Effects of Benzo(e)Pyrene, a Toxic Component of Cigarette Smoke, on Human Retinal Pigment Epithelial Cells In Vitro

Abstract

purpose. To better understand the cellular and molecular basis for the epidemiologic association between cigarette smoke and age-related macular degeneration (AMD), the authors examined the effects of Benzo(e)Pyrene (B(e)P), a toxic element in cigarette smoke, on human retinal pigment epithelial cells (ARPE-19).

methods. ARPE-19 cells were cultured in Dulbecco modified Eagle medium containing 10% fetal bovine serum. Cells were treated for 24 hours with 1000 μM, 400 μM, 200 μM, and 100 μM B(e)P. Cell viability was determined by a trypan blue dye-exclusion assay. Activities of caspase-3/7, caspase-8, caspase-9, and caspase-12 were measured by a fluorescence image scanner, and DNA laddering was evaluated by electrophoresis on 3% agarose gel.

results. The mean percentage of cell viabilities of ARPE-19 cells was decreased in a dose-dependent manner after exposure to B(e)P at the higher concentrations of 1000 μM (20.0 ± 0.4; P < 0.001), 400 μM (35.6 ± 6.4; P < 0.001), and 200 μM (58.7 ± 2.3; P < 0.001) but not at 100 μM (95.9 ± 0.7; P > 0.05) compared with the equivalent dimethyl sulfoxide (DMSO)-treated control cultures. There were significant increases in caspase-3/7, -8, -9, and -12 activities compared with the DMSO-treated controls (P < 0.001). DNA laddering revealed bands at 200-bp intervals.

conclusions. These results show that B(e)P is a toxicant to human retinal pigment epithelial cells in vitro. It causes cell death and induces apoptosis by the involvement of multiple caspase pathways.

Age-related macular degeneration (AMD) is the most important cause of vision loss among the elderly in Western nations. ¹ Smoking is a primary risk factor associated with the prevalence and the incidence of neovascular (wet form) macular degeneration and geographic atrophy (dry form). ^{2 3} The wet and dry forms of AMD are associated with abnormal vascular cell proliferation and RPE cell damage, respectively. A review of 17 studies found a twofold to threefold increased risk for AMD in current smokers compared with those who never smoked. ^{2 3} The association between smoking and AMD has been strengthened recently by various epidemiologic studies, including the Age-Related Eye Disease Study. ^{4 5 6}  

Although cigarette smoke contains more than 4000 toxic agents, the worst may be polycyclic aromatic hydrocarbon (PAH). This is one of the leading causes of the formation of DNA adducts and can lead to more cellular proliferation than any other toxic agent in cigarette smoke. Many in vitro and in vivo studies have been performed to determine the chemical effects of PAHs on cells such as rat renal glomerular mesangial cells, ⁷ human HepG2 cells, ⁸ and mouse preleukemic cells. ⁹ Benzo(a)Pyrene (B(a)P) is one of the better studied PAHs and is known to cause damage to bovine RPE cells. ¹⁰ Among all PAHs, B(a)P and its close derivative, B(e)P, have a maximum ion mass (252.3 m/z for both B(a)P and B(e)P) and a maximum retention time (18.55 minutes for B(e)P and 18.70 minutes for B(a)P), which makes these chemicals very important for diseases related to chronic exposure. Although some studies about the effects of B(a)P on retinal cells have been published, ¹⁰ no information regarding the B(e)P compound, which has maximum retention times and ion mass similar to those of B(a)P, has been published. Therefore, we used a human RPE cell line to examine cytotoxic effects B(e)P might have and to determine which, if any, apoptotic pathways may be involved. The toxic effects and the metabolism of B(a)P depend on the tissue of origin and the species of mammalian cells. In the case of B(a)P, researchers have performed in vitro experiments with various cell types and have routinely used concentrations of 10 to 100 μM. ^{10 11 12 13 14 15 16} In cultures of bovine RPE cells, Patton et al. ¹⁰ found that cells changed their morphology at 50 and 100 μM B(a)P. 

After exposure to PAHs, the uvea undergoes metabolic changes. ¹⁷ The metabolic pathways in the uvea and RPE cells can use the PAH as a substrate; therefore, RPE cells and the uvea are targets for damage caused by PAH. ¹⁷ The strong epidemiologic evidence linking smoking to AMD raises several basic science questions regarding the mechanism of damage at the cellular level. For example, what pathways are associated with different components of cigarette smoke, and what is the association of the components to toxicity in human RPE cells? Many different molecular pathways are associated with apoptotic or necrotic cell death, and these pathways can be triggered by factors such as ischemia, heat shock proteins, deprivation of growth factors, and oxysterols. 

Photoreceptor damage, especially within the macular region, is the major cause of vision loss in AMD. Although the pathogenesis of AMD includes different clinical signs, the degeneration of RPE cells is often observed at early stages of the disease. Abnormalities in the RPE and Bruch membrane are characteristics of the disease. ^{18 19} Oxidative stress involving the mitochondria can lead to mitochondrial DNA (mtDNA) damage, destabilization of mitochondrial function, and induction of apoptosis. ²⁰ Death of RPE, photoreceptors, and inner nuclear layer cells through apoptosis has also been demonstrated by some of the studies on postmortem eyes of patients with AMD. ²¹  

All apoptotic pathways converge on a family of cysteine-aspartase, named caspases, whose activity drives the biochemical events leading to cellular disassembly and death. Caspases are present as inactive precursors. “Initiator” caspases are thought to autoactivate themselves proteolytically, whereas “effector” caspases are activated by initiator caspases and are responsible for execution of cell death. ²²  

Activation of caspase-3/7 represents a commitment for cell disassembly and is a hallmark for apoptosis. Three different pathways are recognized as the initiator caspases involved in activating apoptosis. ²³ The extrinsic (receptor-mediated) pathway includes the Fas/Fas-ligand cell-surface receptors, which are activated and subsequently lead to caspase-8 activation. ²⁴ Alternatively, the intrinsic (mitochondrial) pathway, which is associated with mitochondrial stress, involves the activation of caspase-2 and -9 and the release of cytochrome c. ^{25 26} The newly recognized caspase-12 is activated by endoplasmic reticulum stress and induces the cleavage of caspase-3/7 in a cytochrome c-independent manner. ²⁷ The caspase-12 pathway has a pathologic basis in some neurodegenerative disorders. ²⁸ To date no studies have examined the caspase pathways involved in B(e)P-related cell death on ocular cells. 

In the present study with human ARPE-19 cells, B(e)P increased the activation of caspase-3/7, -8, -9, and -12 and decreased cell viability. This is the first study demonstrating caspase-12 activation with any of the cigarette smoke constituent on ARPE-19 cells. Our findings demonstrate that B(e)P, an element associated with smoking, causes caspase-dependent apoptosis, which may be a major factor in promoting the onset and progression of AMD. 

Materials and Methods

Cell Culture

ARPE-19 cells were obtained from ATCC (Manassas, VA). Cells were grown in a 1:1 mixture (vol/vol) of Dulbecco modified Eagle and Ham nutrient mixture F-12 medium (DMEM F-12; Invitrogen-Gibco, Carlsbad, CA), nonessential amino acids 10 mM 1×, 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 (5.5 × 10⁵ cells/well) and caspase (1.5 × 10⁵ cells/well) assays. When ARPE-19 cells became confluent, they were incubated for 24 hours in serum-free medium so that they would be relatively nonproliferating. This simulated the natural human RPE cells, which remained in a nonproliferating phase and which were not exposed to the circulation because of the blood-retinal barrier. The cells were exposed to B(e)P. Passage numbers 12 to 15 were used for the experiments. Keeping in mind the fact that enzyme activity levels can vary with time, experiments were performed in triplicate, and the entire experiment was repeated three different times. Then the values were combined and calculated statistically to show a consistent increase of caspase activity. 

Exposure to B(e)P

B(e)P is commercially available as a powder. We received 0.0252 g B(e)P powder, and 1000-μM, 400-μM, 200-μM, and 100-μM concentrations of B(e)P were made by solubilizing it into dimethyl sulfoxide (DMSO). The stock solution (100 mM B(e)P) was prepared by dissolving 0.0252 g B(e)P in 1 mL DMSO. Then we prepared our diluted concentrations by adding them to the culture media. Cells were treated with 1000 μM, 400 μM, 200 μM, and 100 μM B(e)P for 24 hours or with the equivalent amounts of DMSO, which served as control cultures. 

Cell Viability Assay

Cell viability 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 were incubated at 37°C for 5 minutes. Cells were centrifuged at 1000 rpm for 5 minutes and resuspended in 1 mL culture medium. Cell viability was analyzed with the use of an automated analyzer (Vi Cell; Beckman Coulter, Fullerton, CA). The analyzer performs an automated trypan blue dye-exclusion assay and gives the percentage of viable cells. 

Caspase Detection

Caspase-3/7, -8, -9, and -12 activities were detected with the use of detection kits (Carboxyfluorescein FLICA Apoptosis Detection kits; Immunochemistry Technologies LLC, Bloomington, MN). The FLICA 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 FLICA probes bound to caspases. Nonapoptotic cells appeared unstained, whereas cells undergoing apoptosis fluoresced brightly. 

At the designated time period, the wells were rinsed briefly with fresh culture media, replaced with 300 μL/well of 1× FLICA solution in culture media, and incubated at 37°C for 1 hour under 5% CO₂. Cells were washed with phosphate-buffered saline (PBS). The following controls were included: untreated ARPE-19 cells without FLICA were used as a background control; untreated ARPE-19 cells with FLICA for comparison of caspase activity of treated cells; wells without cells with buffer alone; tissue culture plate wells without cells with culture media + DMSO to exclude cross-reaction of FLICA with DMSO + culture media; and ARPE-19 cells with DMSO and FLICA 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 average signal intensity of the fluorescence of the pixels in a designated spot (mean signal intensity). 

DNA Fragmentation Assay

ARPE-19 cells (5 × 10⁶) were plated overnight in 100-mm dishes and then incubated for another 24 hours with B(e)P in serum-free medium. DNA was extracted (QIAamp DNA Micro kit; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Samples were separated by electrophoresis on 3% agarose gels and were stained with 5% ethidium bromide. Marker (100 bp) was used, and images were captured with a fluorescence image scanning instrument (FMBIO III; Hitachi). 

Statistical Analysis

Data were subjected to statistical analysis by ANOVA (Prism, ver. 3.0; GraphPad Software, 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 the SEM of triplicate experiments. 

Results

Cell Viability Studies

ARPE-19 cells showed a concentration-dependent decrease in cell viability after exposure to B(e)P for 24 hours (Fig. 1) . Mean cell viabilities of DMSO-treated equivalent cultures of 1000 μM (91.1 ± 3.6), 400 μM (97.3 ± 1.5), 200 μM (98.3 ± 1.7), and 100 μM (98.4 ± 0.6) were similar to those of the untreated ARPE-19 cultures (99.0 ± 0.4). Cell viabilities were 20.0 ± 0.4 (P < 0.001), 35.6 ± 6.4 (P < 0.001), and 58.7 ± 2.3 (P < 0.001) at doses of 1000 μM, 400 μM, and 200 μM B(e)P, respectively. At the lower concentrations of 100 μM B(e)P, cell viability was 95.9 ± 0.7 (P > 0.05). 

Caspase-3/7 Activity

Caspase-3/7 activity in ARPE-19 increased significantly after treatment with B(e)P for 24 hours (Fig. 2A) . Values for untreated cells and for DMSO-equivalent cultures of 1000 μM, 400 μM, 200 μM, and 100 μM were 1607.5 ± 208.6, 4098.0 ± 582.8, 1890.6 ± 571.9, 2297.9 ± 4.1, and 2284.7 ± 254.9, respectively. Cells treated with B(e)P 1000 μM, 400 μM, 200 μM, and 100 μM showed mean fluorescence of 13,403.8 ± 650.1, 30,168.4 ± 1577.8, 22,323.3 ± 419.7, and 18,641.3 ± 1303.9, respectively. Caspase-3/7 is the hallmark of apoptosis because it is the final common pathway of apoptosis. To verify apoptotic activity, DNA fragmentation analysis was performed showing DNA bands that laddered in approximately 200-bp increments, consistent with apoptosis (Fig. 2B) . 

Caspase-8 Activity

Caspase-8 activity in ARPE-19 increased significantly after treatment with B(e)P for 24 hours (Fig. 3) . Values for untreated cells and for DMSO-equivalent cultures of 1000 μM, 400 μM, 200 μM, and 100 μM were 3865.0 ± 176.8, 3655.7 ± 360.8, 3805.4 ± 685.5, 4166.9 ± 381.6, and 4280.1 ± 438.3, respectively. Cells treated with 1000 μM, 400 μM, 200 μM, and 100 μM B(e)P showed mean fluorescence of 28,277.7 ± 813.9, 31,199.8 ± 331.7, 29,728.3 ± 1426.9, and 18,012.9 ± 169.0, respectively. 

Caspase-12 Activity

Caspase-12 activity in ARPE-19 increased significantly after treatment with B(e)P for 24 hours (Fig. 4) . Values for untreated cells and for DMSO-equivalent cultures of 1000 μM-, 400 μM-, 200 μM-, and 100 μM-treated cultures were 2050.5 ± 211.4, −1739.5 ± 351.0, 561.0 ± 223.0, −471.0 ± 3803.3, and −1445.4 ± 596.7, respectively. Cells treated with 1000 μM, 400 μM, 200 μM, and 100 μM B(e)P showed mean fluorescence of 25,769.6 ± 2224.8, 29,297.3 ± 468.7, 27,486.08 ± 1796.1, and 14,525.2 ± 955.7, respectively. 

Caspase-9 Activity

Caspase-9 activity in ARPE-19 increased significantly after treatment with B(e)P for 24 hours (Fig. 5) . Values for untreated cells and for DMSO-equivalent cultures of 1000 μM-, 400 μM-, 200 μM-, and 100 μM-treated cultures were −3884.8 ± 119.0, −3.141.8 ± 173.4, −2208.7 ± 132.9, −3743.2 ± 1547.5, and −3848.2 ± 212.1, respectively. Cells treated with 1000 μM, 400 μM, 200 μM, and 100 μM B(e)P showed mean fluorescence of 25,503.2 ± 1951.5, 27,370.7 ± 633.0, 24,084.3 ± 423.6, and 16,690.3 ± 18.4, respectively. 

Discussion

Among all environmental risk factors for AMD, cigarette smoking is known to be the strongest and to be associated with substantially higher incidences of geographic atrophy and choroidal neovascularization. ^{3 30} Since 1992, five cross-sectional or case-control studies ^{31 32 33 34 35} and two prospective studies ^{36 37} have consistently shown an adverse effect of smoking on AMD. One recent study revealed the formation of sub-RPE deposits and diffuse thickening of Bruch membrane in mice after they were exposed to cigarette smoke or hydroquinone, a cigarette smoke oxidant, ³⁸ suggesting that oxidative stress promotes phenotypic characteristics similar to those found in AMD. Although it is recognized that many oxidants are present in cigarette smoke, the mechanisms by which smoking increases the incidence and severity of AMD is not yet clear. ³⁹  

ARPE-19 is a human diploid RPE cell line that displays many differentiated properties typical of RPE in vivo. Many clinically relevant studies related to cell death and apoptosis using ARPE-19 cultures have been published in the past few years. ^{40 41 42 43 44} Therefore, the ARPE-19 cell line is a commonly used RPE cell in eye research. ⁴⁵ Our data showed that the endoplasmic reticulum pathway (caspase-12), extrinsic Fas/Fas ligand (Fas-L) pathway (caspase-8), and mitochondrial (caspase-9) pathway were activated by B(e)P treatment. These findings support our hypothesis that PAHs, such as B(e)P, can lead to decreased cell viability through caspase-dependent apoptosis and may lead to retinal cell atrophy. 

B(e)P is a high-molecular weight, five-ring PAH found in cigarette smoke (5–40 ng/cigarette). In addition to smoking component and ubiquitous pollutant in the environment, B(e)P can be generated by incomplete combustion of organic materials, automobile exhaust, charcoal-broiled foods, and industrial waste byproducts. ⁴⁶ Another PAH, B(a)P, is an isomer of B(e)P, and both are present in mainstream cigarette smoke. B(a)P disrupts the retinal lysosomes that precede experimental retinal degeneration in mice. ⁴⁷ Many studies have demonstrated the toxic effects of B(a)P in different cells, such as human bronchial epithelial cells ⁴⁸ and human placental tissues, ⁴⁹ but few have been published on the effects of B(e)P. ^{50 51} To the best of our knowledge, our study is the first to examine the effects of B(e)P on ARPE-19 cells, an important retinal cell type that undergoes atrophic changes in AMD. 

Cell viability assays demonstrated that B(e)P was cytotoxic to the ARPE-19 cells in a concentration-dependent fashion. Cells lost 80%, 65%, and 41% of viability after ARPE-19 cells were exposed to, 1000 μM, 400 μM, and 200 μM B(e)P for 24 hours. Interestingly, the 100 μM concentrations of B(e)P had no apparent effect on ARPE-19 cell viability. This is in agreement with a study on human Caco-2 cells that showed no significant change in cell viability with a dose of 50 μM B(e)P after 72 hours of incubation. ⁵⁰ Human hepatoma HepG2 cells and breast carcinoma MCF-7 cells also showed no significant effects on cell viability with concentrations as low as 2.5 μM and 5 μM of B(e)P after 48 hours of incubation. However, using the identical concentrations of B(a)P, they found significant decreases in cell viability, which suggests that B(a)P is more toxic than B(e)P on these cell lines. ⁵¹  

Although hydrophilic compounds such as B(e)P cross the cell membrane within 6 hour, more time is required for its conversion to active compounds within the cell and to stimulate the cellular response. Therefore, we used a 24-hour time period for these experiments because some of our previous studies with ARPE-19 cultures treated with 7-ketocholesterol showed that increased caspase activities could be found within this time frame. ⁵² However, we are also testing 6-hour to 72-hour time periods for the other compounds in cigarette smoke and will present these findings in future studies. 

Apoptosis plays a pivotal role in normal retinal development and in the molecular pathophysiology of various retinal conditions. Apoptosis is not frank cell death. Rather, it is a cascade that begins before frank cell death and cell membrane rupture. Trypan blue dye exclusion assay detects cell death after breach of the cell membrane, whereas our caspase assay can detect apoptosis before cells are dead (i.e., before cell membranes are ruptured). 

Specific apoptotic pathways have been examined in multiple retinal conditions, such as AMD, retinal detachment, diabetic retinopathy, proliferative vitreoretinopathy, cytomegalovirus retinitis, and cancer-associated retinopathy. ^{53 54 55 56 57 58 59} Our study shows that PAHs such as B(e)P can damage RPE cells through apoptosis, and this may play a role in retinal degeneration. It is proposed that RPE cells undergo apoptosis, leading to regional atrophy and affecting the adjacent retina. Dissecting the mechanism(s) of specific apoptotic pathways involved may allow us to identify a therapeutic target for AMD and other retinal disorders. 

In response to B(e)P, human ARPE-19 cells undergo apoptosis, as reflected by increased caspase-3/7 activity and DNA laddering. This is in agreement with studies of cultured human CD34⁺ cells showing caspase-3 activation ⁶⁰ and HepG2 cells showing apoptosis by DNA laddering ⁶¹ after treatment with 10 μM B(a)P. Our findings were also similar to those of Chang et al., ²⁴ who demonstrated caspase-3/7 activation, apoptosis, and loss of cell viability in B(a)P-treated mouse hepatoma Hepa1c1c7 cells. In contrast, other cell types, such as human endothelial cells, respond to B(a)P by undergoing necrotic cell death without caspase-3 activation. ⁶²  

In human ARPE-19 cultures, caspase-8 was activated after B(e)P treatment. This agrees with previous findings that Fas-mediated apoptosis plays a role in oxidant-induced cell death in human RPE cells. ⁶³ This signaling pathway of TNF-α or Fas-mediated apoptosis has been described in numerous cell types, including RPE cells. ^{64 65 66} Our data show that B(e)P-induced cell death occurred not only through the cell-surface death receptors (caspase-8 pathway) but that it also involved caspase-12 activation, which is the endoplasmic reticulum stress-induced cell-signaling pathway. Inactive caspase-12 is localized to the cytosolic face of the endoplasmic reticulum and, once activated, can trigger caspase-3 activation. ⁶⁷ Caspase-12 is closely related to caspase-1 and other members of the caspase family, known as inflammatory caspases, which process and activate inflammatory cytokines such as IL-1 and IL-18. The CASP12 gene is subject to polymorphisms that can generate a full-length caspase protein (Csp12L) or an inactive truncated form (Csp12S). The functional form appears to be confined to people of African decent and is linked with susceptibility to sepsis. Persons carrying the functional gene have decreased responses to bacterial molecules such as lipopolysaccharide. ^{68 69} The gene of caspase-12 is found on chromosome 11 in humans, in a locus with other inflammatory caspases. ⁷⁰ To our best knowledge, this is the first study showing the involvement of caspase-12 by any of the cigarette smoke constituents on ARPE. Interactions between caspase-8 and caspase-12 pathways that caused apoptosis in ARPE-19 cells still must be determined. 

Caspase-9 activities were also increased in B(e)P-treated ARPE-19 cultures, indicating that the mitochondrial pathway for apoptosis was also involved. This is consistent with studies of CD34⁺ cells showing caspase-9 activation after treatment with 10 μM B(a)P. ⁶⁰ Caspase-9 activity of represents important steps in numerous mitochondrion-related apoptotic processes. ⁷¹ Interestingly caspase-9 was not activated in ARPE-19 cultures after treatment with the oxysterol, 7-ketocholesterol, though caspase-8 and caspase-12 pathways were activated. ⁷² This indicates that activation of caspase pathways is related to the specific agent, not necessarily to the cell type. In addition, it suggests that inhibition of the caspase pathways requires a broad inhibitor given that all three initiator caspases are activated by the B(e)P. 

As noted previously, B(e)P is a major PAH component of cigarette smoke. However, because this study was performed in vitro, the B(e)P concentrations used cannot be directly extrapolated to clinical practice. In addition, the levels of B(e)P in the retinas of smokers are unknown. However, our data suggest that if levels of 200 μM B(e)P are reached in the retina, significant cell damage may occur. Given that smoking is a habitual practice, the concentrations of B(e)P may accumulate in tissues during high volume, chronic cigarette use, making the cells more susceptible to oxidative damage. Eventually even lower B(e)P concentrations may cause cell damage. Our data provide a possible mechanism by which smoking may cause retinal cell damage, as seen in AMD. 

 Supported by the Discovery Eye Foundation, Iris and B. Gerald Cantor Foundation, Gilbert Foundation, Ko Family Foundation, the Skirball Molecular Ophthalmology Program, the Research to Prevent Blindness Foundation, and Henry L. Guenther Foundation.

 Submitted for publication March 20, 2008; accepted September 2, 2008.

 Disclosure: A. Sharma, None; A. Neekhra, None; A.L. Gramajo, None; J. Patil, None; M. Chwa, None; B.D. Kuppermann, None; M.C. Kenney, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

ARPE-19 cells showed a dose-dependent decrease in cell viability after B(e)P treatment for 24 hours compared with DMSO-treated control cultures. Cell viability for cultures treated with 100 μM B(e)P were similar to DMSO-equivalent control. Cell viability was decreased significantly with 1000 μM (P < 0.001), 400 μM (P < 0.001), and 200 μM (P < 0.001) B(e)P. ***Statistically significant (P < 0.001).

Figure 2.

 

(A) ARPE-19 cells treated for 24 hours with 400 μM B(e)P had maximum caspase-3/7 activity compared with DMSO-equivalent control. DMSO-treated control cultures and untreated ARPE-19 control cultures showed minimal caspase-3/7 activity. After treatment with 1000 μM, 400 μM, 200 μM, and 100 μM B(e)P, ARPE-19 cells had an increased mean fluorescence compared with DMSO-treated cultures (P < 0.001). ***Statistically significant (P < 0.001). (B) DNA fragmentation analysis showing bands at 200-bp intervals in B(e)P-treated cultures.

Figure 3.

 

ARPE-19 cells treated for 24 hours with 400 μM B(e)P had maximum caspase-8 activity compared with DMSO-treated cultures. DMSO-treated control cultures and untreated ARPE-19 control cultures showed minimal caspase-8 activity. After treatment with 1000 μM, 400 μM, 200 μM, and 100 μM B(e)P, ARPE-19 cells had an increased mean fluorescence compared with DMSO-treated cultures (P < 0.001). ***Statistically significant (P < 0.001).

Figure 4.

 

ARPE-19 cells treated for 24 hours with 400 μM B(e)P had maximum caspase-12 activity compared with DMSO-treated cultures. Caspase-9 activity was nondetectable in DMSO-treated control cultures and minimal in untreated ARPE-19 control cultures. After treatment with 1000 μM, 400 μM, 200 μM, and 100 μM B(e)P, ARPE-19 cells had an increased mean fluorescence compared with DMSO-treated cultures (P < 0.001). ***Statistically significant (P < 0.001).

Figure 5.

 

ARPE-19 cells treated for 24 hours with 400 μM B(e)P had maximum caspase-9 activity compared with DMSO-treated cultures. Caspase-9 activity was nondetectable in DMSO-treated control cultures and untreated ARPE-19 control cultures. After treatment with 1000 μM, 400 μM, 200 μM, and 100 μM B(e)P, ARPE-19 cells had an increased mean fluorescence compared with DMSO-treated cultures (P < 0.001). ***Statistically significant (P < 0.001).