Preventive effect of <Emphasis Type="Italic">Vaccinium uliginosum</Emphasis> L. extract and its fractions on age-related macular degeneration and its action mechanisms

Age-related macular degeneration (AMD) is the leading cause of vision loss and blindness among the elderly. Although the pathogenesis of this disease remains still obscure, several researchers have report that death of retinal pigmented epithelium (RPE) caused by excessive accumulation of A2E is crucial determinants of AMD. In this study, the preventive effect of Vaccinium uliginosum L. (V.U) extract and its fractions on AMD was investigated in blue light-irradiated human RPE cell (ARPE-19 cells). Blue light-induced RPE cell death was significantly inhibited by the treatment of V.U extract or its fraction. To identify the mechanism, FAB-MS analysis revealed that V.U inhibits the photooxidation of N-retinyl-N-retinylidene ethanolamine (A2E) induced by blue light in cell free system. Moreover, monitoring by quantitative HPLC also revealed that V.U extract and its fractions reduced intracellular accumulation of A2E, suggesting that V.U extract and its fractions inhibit not only blue light-induced photooxidation, but also intracellular accumulation of A2E, resulting in RPE cell survival after blue light exposure. A2E-laden cell exposed to blue light induced apoptosis by increasing the cleaved form of caspase-3, Bax/Bcl-2. Additionally, V.U inhibited by the treatment of V.U extract or quercetin-3-O-arabinofuranoside. These results suggest that V.U extract and its fractions have preventive effect on blue light-induced damage in RPE cells and AMD.     

Paeoniflorin attenuates atRAL-induced oxidative stress,mitochondrial dysfunction and endoplasmic reticulum stress in retinal pigment epithelial cells via triggering Ca<Superscript>2+</Superscript>/CaMKII-dependent activation of AMPK

Abnormal accumulation of the free-form all-trans-retinal (atRAL), a major intermediate of human visual cycle, is considered to be a key cause of retinal pigment epithelial (RPE) dysfunction in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration (AMD). Paeoniflorin (PF), a monoterpene glucoside isolated from Paeonia lactiflora Pall., has been used in clinical treatment of retinal degenerative diseases in China for several years; however, the underlying mechanism remains unclear. The aim of this study is to investigate the protective effect of PF against atRAL toxicity in human ARPE-19 cells and its molecular mechanism. The results of our study showed that the pre-treatment of PF dose-dependently attenuated atRAL-induced cell injury by the reduction of Nox1/ROS-associated oxidative stress, mitochondrial dysfunction and GRP78-PERK-eIF2α-ATF4-CHOP-regulated endoplasmic reticulum (ER) stress in ARPE-19 cells. Additionally, our data showed that PF mainly exerted its activity via triggering calcium-calmodulin dependent protein kinase II (CaMKII)-mediated activation of AMP-activated protein kinase (AMPK). AMPK inhibition significantly reversed the protective effect of PF against atRAL toxicity in ARPE-19 cells. Overall, our findings provided the novel mechanism of PF protecting human RPE cells, which may prevent the progression of retinal degenerative diseases.     

Lutein improves cell viability and reduces Alu RNA accumulation in hydrogen peroxide challenged retinal pigment epithelial cells

Purpose: Dysfunction of the microRNA (miRNA)-processing enzyme DICER1 and Alu RNA accumulation are linked to the pathogenesis of age-related macular degeneration (AMD). This study determined the optimal dose of lutein (LUT) and zeaxanthin (ZEA) to protect human retinal pigment epithelium (RPE) cells against hydrogen peroxide (H₂O₂). The effect of the optimal dose of LUT and ZEA as DICER1 and Alu RNA modulators in cultured human RPE cells challenged with H₂O₂ was investigated.

Materials and methods: ARPE-19 cells were pre-treated with LUT, ZEA, or both for 24?h before 200?μM H₂O₂ challenge. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. DICER1 and Alu RNA were quantified by western blotting and real-time polymerase chain reaction, respectively.

Results: H₂O₂ increased cell Alu RNA expression and decreased cell viability of ARPE-19, but had no significant impact on the DICER1 protein level. LUT, alone and in combination with ZEA pre-treatment, prior to H₂O₂ challenge significantly improved cell viability of ARPE-19 and reduced the level of Alu RNA compared to the negative control.

Conclusions: These results support the use of LUT alone, and in combination with ZEA, in AMD prevention and treatment. This study is also the first to report LUT modulating effects on Alu RNA.     

腺苷受体A1亚型对视网膜色素上皮细胞 免疫调节功能的影响

目的 研究视网膜色素上皮细胞（RPE）主要是通过何种亚型的腺苷受体（ARs）来结合腺苷,及其对 RPE 功能的影响。方法 体外培养人 ARPE-19 细胞系,定量 PCR 检测 4 种腺苷受体（ARA1、ARA2A、ARA2B、ARA3）基 因的表达;提取细胞膜蛋白,Western blot 检测 4 种腺苷受体在 RPE 细胞膜上的存在。体外培养 ARPE-19 细胞至 80% 融合后随机分为 A~E 组。其中,A 组为无干预对照组,B～E 组分别给予 ARA1 拮抗剂 DPCPX（50 nmol/L）、 ARA2A 拮抗剂 SCH58261（100 nmol/L）、ARA2B 拮抗剂 MRS1754（100 nmol/L）及 ARA3 拮抗剂 MRS1220（5 μmol/L） 干预。利用 H3-腺苷进行放射性配体结合实验,计算各组细胞对腺苷的最大结合容量（Bmax）。以肿瘤坏死因子 α （TNF-α）10 μg/L 及 γ 干扰素（IFN-γ）1 000 U/mL 联合干预体外培养的 ARPE-19 细胞,给予或不予 ARA1 激动剂 （CCPA）,酶联免疫吸附试验（ELISA）测定培养上清中白细胞介素（IL）-6、IL-10、转化生长因子 β（TGF-β）、单核细胞 趋化因子（MCP）-1、趋化因子 C-X-C 配体 10（CXCL10,IP-10）的含量。结果 在 ARPE-19 细胞中即可检测到 4 种 腺苷受体基因的表达,也可探测到其分子在细胞膜上的存在。A~E 组 ARPE-19 细胞结合腺苷的 Bmax （单位：fmol）分 别为 2.04±0.31、0.44±0.06、1.82±0.28、2.01±0.42 及 2.06±0.44,其中 B 组较其他各组 Bmax均降低（P＜0.01）。以 TNF- α 及 IFN-γ 激活 ARPE-19 细胞,与对照 RPE 组比较,CCPA 干预 RPE 组 IL-6、MCP-1 及 IP-10 的含量降低、IL-10 的含量增加（P＜0.01）。2 组 TGF-β 的含量差异无统计学意义。结论 ARA1 对 ARPE-19 细胞结合腺苷的能力具 有重要的调控作用,ARA1 受体介导的信号可抑制 ARPE-19 细胞分泌促炎因子及驱化因子,具有潜在的免疫抑制 作用。     

Preliminary Investigation into the Expression of Proton-Coupled Oligopeptide Transporters in Neural Retina and Retinal Pigment Epithelium (RPE): Lack of Functional Activity in RPE Plasma Membranes

Purpose. To determine the expression and functional activity of proton-coupled oligopeptide transporters (POT) in retinal pigment epithelial (RPE) cells. Methods. RT-PCR was used to probe the presence of POT mRNA in freshly isolated bovine RPE (BRPE) and human RPE (HRPE) cells, a human RPE cell line (ARPE-19), and human and bovine neural retina. [¹⁴C]GlySar uptake was used to characterize POT activity in cultured ARPE-19 cells and freshly isolated BRPE cell sheet suspensions. Results. PHT1 mRNA was expressed in BRPE, HRPE, ARPE-19, and bovine and human neural retina. In contrast, PEPT2 and PHT2 were expressed only in bovine and human retina, and PEPT1 could not be detected. GlySar exhibited a linear uptake over 6 h at pH values of 6.0 and 7.4, with greater uptake at pH 7.4 (p < 0.01). GlySar uptake did not exhibit saturability (5-2000 M) and was unchanged when studied in the presence of 1 mM L-histidine. In contrast, GlySar uptake was significantly decreased when studied at 4°C or in the presence of endocytic inhibitors at 37°C (p < 0.01). Studies in BRPE cell sheet suspensions validated the results obtained in ARPE-19 cells and strongly suggested the absence of POT on the apical and basolateral membranes of RPE. Conclusions. PHT1 mRNA is present in native bovine and human RPE and a human RPE cell line. However, the data argue against PHT1 being expressed on plasma membranes of RPE. Overall, GlySar appears to be taken up by RPE cells via a low-affinity, endocytic process.     

Protective effects of melatonin and memantine in human retinal pigment epithelium (ARPE-19) cells against 2-ethylpyridine-induced oxidative stress: implications for age-related macular degeneration

Purpose: To investigate the possible protective effects of melatonin and memantine (MMT) against 2-ethylpyridine (2-EP)-induced oxidative stress and mitochondrial dysfunction in human RPE (ARPE-19) cells in vitro.

Materials and methods: The ARPE-19 cells were divided into seven groups. Oxidative stress was triggered by incubating the ARPE-19 cells with 30?μM of 2-EP for 24?h. Then, 200?μM of melatonin was administered over three days and 20?μM of MMT over six hours prior to the experiment. The effects of melatonin and MMT on the intracellular calcium release mechanism, reactive oxygen species production, caspase-3 and caspase-9 activities, as well as vascular endothelial growth factor levels were measured.

Results: Melatonin and MMT were found to significantly decrease apoptosis levels. The intracellular calcium release was regulated by both melatonin and MMT. Further, melatonin and MMT significantly decreased both caspase-3 and caspase-9 activities, as well as pro-caspase and poly(ADP-ribose) polymerase expression, in ARPE-19 cells. Moreover, melatonin significantly increased the protective effect of MMT. The combination of melatonin and MMT significantly decreased 2-EP-induced oxidative toxicity and apoptosis by inhibiting the intracellular reactive oxygen species production and mitochondrial depolarization levels.

Conclusions: These notable findings are the first to demonstrate the synergistic protective effects of melatonin and MMT against 2-EP-induced oxidative stress in ARPE-19 cells.     

Photoprotective effects of cranberry juice and its various fractions against blue light-induced impairment in human retinal pigment epithelial cells

Context: Cranberry has numerous biological activities, including antioxidation, anticancer, cardioprotection, as well as treatment of urinary tract infection (UTI), attributed to abundant phenolic contents.

Objective: The current study focused on the effect of cranberry juice (CJ) on blue light exposed human retinal pigment epithelial (ARPE-19) cells which mimic age-related macular degeneration (AMD).

Materials and methods: Preliminary phytochemical and HPLC analysis, as well as total antioxidant capacity and scavenging activity of cranberry ethyl acetate extract and different CJ fractions (condensed tannins containing fraction), were evaluated. In cell line model, ARPE-19 were irradiated with blue light at 450?nm wavelength for 10?h (mimic AMD) and treated with different fractions of CJ extract at different doses (5–50?μg/mL) by assessing the cell viability or proliferation rate using MTT assay (repairing efficacy).

Results: Phytochemical and HPLC analysis reveals the presence of several phenolic compounds (flavonoids, proanthocyanidin, quercetin) in ethyl acetate extract and different fractions of CJ. However, the condensed tannin containing fraction of ethyl acetate extract of CJ displayed the greater (p?p?p?Discussion and conclusion: In conclusion, this study distinctly proved that condensed tannin containing fraction of CJ probably exhibits better free radicals scavenging activity and thereby effectively protected the ARPE-19 cells and thus, hampers the progress of AMD.     

Diarylheptanoid 7-(3,4 dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene from Curcuma comosa Roxb. protects retinal pigment epithelial cells against oxidative stress-induced cell death

Chronic exposure to oxidative stress causes damage to retinal pigment epithelial cells which may lead to the development of age-related macular degeneration, the major cause of vision loss in humans. Anti-oxidants provide a natural defense against retinal cell damage. The present study was designed to evaluate the potential anti-oxidant activity and protective effect of two diarylheptanoids isolated from a medicinal herb Curcuma comosa; 7-(3,4 dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene (compound A), and 1,7-diphenyl-4(E),6(E)-heptadien-3-ol (compound B) against oxidative stress (H₂O₂)-induced human retinal pigment epithelial (APRE-19) cell death. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay indicated that the anti-oxidant activity (IC₅₀) of compound A was similar to that of vitamin C. Pre-treatment of ARPE-19 cells with 20 μM compound A for 4 h afforded greater protection against the insult from 500 μM H₂O₂, compared to a similar protection period for compound B. Compound A lowered H₂O₂-induced lipid peroxidation, malondialdehyde formation and intracellular reactive oxygen species. Furthermore, compound A ameliorated the H₂O₂-induced decrease in anti-oxidant enzyme activities and subsequent apoptotic cell death in ARPE-19 cells in a dose and time-dependent manner. These results suggest that compound A protects ARPE-19 cells against oxidative stress, in part, by enhancing several anti-oxidant defense mechanisms. Therefore, compound A may have therapeutic potential for diseases associated with oxidative stress, particularly degenerative retinal diseases.     

Functional activity of a monocarboxylate transporter, MCT1, in the human retinal pigmented epithelium cell line, ARPE-19

The purpose of this study was to identify and characterize the functional activity of monocarboxylic acid transporter 1 (MCT1) on the human retinal pigmented epithelium (RPE) cell line, ARPE-19, and to evaluate whether the cell line can function as an in vitro screening tool for intravitreally administered drugs/prodrugs targeted to the MCT1 expressed in RPE. Uptake studies were carried out at 37 degrees C, for 30 s, with ARPE-19 cells. [(14)C]l-Lactic acid was selected as a substrate for this transporter. Uptake of [(14)C]L-lactic acid by ARPE-19 cells was found to exhibit saturable kinetics (K(m) = 3.1 +/- 0.6 mM and V(max) = 63.1 +/- 4.1 pmol/min/mg of protein). Monocarboxylic acids, such as benzoic acid, salicylic acid, and pyruvic acid, inhibited the uptake of [(14)C]L-lactic acid whereas di- and tricarboxylic acids, such as phthalic, succinic, and citric acids, did not demonstrate any inhibitory effect. Uptake was stereospecific where D-lactic acid was less effective in inhibiting [(14)C]L-lactic acid uptake than unlabeled L-lactic acid. ELISA indicated the expression of only MCT1, MCT4, and MCT8 isoforms by ARPE-19 cells. Increase in [(14)C]L-lactic acid uptake was observed as the uptake medium pH was lowered from 7.4 to 5.0. Moreover, inhibition of [(14)C]L-lactic acid uptake was observed in the presence of the protonophore 2,4-dinitrophenol. Uptake was significantly decreased in the presence of sodium azide, ouabain, p-chloromercuribenzoic acid (pCMBA), N-ethylmaleamide, dithiothreitol, and p-chloromercuribenzene sulfonate (pCMBS). However, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and L-thyroxine did not inhibit [(14)C]L-lactic acid. RT-PCR studies and sequence analysis of the PCR product confirmed the expression of MCT1 by ARPE-19 cells. Our results indicate that MCT1 is functionally active and is the only MCT isoform involved in the apical uptake of monocarboxylates by ARPE-19 cells. This cell line may thus be used as an effective screening tool for intravitreally administered drugs/prodrugs targeted toward MCT1 expressed on the RPE.     

A novel polysaccharide compound derived from algae extracts protects retinal pigment epithelial cells from high glucose-induced oxidative damage in vitro

Diabetic retinopathy is a common complication of diabetes mellitus (DM). The oxidative damage inflicted on retinal pigment epithelial (RPE) cells by high glucose closely approximates the molecular basis for the loss of vision associated with this disease. We investigate a novel algae-derived polysaccharide compound for its role in protecting ARPE-19 cells from high glucose-induced oxidative damage. ARPE-19 cells were cultured for 4?d with normal concentration of D-glucose, and exposed to either normal or high concentrations of D-glucose in the presence or absence of the polysaccharide compound at variety of concentrations for another 48?h. Taurine was used as a positive control. Activity of super oxide dismutase (SOD) and concentration of glutathione (GSH) were measured as well as cytotoxicity of high glucose and the polysaccharide compound. To analyse cellular damage by high glucose, activation of Annexin V and p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) were examined. Our results showed that a significant cellular damage on ARPE-19 cells after 48?h treatment with high glucose, accompanied by a decrease in SOD activity and GSH concentration; high glucose also caused ARPE-19 cell apoptosis and activation of p38MAPK and ERK. As the non-toxic polysaccharide compound protected ARPE-19 cells from high glucose-induced cellular damage, the compound recovered SOD activity and concentration of GSH in the cells. The compound also abrogated the cell apoptosis and activation of p38MAPK and ERK. Therefore, the polysaccharide compound derived from algae extracts could be unique candidate for a new class of anti-DM and anti-oxidative damage.     

Knockdown of GCN2 inhibits high glucose-induced oxidative stress and apoptosis in retinal pigment epithelial cells

Increasing evidence suggests that general control nonderepressible 2 (GCN2) is a critical regulator of oxidative stress and cell apoptosis in response to various stimuli. However, the role of GCN2 in diabetic retinopathy remains unclear. The aim of the present study was to investigate the effects of GCN2 on oxidative stress and apoptosis in ARPE-19 cells exposed to high glucose. The results showed that GCN2 was highly expressed in high glucose-induced ARPE-19 cells. Moreover, knockdown of GCN2 greatly improved ARPE-19 cell viability in response to high glucose. In addition, GCN2 knockdown significantly suppressed the production of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as increased superoxide dismutase (SOD) activity in high glucose-stimulated ARPE-19 cells. Furthermore, GCN2 knockdown reduced cell apoptosis and enhanced the activation of nuclear factor E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway in high glucose-stimulated ARPE-19 cells. However, knockdown of Nrf2 reversed the effects of GCN2 on oxidative stress and cell apoptosis. Taken together, our findings suggest that knockdown of GCN2 inhibits high glucose-induced oxidative stress and apoptosis in ARPE-19 cells through activation of the Nrf2/HO-1 pathway.     

Transport of thiol-conjugates of inorganic mercury in human retinal pigment epithelial cells

Inorganic mercury (Hg(2+)) is a prevalent environmental contaminant to which exposure to can damage rod photoreceptor cells and compromise scotopic vision. The retinal pigment epithelium (RPE) likely plays a role in the ocular toxicity associated with Hg(2+) exposure in that it mediates transport of substances to the photoreceptor cells. In order for Hg(2+) to access photoreceptor cells, it must first be taken up by the RPE, possibly by mechanisms involving transporters of essential nutrients. In other epithelia, Hg(2+), when conjugated to cysteine (Cys) or homocysteine (Hcy), gains access to the intracellular compartment of the target cells via amino acid and organic anion transporters. Accordingly, the purpose of the current study was to test the hypothesis that Cys and Hcy S-conjugates of Hg(2+) utilize amino acid transporters to gain access into RPE cells. Time- and temperature-dependence, saturation kinetics, and substrate-specificity of the transport of Hg(2+), was assessed in ARPE-19 cells exposed to the following S-conjugates of Hg(2+): Cys (Cys-S-Hg-S-Cys), Hcy (Hcy-S-Hg-S-Hcy), N-acetylcysteine (NAC-S-Hg-S-NAC) or glutathione (GSH-S-Hg-S-GSH). We discovered that only Cys-S-Hg-S-Cys and Hcy-S-Hg-S-Hcy were taken up by these cells. This transport was Na(+)-dependent and was inhibited by neutral and cationic amino acids. RT-PCR analyses identified systems B(0,+) and ASC in ARPE-19 cells. Overall, our data suggest that Cys-S-Hg-S-Cys and Hcy-S-Hg-S-Hcy are taken up into ARPE-19 cells by Na-dependent amino acid transporters, possibly systems B(0,+) and ASC. These amino acid transporters may play a role in the retinal toxicity observed following exposure to mercury.     

New therapeutic targets in atrophic age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. There is no effective treatment for the most prevalent atrophic (dry) form of AMD. Atrophic AMD is triggered by abnormalities in the retinal pigment epithelium (RPE) that lies beneath the photoreceptor cells and normally provides critical metabolic support to these light-sensing cells. Secondary to RPE dysfunction, macular rods and cones degenerate leading to the irreversible loss of vision. Oxidative stress, formation of drusen, accumulation of lipofuscin, local inflammation and reactive gliosis represent the pathologic processes implicated in pathogenesis of atrophic AMD. This review discusses potential target areas for small-molecule and biologic intervention, which may lead to development of new therapeutic treatments for atrophic AMD.     

New therapeutic targets in atrophic age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. There is no effective treatment for the most prevalent atrophic (dry) form of AMD. Atrophic AMD is triggered by abnormalities in the retinal pigment epithelium (RPE) that lies beneath the photoreceptor cells and normally provides critical metabolic support to these light-sensing cells. Secondary to RPE dysfunction, macular rods and cones degenerate leading to the irreversible loss of vision. Oxidative stress, formation of drusen, accumulation of lipofuscin, local inflammation and reactive gliosis represent the pathologic processes implicated in pathogenesis of atrophic AMD. This review discusses potential target areas for small-molecule and biologic intervention, which may lead to development of new therapeutic treatments for atrophic AMD.     

Natural polyphenols may ameliorate damage induced by copper overload

The effect of the simultaneous exposure to transition metals and natural antioxidants frequently present in food is a question that needs further investigation. We aimed to explore the possible use of the natural polyphenols caffeic acid (CA), resveratrol (RES) and curcumin (CUR) to prevent damages induced by copper-overload on cellular molecules in HepG2 and A-549 human cells in culture. Exposure to 100μM/24h copper (Cu) caused extensive pro-oxidative damage evidenced by increased TBARS, protein carbonyls and nitrite productions in both cell types. Damage was aggravated by simultaneous incubation with 100μM of CA or RES, and it was also reflected in a decrease on cellular viability explored by trypan blue dye exclusion test and LDH leakage. Co-incubation with CUR produced opposite effects demonstrating a protective action which restored the level of biomarkers and cellular viability almost to control values. Thus, while CA and RES might aggravate the oxidative/nitrative damage of Cu, CUR should be considered as a putative protective agent. These results could stimulate further research on the possible use of natural polyphenols as neutralizing substances against the transition metal over-exposure in specific populations such as professional agrochemical sprayers and women using Cu-intrauterine devices.     

Amiodarone increases the accumulation of DEA in a human alveolar epithelium-derived cell line

Amiodarone (AMD)-induced pulmonary toxicity (AIPT) is the most life-threatening side-effect of AMD treatment. N-Monodesethylamiodarone (DEA), an active metabolite of AMD, also exhibits cytotoxicity and tends to accumulate in the lung more intensively than AMD. In this study, we characterized the mechanism of DEA accumulation using A549 cells as a model of the alveolar epithelium. Typical ATP-depletion compounds caused an approximately 30% increase in the accumulation of DEA in A549 cells, although these effects were less than those in Caco-2 cells. Triiodothyronine (T(3)), which exhibited an inhibitory effect on DEA efflux in Caco-2 cells, did not affect the accumulation of DEA in A549 cells. On the other hand, 100 microM AMD caused an approximately 200% increase in DEA content in A549 cells, although AMD accumulation was not affected by 100 microM DEA. Since the reducing effect of AMD on cellular ATP levels and that of FCCP were similar, the mechanism by which DEA accumulation is increased by AMD might be different from the ATP-dependent DEA efflux mechanism. The decrease in cell viability by DEA in the presence of AMD (IC(50) value of DEA for A549 cell viability: 25.4+/-2.4 microM) was more pronounced than that by DEA alone (IC(50) value: 11.5+/-3.0 microM). This further DEA accumulation by AMD might be a factor responsible for the greater accumulation of DEA than that of AMD in the lung in long-term AMD-treated patients.     

Development of an in vitro blood-brain barrier model-cytotoxicity of mercury and aluminum

In this study, in vitro blood-brain barrier (BBB) models composed of two different cell types were compared. The aim of our study was to find an alternative human cell line that could be used in BBB models. Inorganic and organic mercury and aluminum were studied as model chemicals in the testing of the system. BBB models were composed of endothelial RBE4 cell line or retinal pigment epithelial (RPE) cell line ARPE-19 and neuronal SH-SY5Y cells as target cells. Glial U-373 MG cells were included in part of the tests to induce the formation of a tighter barrier. Millicell CM filter inserts were coated with rat-tail collagen, and RBE4 or ARPE-19 cells were placed on the filters at the density of 3.5-4 × 10⁵ cells/filter. During culture, the state of confluency was microscopically observed and confirmed by the measurement of electrical resistance caused by the developing cell layer. The target cells, SH-SY5Y neuroblastoma cells, were plated on the bottom of cell culture wells at the density of 100 000 cells/cm². In part of the studies, glial U-373 MG cells were placed on the under side of the membrane filter. When confluent filters with ARPE-19 or RBE4 cells were placed on top of the SH-SY5Y cells, different concentrations of mercuric chloride, methyl mercury chloride, and aluminum chloride were added into the filter cups along with a fluorescent tracer. Exposure time was 24 h, after which the cytotoxicity in the SH-SY5Y cell layer, as well as in the ARPE-19 or RBE4 cell layer, was evaluated by the luminescent measurement of total ATP. The leakage of the fluorescent tracer was also monitored. The results showed that both barrier cell types were induced by glial cells. Inorganic and organic mercury caused a leakage of the dye and cytotoxicity in SH-SY5Y cells. Especially, methyl mercury chloride could exert an effect on target cells before any profound cytotoxicity in barrier cells could be seen. Aluminum did not cause any leakage in the barrier cell layer, and even the highest concentration (1 mM) of aluminum did not cause any cytotoxicity in the SH-SY5Y cells. In conclusion, BBB models composed of RBE4 and ARPE-19 cells were able to distinguish between different toxicities, and ARPE-19 cells are thus promising candidates for studies of drug penetration through the blood-brain barrier.