Evaluation of the selected barrier properties of retinal pigment epithelial cell line ARPE-19 for an in-vitro blood-brain barrier model.

In-vitro models that maintain complex transport mechanisms and structural properties associated with the blood-brain barrier in vivo would be useful in drug permeability and neurotoxicological studies. To evaluate the suitability of a human retinal pigment epithelial cell line for a blood-brain barrier model, we have compared the barrier properties of the human retinal pigment epithelial cell line ARPE-19, the human colonic adenocarcinoma cell line Caco-2, and primary porcine microvessel endothelial cells. The tight junction proteins occludin and ZO-1 were stained immunocytochemically. The paracellular ionic permeability was evaluated by measuring the trans-epithelial or trans-endothelial electric resistance. To evaluate the active transport mechanisms, the existence and the activity of the efflux transporters, P-glycoprotein and multidrug resistance-associated proteins, were studied. All the cell types in this study stained positively for occludin and ZO-1. However, the trans-endothelial electric resistance of ARPE-19 cells was low compared with that of primary porcine microvessel endothelial cell and Caco-2 cells. In addition, both the P-glycoprotein expression and its activity in ARPE-19 cells were low. In conclusion, the barrier properties of the human ARPE-19 cell line were not satisfactory for a blood-brain barrier model. For future studies, it is important to develop a human brain endothelial cell line with expression of the complex in-vivo properties of the blood-brain barrier.

Astrocyte membrane Na+, K(+)-ATPase and Mg(2+)-ATPase as targets of organic solvent impact.

Astrocytes serve to maintain the proper homeostatic environment for neurons. In these regulations, the astrocyte membrane ATPase plays an important role. In the present study, the astrocyte were cultured in the modified Minimum Essential Medium (MEM), and the isolated cell membranes were exposed to solvents at different concentrations (3, 6 and 9 mM) for one hour. The activities of the membrane-bound Na+, K(+)-ATPase and Mg(2+)-ATPase were determined after exposure to aromatic, alicyclic and aliphatic hydrocarbons. Aromatic hydrocarbons decreased the enzyme activities dose-dependently according to their lipid solubilities. The slight enzyme-inhibiting effect of aliphatic and alicyclic hydrocarbons was not dependent on the lipophilicity of these solvents. Their molecular structure may have determined the solvent impact. In the present study, the activity of ouabain sensitive Na+, K(+)-ATPase in astrocytes was only 20-15% of the total ATPase activity, and in neurons 45-55%. The enzyme inhibition caused by organic solvents may disturb the homeostatic regulatory functions of astrocytes and thus have a toxic effect on the CNS.

Astrocytes as targets for CNS effects of organic solvents in vitro.

The aim of the study was to evaluate astrocytes in vitro as a model for studies of solvent neurotoxicity. Primary astrocyte cultures were established from newborn rat cerebella. The cells were cultured in the modified Minimum Essential Medium (MEM), and the neural membranes isolated from cultures were exposed to solvents in incubation mixtures containing different solvent concentrations (3, 6, and 9 mM) for one hour. The activity of membrane-bound total ATPase was determined after exposure to aromatic hydrocarbons (benzene, toluene, xylene, styrene and ethylbenzene), and to n-hexane and cyclohexane. The enzyme activities were decreased by aromatic hydrocarbons linearly according to the log dose and in order to the log lipid/water partition coefficients, benzene having the smallest and ethylbenzene the greatest effect in all concentrations studied. Cyclohexane caused much smaller enzyme inhibition (18% of control activity in 9 mM concentration) than ethylbenzene (67% in 9 mM), in spite of very similar partition coefficients. N-hexane had clearly slighter enzyme inhibiting effect than aromatic hydrocarbons, in spite of its markedly greater lipophilicity. In addition to lipophilicity, the structure of solvent molecule seems to be important when considering the CNS toxicity. These results suggest that organic solvents exert their toxic effects on CNS, at least in part, by disturbing ATPase-dependent astrocytic regulatory functions.

Follow-up of the antihemolytic effect of toluene inhalation in rats.

The antihemolytic effects of toluene were studied with inhalation exposures in vivo. At a concentration of 2000 ppm the effect seemed to begin 0.5 h after the onset of exposure, and it ended completely 0.5 h after the end of the exposure. There was no significant change in the mean corpuscular volume (MCV), although it increased at first during and also following the exposure. The antihemolytic effect of different toluene concentrations was also tested with inhalation exposures. The inhalation of 300-2000 ppm of toluene caused a significant antihemolytic effect in a 1-h exposure, but it did not change the MCV. The greatest antihemolytic effect was obtained with a concentration of 1500 ppm of toluene. Our results confirm the assumption that the effect of toluene as a membrane-stabilizing agent is a reversible phenomenon that requires the presence of the agent molecules during the hemolytic process. The antihemolytic effect does not seem to be caused by an increase in the MCV.

Effect of toluene on the hemolytic resistance of rat erythrocytes.

The effect of toluene on rat erythrocyte hemolytic resistance was studied both in vitro and in vivo. In vitro, toluene at concentrations up to 1000 ppm showed a marked antihemolytic effect, the maximum being at 300 ppm. Above 1000 ppm, an increase in the hypotonic hemolysis was seen. The antihemolytic effect of toluene was temperature-dependent. Elevation of temperature diminished the ability of toluene to protect erythrocytes. In the in vivo experiments, when the rats breathed 2000 ppm of toluene in an inhalation chamber for 7 days and for 21 days (6 h/day), the antihemolytic effect of toluene was evident. Our results demonstrate that toluene, at moderate concentrations, increases the hemolytic resistance of rat erythrocytes in hypotonic media both in vitro and in inhalation exposures in vivo.

Human and rat erythrocyte membranes as a model for studying the effects of organic solvents on membrane-bound acetylcholinesterase in vitro.

The effects of toluene, trichloroethylene and ethanol on membrane acetylcholinesterase (AChE) activity in human and rat intact erythrocytes and erythrocyte ghosts were studied in vitro over a range of concentrations (300-3000 ppm) and at three different incubation temperatures (37, 15 and 5 degrees C). Toluene and trichloroethylene decreased the AChE activity in intact erythrocytes and in erythrocyte ghosts in both species. Trichloroethylene had a more marked effect on erythrocyte ghosts than on intact cells. At low concentrations, toluene generally had greater AChE-inhibiting potency than trichloroethylene. At the concentrations studied, ethanol had no effect on AChE activity. The effects of decreases in incubation temperature on solvent-induced AChE inhibition were very variable. We consider both the human and rat erythrocyte membrane to be a good model for studies of the anaesthetic effects of organic solvents on cell membranes. The effects of organic solvents on AChE, an integral component of the cell membrane, may be one of the critical factors determining the membrane effects of organic solvents even in nerve cells.

The neurotoxic effect of carbon disulphide, N-hexane and its metabolites studied with erythrocyte and synaptosome membranes in vitro.

The aim of the present study was to find a method for in vitro studies of the neurotoxic mechanism of industrial solvents. The effects of carbon disulphide and n-hexane and its metabolites were studied. Cell membrane changes were studied by measuring changes in the activity of the integral cell membrane enzymes acetylcholinesterase (AChE) and adenosinetriphosphatase (ATPase). The erythrocyte membranes were isolated from human peripheral blood samples and the synaptosome membranes from rat brain by using the non-toxic iso-osmotic Percoll gradient system. The cell membrane samples were incubated at +37 degrees C in an incubation mixture with known solvent concentrations. In erythrocyte membranes, n-hexane decreased the activity of AChE more than did carbon disulphide. In synaptosome membranes, 2-hexanone and 2,5-hexanedione inhibited AChE significantly more than did n-hexane. On ATPase, n-hexane, as well as the metabolites of n-hexane, had a slightly activating effect in erythrocyte membranes, and no or a slightly inactivating effect in synaptosome membranes. Carbon disulphide had a clear inactivating effect on ATPase in synaptosome membranes. The action of the neurotoxic organic solvents may be mediated by way of the integral membrane proteins, especially AChE. Thus, in neural synaptosome membranes, the metabolites of n-hexane had a greater effect on AChE activity than did n-hexane. The in vitro membrane model can be applied in studies of the neurotoxicity of organic solvents, and in predicting their neurotoxic potency.

Effects of mercuric chloride exposure on the glutamate uptake by cultured retinal pigment epithelial cells.

The cytotoxicity of mercuric chloride and the effects of mercuric chloride on glutamate and calcium uptake and the factors regulating glutamate uptake were studied in retinal pigment epithelium (RPE) cell cultures. RPE cells isolated from pig eyes and human RPE cell line (D407) cells were cultured to confluency and further subcultured according to the test protocol in question. The cytotoxicity caused by 15 min of exposure to mercuric chloride (0.01--1000 microM) was evaluated by WST-1 assay based on the activity of mitochondrial dehydrogenases. [(3)H]Glutamate uptake was measured after the cells were exposed to 0.1--100 microM mercuric chloride and the selected regulators of protein kinase C (PKC) pathway: PKC activator SC10, PKC inhibitor chelerythrine chloride, phospholipase A(2)/C inhibitor manoalide, tyrosine kinase inhibitor lavendustin A, competitive NMDA receptor antagonist AP7 and IP(3) receptor antagonist heparin. Intracellular calcium was monitored with Fluo-3 probe starting immediately after the exposure to 1--1000 microM mercuric chloride. Mercuric chloride showed concentration-dependent effects on cell viability, on glutamate uptake and on intracellular calcium concentration. The results give some support to the concept that glutamate uptake is affected by PKC. The PKC inhibitor chelerythrine chloride decreased glutamate uptake by 25%, but the PKC activator SC10 could partly prevent the inhibitory effect of mercuric chloride. Lavendustin A, manoalide and heparin had smaller, but statistically significant, effects. All these substances act on mediators which can regulate the activity of PKC. However, PKC is not likely to be the only regulator of glutamate uptake. The rise observed in [Ca(2+)](i) may initiate various cellular events during mercury intoxication.

Perturbation of artificial and biological membranes by organic compounds of aliphatic, alicyclic and aromatic structure.

Aliphatic, alicyclic and aromatic hydrocarbons interact with biological membranes. Until now little has been known about their mode of interaction with the membrane bilayer and membrane integral proteins with toxic effects to cells. The lipid theory hypothesis explains the toxic effects by the organic solvent-induced disorder in the lipid bilayer, which indirectly affects the function of membrane-embedded proteins. The extent of bilayer perturbations is ascribed to the solvent accumulation in the bilayer, which is related only to the lipophilicity of the molecule, independent of the chemical structure. In this study the fluidizing effects of aliphatic, alicyclic and aromatic hydrocarbons were compared. Membrane fluidity changes were estimated from the pyrene excimer formation, using pyrene and pyrene derivatives to label specifically the localization of solvent molecules in the transverse plane of the bilayer. Liposomal, microsomal and synaptosomal membrane preparations were evaluated because proteins and cholesterol, as natural membrane components, increase the bilayer order and reduce the organic solvent membrane/buffer partition. In the concentration range investigated, only the aromatic solvents disorder the lipid bilayer, with the greatest perturbation in the centre of the bilayer. These results are related to structural properties of the organic solvents investigated.

Effects of mercury, methylmercury and aluminium on glial fibrillary acidic protein expression in rat cerebellar astrocyte cultures.

The effects of mercuric chloride, methylmercury chloride and aluminium chloride on glial fibrillary acidic protein (GFAP) expression in primary cerebellar astrocyte cultures were studied. GFAP has been found to be a quantitative marker of neuronal injuries on the central nervous system in vivo. The GFAP content of the astrocytes was examined by sandwich ELISA after exposure for 1 wk to various metal concentrations in the culture medium. Cellular injuries were assessed by quantifying the lactate dehydrogenase (LDH) leakage into the culture medium. The expression of GFAP was found at lower dose levels than the leakage of LDH, indicating that the expression of GFAP could be a more sensitive marker of neurotoxicity than LDH leakage. In morphological examination, staining with monoclonal antibody showed GFAP induction after mercury exposure.

Evaluation of the cytotoxicity of selected systemic and intravitreally dosed drugs in the cultures of human retinal pigment epithelial cell line and of pig primary retinal pigment epithelial cells.

The cytotoxicity of the selected systemic and intravitreally dosed drugs tamoxifen, toremifene, chloroquine, 5-fluorouracil, gentamicin and ganciclovir was studied in retinal pigment epithelium (RPE) in vitro. The cytotoxicity was assayed in the human RPE cell line D407 and the pig RPE cell culture using the WST-1 test, which is an assay of cell proliferation and viability. The effects of experimental conditions on the WST-1 test (cell density, serum content in the culture medium, the exposure time) were evaluated. The EC50 values in tamoxifen-treated D407 cells ranged between 6.7 and 8.9 micromol/l, and in pig RPE cells between 10.1 and 12.2 micromol/l, depending on the cell density used. The corresponding values for toremifene were 7.4 to 11.1 micromol/l in D407 cells and 10.0 to 11.6 micromol/l in pig RPE cells. In chloroquine-treated cells, the EC50 values were 110.0 micromol/l for D407 cells and 58.4 micromol/l for pig RPE cells. Gentamicin and ganciclovir did not show any toxicity in micromolar concentrations. The exposure time was a significant factor, especially when the drug did not induce cell death, but was antiproliferative (5-fluorouracil). Serum protected the cells from the toxic effects of the drugs. Both cell cultures were most sensitive to tamoxifen and toremifene, and next to chloroquine. The drug toxicities obtained in the present study were quite similar in both cell types; that is, the pig RPE cells and the human D 407 cell line, despite the differences in, for example, the growth rate and melanin contents of the cell types. Owing to the homeostatic functions important for the whole neuroretina, RPE is an interesting in vitro model for the evaluation of retinal toxicity, but, in addition to the WST-1 test, more specific tests and markers based on the homeostatic functions of the RPE are needed.

Effects of industrial organic solvents on human erythrocyte membrane adenosine triphosphatase activities in vitro.

The effects of some aromatic hydrocarbons, aliphatic chlorinated hydrocarbons, and alcohols on adenosine triphosphatase (ATPase) activity in human erythrocyte ghost membrane were studied in vitro. Both aromatic and chlorinated aliphatic hydrocarbons inhibited this activity dose-dependently, the inhibition of total ATPase activity being clearer than that of magnesium-activated ATPase. Of the alcohols studied, methanol had no effect on the ATPase activity, but ethanol, propranolol, and butanol were slightly enzyme-activating at high concentrations. The enzyme-inhibiting potency of organic solvents was generally related to their lipid solubilities, but 1,1,2,2-tetrachloroethane was a potent enzyme inhibitor despite its low lipid solubility. This findings indicates that, eg, the molecular structure of solvents may modulate their enzyme inhibition. In the presence of Triton-X-100, toluene did not cause any changes in the activity of total ATPase, and the combined effect of the two compounds was slight. Triton-X-100 also caused a significant solubilization of membrane proteins although even the highest toluene concentrations did not. These results show that organic solvents may cause their membrane effects by acting directly on membrane-bound integral proteins such as ATPase. This action is not only dependent on the lipid solubility of the compounds, but also on their molecular structure.

Glutamate and calcium uptake in astrocytes after acute lead exposure.

The neurotoxicity of lead is well known, but the exact mechanisms of its toxicity are not solved yet. Disturbances in glutamate homeostasis of neural tissue and interactions of lead with calcium metabolism have been considered as a potential mechanism. In the present study, we evaluate the acute effect of lead exposure by studying glutamate uptake, as well as calcium influx and intracellular lead in cultured rat cerebellar astrocytes. Glutamate uptake and calcium influx were evaluated with radioactive probes by liquid scintillation counting, and intracellular lead was measured by atomic absorption method. Lead increased calcium influx dose dependently, but in an acute 15-min exposure it had no statistically significant effect on glutamate uptake into astrocytes. Calcium decreased at high concentrations but did not hamper lead uptake. Lead uptake rate into astrocytes was high compared to calcium.

Piperonyl butoxide potentiates the synaptosome ATPase inhibiting effect of pyrethrin.

Pyrethrins are widely used insecticides in both agriculture and households. In many commercial formulations piperonyl butoxide (PBO) is used with pyrethrins. PBO is a well-known synergist of pyrethrins, used to intensify their effects. One of the cellular targets of pyrethrins is the sodium channel in the membrane. In the present study, the activity of the membrane-bound integral protein ATPase was studied as a biomarker for the membrane effects of pyrethrin and PBO. Cerebral synaptosomes of rat brain were used in the study. The isolation of synaptosomes was performed with the Percoll gradient method. Both total ATPase and Mg2+ activated ATPase were studied by determining inorganic phosphate. Exposure to 0.1-1000 microM of pyrethrin and to 0.4-4000 microM of PBO decreased ATPase activity dose-dependently. The most efficient mixture was the one consisting of one part of pyrethrin and four parts of PBO. The activity of total ATPase decreased 15% in concentrations of 0.1-10 microM pyrethrin, and a 50% decrease was found at 100 microM pyrethrin. The mixture of pyrethrin and PBO caused a 15-60% decrease in the total ATPase activity at 0.1-10 microM pyrethrin and 0.4-40 microM PBO. A 85% decrease was found after exposure to the mixture of 100 microM pyrethrin and 400 microM PBO. PBO alone had no effect at 0.4-40 microM concentrations, but a marked effect was seen at over 40 microM concentrations. The results indicate that PBO is an effective synergist of pyrethrin and that it is very toxic in high concentrations. The results also confirm that neuronal sodium homeostasis is one target of the neurotoxic effect of pyrethroid compounds.

An inhalation method for testing the toxicity of volatile compounds in small laboratory animals. A study on short-term and long-term toluene inhalation in rats.

A coupola-shaped Plexiglass inhalation chamber (volume 190 I) with continuous infusion of toxicant and air flow was constructed for small laboratory animals. The method guarantees an even distribution of vapourized toxicant to 16 animals at the same time, and is convenient both in short-term and long-term experiments. As a volatile toxicant, toluene was used. The short-term and long-term effects of toluence on rats were studied using psychomotor tests, blood glucose, serum ALAT and ASAT values as well as hematocrit. Short-term exposure, but not long-term, impaired the performance in psychomotor tests, elevated blood glucose and serum ALAT and ASAT levels, and slightly increased hematocrit. Body weight decreased as compared to controls in short-term toluene exposure, but showed no difference in long-term treatment between toluene-and control groups at the end of exposure. The differences between the two exposures are probably due to the development of tolerance during long-term exposure either on receptor or metabolic level.

Retinal müller cell culture.

A mini-review is presented of the current techniques for maintaining Müller cells in a culture. Within the retina, Müller cells are the predominant glial cells. These highly specialised cells extend over the entire neural retina. One of the most important of the various physiological functions of Müller cells is to regulate the balance of ions and neurotransmitters in the retina. Disturbance of these regulatory functions may lead to toxic effects on receptor and other neural cells in the neuroretina, and may be a common mechanism of clinical retinal neuropathy. The main excitatory neurotransmitter in the retina is glutamate. Müller cells regulate the amount of glutamate in the synaptic regions of the neural network in the retina. Accumulation of extra glutamate seems to be an important mechanism for initiating pathological changes leading to retinal damage. Many previous in vitro studies on the role of Müller cells in retinal toxicology have been based on the use of morphological and histochemical methods. In cell toxicology studies, it is important to develop culture techniques able to provide more cells for biochemical determinations.

Retinal pigment epithelial cell cultures as a tool for evaluating retinal toxicity in vitro.

This article reviews in vitro testing of retinal toxicity in retinal pigment epithelium (RPE) cell cultures. It is based on the literature on RPE cell cultures and on our recent studies on the retinal toxicity of selected amphiphilic drugs. The RPE plays a major role in maintaining the homeostasis and health of the retina. Various pharmacological agents are known to cause adverse effects in RPE cells. For example, long-term treatment with chloroquine in patients with rheumatoid arthritis has induced retinopathy, and tamoxifen, a drug that is commonly used in the treatment of advanced breast cancer and in the prevention of breast cancer among high-risk women, has been reported to cause retinal changes and impaired vision. During our research, we have developed novel in vitro methods for evaluating the retinal toxicity of xenobiotics. We have used a pig RPE primary culture and a human RPE cell line (D407), which retain epithelial cell characteristics. They form a layer of hexagonal cells with intercellular junctions, and possess a keratin-containing cytoskeleton. They are both good models for determining the retinal cell toxicity of test compounds. Further studies on phagocytic activity, lysosomal enzyme activity and glutamate uptake might generate new methods for the toxicological evaluation of the retinal side-effects of drugs in vitro.

The effect of selected organic solvents on intact human red cell membrane acetylcholinesterase in vitro.

Human erythrocytes were exposed to different concentrations of aromatic hydrocarbons, chlorinated aliphatic hydrocarbons, and alcohols in vitro to study the effects of these agents on the activity of acetylcholinesterase (AchE), a membrane integral protein. Aromatic hydrocarbons were in general more potent AchE inhibitors than chlorinated aliphatic hydrocarbons and alcohols at +37 degrees C. The influence of decreasing the temperature to +15 degrees C and +5 degrees C was more prominent on the effect of aromatic hydrocarbons than on the effect of chlorinated aliphatic hydrocarbons and alcohols. In general, however, the decrease in the incubation temperature increased the AchE-inhibiting effect of organic solvents. The lipid solubility and molecular structure, among other factors, may determine the AchE inhibitory potency of organic solvents. Changes in membrane AchE may be one of the factors affecting membrane fluidity, which is considered to determine membrane stabilization. The primary site of action of the membrane-stabilizing agents may involve a membrane protein.

Effect of organic solvents on human erythrocyte membrane acetylcholinesterase activity in vitro.

To study the effects of aromatic hydrocarbons, chlorinated aliphatic hydrocarbons, and alcohols on erythrocyte membrane, changes in the membrane-bound acetylcholinesterase (AchE) activity were tested after solvent treatment by the Ellman method. When the maximal effects of these solvents were tested at +37 degrees C, they diminished the AchE activity: benzene 75%, xylene 65%, styrene 80%, trichloroethylene 55%, tetrachloroethylene 60%, 1,1,1-trichloroethane 25%, and 1,1,2,2-tetrachloroethane 75%. Of the alcohols studied, only ethanol had a slight AchE inhibiting effect at +37 degrees C. The decrease in the incubation temperature increased the AchE inhibiting potency of aromatic hydrocarbons more than that of chlorinated aliphatic hydrocarbons and alcohols. The differences in inhibition may be due to the lipid solubility of the solvents, but also other factors, e.g. molecular structure, may determine it. Changes in the membrane-bound AchE may be one of the factors determining membrane fluidity.