0.01 μM, 1 μM, and 10 μM; epicatechin: 5 μM, 10 μM, and 20 μM; ALLN Ac-Leu-Leu-Nle-H (aldehyde): 5 μM, 12.5 μM, and 25 μM; ALLM Ac-Leu-Leu-Met-H (aldehyde): 5 μM, 12.5 μM, and 25 μM; BITC: 15 μM, 30 μM, and 45 μM); genistein: 20 μM, 30 μM, 40 μM, and 50 μM; and resveratrol: 20 μM, 30 μM, 40 μM, and 50 μM. 

Exposure to B(e)P

Powder B(e)P was purchased from Sigma Aldrich Inc. (St. Louis, MO), and the stock solution (100 mM B(e)P) was prepared by solubilizing 0.0252 g B(e)P in 1 mL dimethyl sulfoxide (DMSO). Then 200 μM B(e)P was added for 24 hours to the culture media. Equivalent amounts of DMSO served as control cultures. 

Cell Viability Assay: Screening of Inhibitors

After 6 hours of pretreatment with the different inhibitors, 200 μM B(e)P was added to ARPE-19 cultures incubated overnight, and the cell viability (CV) assay was performed to measure the percentage of viable cells. The optimum inhibitory effect of each inhibitor at a particular concentration was determined on the basis of mean percentage of viable cells. 

The CV assay was performed as described by Narayanan et al. ²⁹ Briefly, cells were harvested from the 6-well plates by treatment with 0.2% trypsin-EDTA and then incubated at 37°C for 5 minutes. The cells were centrifuged at 1000 rpm for 5 minutes and resuspended in 1 mL culture medium. CV was analyzed with an automated analyzer (Vi Cell; Beckman Coulter Inc., Fullerton, CA). The analyzer performs an automated trypan blue dye-exclusion assay and gives the percentage of viable cells. 

Caspase-3/7 and Caspase-9 Assays

Caspase-3/7 and caspase-9 activities were detected with the use of carboxyfluorescein apoptosis detection kits (FLICA; Immunochemistry Technologies LLC, Bloomington, MN). The detection kit reagent has an optimal excitation range from 488 to 492 nm and an emission range from 515 to 535 nm. Apoptosis was quantified as the level of fluorescence emitted from the detection kit probes bound to caspases. Nonapoptotic cells appeared unstained, whereas cells undergoing apoptosis fluoresced brightly. 

After 6 hours of pretreatment with different inhibitors and overnight incubation with B(e)P + inhibitor, the wells were rinsed briefly with fresh culture media, replaced with 300 μL/well of 1× reagent in culture media, and incubated at 37°C for 30 minutes under 5% CO₂. Cells were washed with phosphate-buffered saline (PBS). The following controls were included: untreated ARPE-19 cells without reagent as background control; untreated ARPE-19 cells with reagent for comparison of caspase activity of treated cells; wells without cells with buffer alone; tissue culture plate wells without cells with culture media plus DMSO to exclude cross-reaction of reagent with DMSO plus culture media; ARPE-19 cells with DMSO and reagent to account for any cross-fluorescence between untreated cells and DMSO. 

Quantitative calculations of caspase activities were performed with a fluorescence image scanning unit instrument (FMBIO III; Hitachi, Yokohama, Japan). Caspase activity was measured as an average signal intensity of the fluorescence of the pixels in a designated spot (mean signal intensity [msi]). 

Detection of ROS/RNS Production

ROS/RNS production was measured with the fluorescent dye 2′-,7′-dicholorodihydrofluorescein diacetate assay (H₂DCFDA; Invitrogen), ³⁰ which detects hydrogen peroxide, peroxyl radicals, and peroxynitrite anions. The cells were washed with sterile PBS and incubated with 500 μL of 10 μM H₂DCFDA for 30 minutes at 37°C and again were washed with PBS. H₂DCFDA (10 μM) was prepared by adding 2 μL of 5 mM (H₂DCFDA) stock/mL in serum-free culture media. The 5-mM H₂DCFDA stock solution was prepared fresh by mixing 0.005 g H₂DCFDA in 2.05 mL DMSO. ROS/RNS production was measured with the scanning unit (excitation 488 nm, emission 520 nm, FMBIO III; Hitachi). 

Statistical Analysis

Data were subjected to statistical analysis by ANOVA (Prism, version. 3.0; GraphPad Software Inc., San Diego, CA). Newman-Keuls multiple-comparison test was performed to compare the data within each experiment. P < 0.05 was considered statistically significant. Error bars in the graphs represent SEM, with experiments performed in triplicate. 

Results

Cell Viability Assay: Screening of Inhibitors

Pretreatment of ARPE-19 cells with different concentrations of ALLN, ALLM, BITC, epicatechin, and simvastatin did not significantly reverse the mean CV percentage CV (Fig. 1a). The B(e)P-treated cultures showed 64.1% ± 1.1% cell viability, whereas the optimum mean CV percentages after B(e)P + inhibitors treatment were as follows: 65% ± 1% (by ALLN 12.5 μM), 58.25% ± 0.75% (by ALLM 12.5 μM), 20.7% ± 1.9% (by BITC 30 μM), 72.7% ± 1.7% (by epicatechin 10 μM), and 66.45% ± 2.15% (by simvastatin 1 μM). However, pretreatment with genistein, resveratrol, and memantine at 30 μM (followed by B(e)P treatment) could fully reverse the effect of B(e)P on mean CV (Fig. 1b). The mean CV percentages due to B(e)P + genistein 30 μM, B(e)P + resveratrol 30 μM, and B(e)P+ memantine 30 μM were 85.8% ± 2.2%, 87% ± 2%, and 94.3% ± 2.5%, respectively, compared to 64.1% ± 1% in B(e)P-treated cells (P < 0.01). Based on the reversed mean CV percentage performance by genistein, resveratrol, and memantine, the 30 μM concentration was used for these inhibitors in other assays. 

Figure 1.

 

(a) Bar graphs showing effect of pretreatment with inhibitors plus 200 μM B(e)P for 24 hours on cell viability in ARPE-19 cells. The inhibitors were ALLN, ALLM, BITC, and epicatechin, tested at different concentrations. None of the inhibitors at any of the studied concentrations were able to reverse the decreased cell viability due to B(e)P. ALLN, Ac-Leu-Leu-Nle-H (aldehyde); ALLM, Ac-Leu-Leu-Met-H (aldehyde); B(e)P, benzo(e)pyrene; BITC, benzyl isothiocyanate; DMSO, dimethylsulfoxide; Epi, epicatechin. (b) Bar graphs showing the effect of pretreatment with inhibitors plus 200 μM B(e)P for 24 hours on cell viability in ARPE-19 cells. The inhibitors were simvastatin, genistein, resveratrol, and memantine. None of the studied concentrations of simvastatin was able to reverse the decreased cell viability caused by B(e)P. However, genistein, resveratrol, and memantine (at 30 μM) were able to reverse the decreased cell viability significantly (compared with B(e)P treatment; P < 0.01) due to B(e)P in the following order compared with DMSO control: genistein < resveratrol < memantine. Simv, simvastatin; Gen, genistein; Res, resveratrol; Mem, memantine. **P < 0.01, statistically significant.

Caspase-3/7 Assay

The ARPE-19 cells treated with B(e)P had increased caspase-3/7 activity compared with the DMSO-treated cultures (20,500 ± 1500 msi vs. 5250 ± 1250 msi, P < 0.05; Fig. 2). Caspase-3/7 activity decreased significantly in the B(e)P + genistein 30 μM (10,750 ± 1750 msi; P < 0.05)–, B(e)P + resveratrol 30 μM (10,000 ± 1000 msi; P < 0.05)–, and B(e)P + memantine 30 μM (9000 ± 1000 msi; P < 0.05)–treated cultures compared with B(e)P-treated culture. The potency of these inhibitors in decreasing caspase-3/7 activity was in the order genistein < resveratrol < memantine. 

Figure 2.

 

Bar graphs showing significantly increased caspase-3/7 activity in B(e)P-treated ARPE-19 cells compared with DMSO-treated controls (P < 0.05). This increased activity was significantly inhibited by pretreatment with genistein, resveratrol, and memantine (P < 0.05) in the following order: genistein < resveratrol < memantine. B(e)P, benzo(e)pyrene; DMSO, dimethylsulfoxide; Gen, genistein; Res, resveratrol; Mem, memantine. *P < 0.05, statistically significant.

Caspase-9 Assay

The ARPE-19 cells treated with B(e)P had increased caspase-9 activity compared with the DMSO-treated cultures (23,000 ± 1000 msi vs. 3650 ± 550 msi; P < 0.001; Fig. 3). Caspase-9 activity decreased significantly in the B(e)P + genistein 30 μM (3500 ± 700 msi; P < 0.001)–, B(e)P + resveratrol 30 μM (2900 ± 600 msi; P < 0.001)–, and B(e)P + memantine 30 μM (2100 ± 300 msi; P < 0.001)-treated cultures compared with B(e)-treated cultures. The potency of these inhibitors in decreasing caspase-9 activity was again genistein < resveratrol < memantine. 

Figure 3.

 

Bar graphs showing increased caspase-9 activity in B(e)P-treated ARPE-19 cells compared with DMSO-treated controls (P < 0.001). This increased activity was significantly inhibited by pretreatment with genistein, resveratrol, and memantine (P < 0.001) in the following order: genistein < resveratrol < memantine. B(e)P, benzo(e)pyrene; DMSO, dimethylsulfoxide; Gen, genistein; Res, resveratrol; Mem, memantine. ***P < 0.001, statistically significant.

ROS/RNS Measurement

The ARPE-19 cells treated with B(e)P had increased ROS/RNS value compared with the DMSO-treated cultures (32,250 ± 750 vs. 2550 ± 750 msi; P < 0.001), as shown in Figure 4. The ROS/RNS levels decreased significantly in the B(e)P + genistein 30 μM (3550 ± 850 msi; P < 0.001)–, B(e)P + resveratrol 30 μM (3100 ± 700 msi; P < 0.001)–, and B(e)P + memantine 30 μM (2200 ± 500 msi; P < 0.001)–treated cultures compared with B(e)P-treated cultures. The potency of these inhibitors in decreasing ROS/RNS level was genistein < resveratrol < memantine, similar to the order found for decreasing caspase-3/7 and caspase-9 activities. 

Figure 4.

 

Bar graphs showing increased ROS/RNS levels in B(e)P-treated ARPE-19 cells compared with DMSO-treated controls (P < 0.001). This increased activity was significantly inhibited by pretreatment with genistein, resveratrol, and memantine (P < 0.001) in the following order: genistein < resveratrol < memantine. B(e)P, benzo(e)pyrene; DMSO, dimethylsulfoxide; Gen, genistein; Res, resveratrol; Mem, memantine. ***P < 0.001, statistically significant.

Discussion

In the United States there are approximately 8 million people 55 years of age or older with early AMD, and of these approximately 1.3 million are expected to develop late AMD. ³¹ The Age-Related Eye Diseases Study (AREDS) has suggested that zinc-antioxidant supplements can prevent or delay the progression of early AMD to late AMD and that high-dose antioxidant vitamin therapy could reduce the advancement of AMD. ³² Although significant progress has been made in the treatment of the neovascular form of the disease using inhibitors of the vascular endothelial growth factor (VEGF), ³³ VEGF inhibitors are not enough to cure the disease. Therefore, searching for inhibitors that have broad inhibitory potency is very important. 

Our present study demonstrated the beneficial effect of several classes of inhibitors on reversing the loss of cell viability, caspase-3/7, and caspase-9 activation as well as increased ROS/RNS level in B(e)P-treated ARPE-19 cells. 

The B(e)P-induced decrease in CV percentage in ARPE-19 cells was not reversed by calpain inhibitors, BITC, simvastatin, or epicatechin at any of the tested concentrations. This suggests that the pathways these inhibitors target are not involved in the B(e)P toxic effects. However, at 30-μM concentrations, three other inhibitors, genistein, resveratrol, and memantine, all reversed the B(e)P toxicity. Memantine showed the most potent effect, and genistein showed the least potent effect, to reverse CV percentage in B(e)P-treated ARPE-19 cells. 

Caspases are present in cytosol as inactive proenzymes. Once activated, caspase-3/7 is capable of initiating DNA fragmentation, which is a key executioner of apoptosis and is critical to the cellular commitment to apoptotic cell death. ^34,35 The mechanism of apoptosis inhibition can occur by either direct caspase-3 inhibition or inhibition of the conversion of procaspase-3 to the active form. ³⁶ The increased caspase-3/7 activities in B(e)P-treated cultures were inhibited by genistein < resveratrol < and memantine. This indicates that genistein, resveratrol, and memantine play biologically important roles in the control of apoptosis, ³⁷ and that they protected ARPE-19 cells from injury in the present study. 

Similarly, increased caspase-9 activity in B(e)P-treated ARPE-19 cells provides evidence for mitochondrial function–related cellular injury. The activation of caspase-9 is associated with mitochondrial stress, ³⁸ which inactivates mitochondrial enzymes and proteins, ^39,40 resulting in mitochondrial dysfunction. However, in the present study, the inhibition of caspase-9 activity by genistein, resveratrol, and memantine suggests these inhibitors act as mitochondrial-protective agents in B(e)P-induced injury. 

In the B(e)P-treated ARPE-19 cell cultures, the generation of ROS/RNS was significantly elevated. Increased levels of ROS, if not scavenged immediately, will cause oxidative damage to macromolecules, such as proteins, enzymes, and DNA, and will weaken antioxidant defenses. ⁴¹ However, the reversal of increased ROS/RNS levels by genistein, resveratrol, and memantine indicates that they function as efficient antioxidants. Such antioxidants would be able to protect proteins, enzymes, and DNA from oxidative damage. 

Several in vitro and animal studies have reported protective effects of genistein, resveratrol, and memantine against damage induced by different agents and chemicals. These inhibitors act through various mechanisms, such as inhibition of free radical generation, DNA oxidation, and suppression of enzymes responsible for the metabolism of agents and chemicals into carcinogenic agents. 

In our study, memantine was the most potent inhibitor for each of the assays used. Memantine is a neuroprotectant with protective actions in models of ischemia of the central nervous system and retina. ^42,43 Ju et al. ⁴⁴ reported that memantine treatment (5 mg/kg) inhibited cytochrome c release, blocked apoptotic cell death, and increased retinal ganglion cell (RGC) survival in glaucomatous retina of mice. Hare et al. ^45,46 reported that memantine (4 mg/kg) given orally was both safe and effective in RGC survival and in reducing tomographic measurements of nerve head topography in experimental glaucoma in monkeys. Studies have also demonstrated the neuroprotective effects of memantine in other animal models. WoldeMussie et al. ⁴⁷ showed that memantine treatment (5–10 mg/kg) increased compound action potential amplitude and RGC survival and prevented further loss after laser treatment in rats. Lagreze et al. ⁴⁸ showed that memantine (10–20 mg/kg) increased vitreal concentrations of glutamate and glycine and reduced ganglion cell loss when given systemically (intraperitoneally) before or within 30 minutes of ischemia in rats. Caumont et al. ⁴⁹ reported that glioma cell cultures treated with memantine showed dose-dependent increased in glial cell line-derived neurotrophic factor (GDNF) and GDNF mRNA levels. Beside neuroprotective effects, memantine (10 mg/kg daily) treatment significantly improved retinal function, protected RGC loss, and decreased vitreoretinal VEGF protein levels and blood-retinal barrier leakage in the diabetic retina of rats. ⁵⁰  

Resveratrol, another effective inhibitor in our study, showed optimum inhibition at 30 μM, but other studies have reported its inhibitory effect at variable concentrations. For example, Anekonda et al. ⁵¹ reported the inhibition of caspase-3 activity and the protection of retinal cells in vitro from apoptosis by resveratrol at 40 μM. Sheu et al. ⁵² reported that resveratrol (10 μM, 20 minutes) significantly reduced oxidative damage in H₂O₂ (hydrogen peroxide)-treated human RPE R-50 cells through the activity of calcium-activated potassium channels (BK_Ca) in RPE cells. Liu et al. ⁵³ reported that pretreatment with resveratrol (40 μM) reduced oxidative damage in RGCs in vitro and prevented pressure-induced neuronal death in a mouse model of glaucoma. Revel ⁵⁴ reported that resveratrol (50 mg/kg per week) protected BaP-induced DNA damage and apoptosis in the lungs of mice through CYP1A1/AHH and BPDE-DNA adduct formation and potentially could be used clinically to decrease the risk for lung cancer. 

Similarly, genistein showed optimum inhibition at 30 μM in our study, but other studies reported its various types of inhibitory effects at different concentrations. Genistein significantly inhibited the upregulation of VEGF protein expression induced by cobalt chloride or hypoxia in rabbit RPE cells at a concentration of 50 μM. ⁵⁵ Li et al. ⁵⁶ reported inhibitory effects of genistein at 50 μM (for 30 minutes) on interleukin-8 (IL-8) expression in ARPE-19 cells under hypoxia or stimulation by potassium chloride, norepinephrine, and glutamate. Leung et al. ⁵⁷ reported that genistein at 5 mM prevented oxidative DNA damage by suppressing cytochrome enzymes (CYP1A1 and CYP1B1) that convert polyaromatic hydrocarbon (PAH) to PAH metabolites, which bind to DNA. Moreover, suppressing these enzymes reduced free radical generation and decreased cellular oxidative stress. Ruwelar et al. ⁵⁸ reported the inhibition of cumene hydroperoxide-induced radical generation by both genistein and resveratrol at 20 μM in C6 astroglioma cells. 

In summary, our results demonstrated genistein, resveratrol, and memantine as the broad inhibitors (and memantine the most potent) by acting through the caspase pathways, particularly mitochondrial caspase-9 pathway and ROS/RNS formation. These inhibitory effects ultimately protected the cell viability of ARPE-19 cells. Therefore, such inhibitors could be beneficial to a number of diseases (such as diabetes, cataracts, HIV activation, neurodegeneration, and radiation injury in animals) associated with mitochondrial dysfunction and oxidative stress. ^59,60 The strong protective effects of these inhibitors against B(e)P-induced cytotoxicity suggest they may be useful for the prevention or treatment of smoking- and age-related degenerative diseases, such as AMD. However, our results are based on in vitro study using a single cell line and would not be appropriate to predict its clinical usefulness. Therefore, further studies using other cell lines and animal models should be conducted with the goal of developing prophylaxis or therapy for the treatment of retinal diseases in humans. 

Footnotes

 Supported by the Discovery Eye Foundation, the Henry L. Guenther Foundation, the Iris and B. Gerald Cantor Foundation, the Ko Family Foundation, the Lincy Foundation, and the Research to Prevent Blindness Foundation.

Footnotes

 Disclosure: S. Mansoor, None; N. Gupta, None; A.J. Patil, None; M.F. Estrago-Franco, None; C. Ramirez, None; R. Migon, None; A. Sapkal, None; B.D. Kuppermann, None; M.C. Kenney, None