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The development of cell methods for the toxicity evaluation of drugs and chemicals started in Scandinavia at the end of the 1970s. The pioneer in vitro toxicologist was Bjorn Ekwall (1940–2000), who published his basal cytotoxicity concept in 1983, and started the MEIC project in 1989, the first international project aiming to evaluate the competence of cell toxicity tests to predict human toxicity. The EU framework project, ACuteTox (2005–10), can be seen as a continuation of the MEIC study. Generally, the early use of in vitro methods in all Scandinavian countries initially began for cell toxicology and genotoxicology and then for neurotoxicology and other tissue-specific toxicities. The Scandinavian Society for Cell Toxicity, established in 1983, played an important role, not only in gathering the MEIC participants, but also by encouraging other in vitro toxicologists from Scandinavia to meet at its annual workshops. The EU framework programmes, Oculotox in vitro, ACuteTox, ReproTect, Sens-it-iv and NanoTest, secured the place of research on alternative methods to replace animal experiments in Scandinavia. After the implementation of Directive 2010/63/EU, governmental measures have promoted research and education on alternative methods in various countries, but without providing sufficient resources, especially for Replacement.

Toxicological and pharmacological information from human cells and tissues provides knowledge readily applicable to human safety assessment and to the efficacy assessment of pharmaceuticals. The 3R principle in animal studies includes the use of human material in the R of Replacement. The Reduction and Refinement Rs are related to animal use. Knowledge of the 3Rs and successful 3R methods are a prerequisite for the Reduction of animal experiments in the future. More collaboration among researchers using experimental animals and those working in vitro is necessary with mutual respect. The OECD Guidelines for the Testing of Chemicals have included the animal-free part of the 3Rs in guidances for the development and reporting of Adverse Outcome Pathways (AOPs), which is to be part of the Integrated Approaches to Testing and Assessment (IATA). The 3R centres established to help fulfil the Directive 2010/63/EU play an important role to promote the 3Rs and in the development of animal-free toxicology. Research centres in each Nordic country are founded upon solid research activities in cell and organ toxicity, including major EU programmes to promote 3Rs and implementation of good practices and methods broadly in all stakeholders of industry, regulators and academia. In the light of this, the Nordic Symposium on Toxicology and Pharmacology without Animal Experiments addressed more adopted/modified test guidelines or new test guidelines for new end-points, or hazard challenges, new in vitro 3D models, speeding up transfer of knowledge from research to regulation to understand AOP and towards IATA.

The Björn Ekwall Memorial Foundation (BEMF) was initiated by the Scandinavian Society for Cell Toxicology in 2001, to honour the memory of Dr Björn Ekwall (1940–2000) and to establish a prize, the Björn Ekwall Memorial Award. The prize is awarded to scientists who have significantly contributed to the field of cell toxicology, and whose work is contributing toward the replacement of animal experiments by alternative toxicity tests. Over the past 10 years, the Björn Ekwall Memorial Award has been presented annually. Björn Ekwall, an outstanding Swedish cell toxicologist, was one of the pioneers in the development and application of alternative methods to animal tests in toxicology. All his scientific work was devoted to in vitro toxicology, and in particular, to the use of cultured human cells for the screening of toxic chemicals. In the middle of the 1980s, he initiated the international Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) project, to evaluate the usefulness of in vitro tests for the estimation of human acute systemic toxicity. To prove his “basal cytotoxicity concept”, he established the MEMO database, in which data on the acutely toxic human blood concentrations of drugs and chemicals were collated from the literature and from clinical studies. He also initiated another project, Evaluation-Guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT). The ideas from the EDIT project, together with those from the MEIC project, became the basis for today's international EU projects, e.g. ACuteTox, Sens-it-iv and ReProTect. In this article, 10 years after the start of the BEMF, the scientific achievements of each of the award winners in the field of in vitro toxicology are presented, together with a brief synopsis of their careers.

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commissions Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 79 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 57 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.

Machine vision is an application of computer vision. It both collects visual information and interprets the images. Although the machine obviously does not ‘see’ in the same sense that humans do, it is possible to acquire visual information and to create programmes to identify relevant image features in an effective and consistent manner. Machine vision is widely applied in industrial automation, but here we describe how we have used it to monitor and interpret data from cell cultures. The machine vision system used (Cell-IQ) consisted of an inbuilt atmosphere-controlled incubator, where cell culture plates were placed during the test. Artificial intelligence (AI) software, which uses machine vision technology, took care of the follow-up analysis of cellular morphological changes. Basic endpoint and staining methods to evaluate the condition of the cells, were conducted in parallel to the machine vision analysis. The results showed that the automated system for pattern analysis of morphological changes yielded comparable results to those obtained by conventional methods. The inbuilt software analysis offers a promising way of evaluating cell growth and various cell phases. The continuous follow-up and label-free analysis, as well as the possibility of measuring multiple parameters simultaneously from the same cell population, were major advantages of this system, as compared to conventional endpoint measurement methodology.

The cytotoxicity of amitriptyline (0-100microM), selegiline (0-4.5microM), carbamazepine (0-420microM) and paracetamol (0-10mM) was studied in metabolically competent mouse hepatocytes, metabolically incompetent human hepatoblastoma (HepG2) cells, and in neuroblastoma (SH-SY5Y) and astrocytoma (U-373 MG) cells, by using luminescence-based ATP measurement as an endpoint of cell toxicity. The aim was to evaluate the potential of the selected cell cultures to recognize metabolism-induced toxicity of the test compounds, and to predict further hepatic and neural toxicity. In SH-SY5Y cells amitriptyline was severely toxic, while selegiline and paracetamol failed to show any toxic effect, and carbamazepine was only slightly toxic at the highest concentration. In U-373 MG cells the onset of amitriptyline toxicity started earlier than in SH-SY5Y cells. However, the highest amitriptyline concentration resulted in approximately 100% decrease in the viability of the SH-SY5Y cells, whereas the decrease in the viability of the U-373 MG cells was only approximately 30%. Selegiline, carbamazepine and paracetamol were toxic in mouse hepatocytes (but not in HepG2 cells), which suggests that these drugs may show metabolism-dependent (neuro)toxicity. In conclusion, compared to the use of neurons alone, better estimations of neurotoxicity can be made by the combined use of metabolically competent hepatocytes and glial cells (e.g. U-373 MG) together with neuronal cells (e.g. SH-SY5Y).

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.

Evaluation of the effects of laser photocoagulation on cultured primary retinal pigment epithelial cells.Cells were treated by a diode laser (678 nm) with 800 and 1600 mW for 0.186 second. Cell toxicity was tested by the WST-1 assay, and the uptakes of glutamate and gamma-aminobutyric acid (GABA) were measured.Laser photocoagulation (1600 mW) caused cell damage and the mitochondrial enzyme activity evaluated by a WST-1 test significantly decreased by 20%-30%. Laser treatment caused a dose-dependent decrease in glutamate uptake but increased GABA uptake.Laser treatment and the laser-induced increase in temperature influence transport processes in retinal pigment epithelial cells and may cause cell damage in the posterior part of the retina.

The effects of aluminium lactate (Al-lactate) on the rat cerebral synaptosome integral proteins adenosinetriphosphatase (ATPase) and acetylcholinesterase (AChE) were studied in vitro and in vivo. Coexposure with ethanol (EtOH) was studied in both situations. Isolation of synaptosomes was carried out using isoosmotic Percoll gradients. In in vitro experiments, the synaptosomes were exposed to different concentrations of Al-lactate in the incubation mixture. Al-lactate caused decreases in total ATPase and AChE activities concentration dependently. The decrease in ATP activity started at 0.2 mM concentration, and concentration for the 50% decrease of the enzyme activity (EC50 ) was 1.1 mM. The decrease in AChE activity started at 5–10 mM concentration, and the EC50 value was 15.8 mM. Coexposure with ethanol (2 mM) increased the EC50 values similarly in both cases. After 90-day oral exposure of rats to Al-lactate (91.8 mg/kg/day), the serum aluminium level was 0.9–1.3 μM/l. Coexposure with EtOH (3.0 g/kg/day) did not significantly increase the blood Al (0.7–2.2 μM/l). Aluminium exposure caused a decrease in the blood EtOH concentration (0.6 mM/l) compared with blood EtOH (12.3 mM/l) in the rats exposed to ethanol only. In the rats studied 2 weeks after the Al exposure, the activities of ATPase and AChE were significantly lower than in the rats studied immediately after the exposure. Correspondingly, a significant decrease in AChE activity was found in Al- and EtOH-exposed rats, but in the control rats there were no differences between the study groups. Immediately after the 90-day dosing, the exposed rats did not differ significantly from the control rats. Based on the in vitro results, the neural membrane integral proteins ATPase and AChE may be considered as targets for the effects of aluminium and ethanol. Ninety-day in vivo exposure of rats to aluminium caused decrease in ATPase and AChE activities, detectable 2 weeks after the exposure.

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 x 10(5) 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 100000 cells/cm(2). 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.

Tamoxifen and toremifene are antiestrogenic drugs successfully used in the therapy of breast cancer. Rheumatoid arthritis and malaria have been treated with chloroquine for decades. Unfortunately, tamoxifen and chloroquine are reported to induce retinal changes as a side effect. We now studied the effects of tamoxifen, toremifene, and chloroquine on the viability of the human retinoblastomal cell line Y79, using the WST-1 test or measurement of the cellular ATP content. The studies were made on Y79 cell cultures and on cocultures of Y79 cells and retinal pigment epithelial cell line ARPE-19. The cocultures were used to clarify the effect of retinal pigment epithelium on toxicity to Y79 cells. In the coculture, the drugs were applied to ARPE-19 cells growing in the culture inserts on top of Y79 cells and the viability of ARPE-19 and Y79 cells was assessed separately. Tamoxifen, toremifene, and chloroquine reduced dose-dependently the viability of Y79 cells after 24-h exposure. The ARPE-19 cells proved to be protective after chloroquine exposure in the coculture. The results shed light on the toxicity of tamoxifen and chloroquine in Y79 cells in vitro. With the coculture we were able to simulate the in vivo route of chloroquine to the retina via the retinal pigment epithelium.

Glutamate is the major excitatory neurotransmitter in the retina and glutamate uptake is essential for normal glutamate signalling. Retinal diseases may induce neurochemical changes which affect retinal cells including retinal pigment epithelium (RPE). The aim of the study was to investigate the expression of glutamate transporter subtypes in RPE and retinoblastoma cells and to clarify the effect of proliferation modulators on the levels of the expressed transporter in the RPE cell line.Cultured pig RPE cells and two human RPE cell lines, D407 and ARPE-19, as well as the human retinoblastoma cell line Y79 were used. Glutamate transporter expression was evaluated with Western blot analysis and immunocytochemistry.The study revealed unexpected expression of neuronal glutamate transporter/chloride channel EAAT4 in these three cell lines, but not in cultured pig RPE cells, whereas another glutamate carrier, EAAC1, was present in all cell types utilized. Other transporter subtypes, GLT1, GLAST and EAAT5 were not found. Neither tamoxifen, known to inhibit both proliferation and glutamate uptake in RPE cells, nor retinoic acid nor insulin, also known to affect cell proliferation rates, were capable of changing the total levels of EAAT4 in APRE-19 cells.Neuronal glutamate transporter EAAC1 is expressed in RPE cells. The robust expression of EAAT4 in cell lines may reflect a role of EAAT4 in cell proliferation and migration. Unaltered steady-state expression of this carrier and chloride-channel protein hints at posttranslational mechanisms of regulation of EAAT4.

The antioestrogens, tamoxifen and its more recent homologue toremifene, are used in the therapy of breast cancer. Tamoxifen has been reported to cause retinal changes as side effects. Both compounds inhibited glutamate uptake in retinal pigment epithelial cells, and the present study was conducted to clarify the mechanism of this inhibition. Retinal pigment epithelial cells are part of the blood-retina barrier, and their glutamate transporters are essential for retinal glutamate homeostasis. Glutamate uptake was investigated in human retinal pigment epithelial cell line D407 and in cultured pig retinal pigment epithelial cells using L-[3H]glutamate as a tracer. The cells were exposed to 7.5 μM tamoxifen and toremifene. β-Hydroxyaspartate, a transportable inhibitor of glutamate transport, was used as a reference compound. In kinetic analyses, β-hydroxyaspartate increased the Km constant for glutamate transport. Tamoxifen and toremifene exhibited the same effect, which indicates that inhibition evoked by them is also competitive in nature. Both drugs were more effective in the human retinal pigment epithelial cell line than in the pig retinal pigment epithelial cells. The results show for the first time that the antioestrogens tamoxifen and toremifene could possibly hamper glutamate transport by replacing glutamate as the substrate.

Pyrethroids are used widely as insecticides both in agriculture and in households. A cellular target of pyrethroids 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 effect of the pyrethroids permethrin and cypermethrin. Male Sprague-Dawley rats were used for cerebral synaptosome preparation. The isolation of synaptosomes was performed with the Percoll gradient method. Both total ATPase and Mg(2+) activated ATPase were studied by determining inorganic phosphate liberated from the substrate ATP. One hour exposure to permethrin (Biokill) and cypermethrin (Ripcord) insecticide products affected ATPase activities. The activity of Na(+), K(+) ATPase decreased dose-dependently in 10-50 microM concentrations of permethrin, and Mg(2+) activated ATPase increased over twofold in the same concentrations of the active components. The effect of the cypermethrin compound Ripcord was not clearly dose-dependent. The activity of total ATPase was almost entirely lost in the concentrations of 100 microM of permethrin and cypermethrin. The results support the idea that membrane ATPases are one target of the neurotoxic effect of pyrethroid compounds.

The cytotoxicity of benzalkonium chloride (BAC) and disodium edetate (EDTA) was evaluated in vitro in rabbit corneal epithelial primary cells and in the immortalized human corneal epithelial cell line SV40. Cell injury was assessed by lactate dehydrogenase (LDH) leakage and by reduction of the tetrazolium salt WST-1 to formazan by mitochondrial metabolic activity. Cell cultures were exposed to test compounds both in serum-free and in serum-containing medium. Although WST-1 and LDH tests measured different physiological endpoints, they yielded comparable results. However, the LDH test seemed less reliable due to great variation. The use of serum was found to result in lower toxicity of the compounds in both tests. The rabbit primary cell culture and the human corneal cell line were quite similar in their responses to BAC and EDTA. The human cell line is a promising in vitro alternative in oculotoxicity testing.

5-Fluorouracil (5-FU) is commonly used in ophthalmology for suppressing fibroblast activity after glaucoma surgery. Adverse effects on corneal epithelial cells have been reported to relate to 5-FU therapy. The effects of 5-FU were evaluated in vitro on SV40-immortalized human corneal epithelial cell (HCE) cultures with two cytotoxicity tests: WST-1 assay as an index of cell proliferation, and lactate dehydrogenase (LDH) assay as an index of plasma membrane integrity. The cells were exposed to 5-FU with various concentrations in serum-free medium and in medium containing 15 % (v/v) fetal bovine serum (FBS) for 1, 24, 48 and 72 hours. One-hour exposure had no effects on HCE cells. Longer exposures caused dose-dependent inhibition of cell proliferation. Exposure to 5 mg/ml 5-FU lowered cell number to 50 % of controls after 24-hour treatment and resulted to complete cell death after 72 hours. Serum protected the cells for 24 hours, but after longer exposure times the protective nature of serum disappeared. 5-...

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.

The effects of tamoxifen, toremifene and chloroquine on the phagocytosis of rod outer segments by retinal pigment epithelium were evaluated in human retinal pigment epithelial cell line D407 and pig retinal pigment epithelial cell culture. Retinal pigment epithelial cells were exposed to different concentrations of tamoxifen (1-20 microM), toremifene (1-20 microM) and chloroquine (1-1000 microM), and challenged with FITC-labeled rod outer segments for 24 hr. The phagocytized (bound and ingested) rod outer segments were measured fluorometrically, and the effect of the drugs on the phagocytosis was determined. The cytotoxicity of the drugs was evaluated by measuring their effects on mitochondrial enzyme activities (WST-1-test). The results showed that the test compounds inhibited the phagocytosis of rod outer segments in both D407 and pig retinal pigment epithelial cells. The phagocytic activity was more sensitive to tamoxifen (EC(50) 7.2 microM for D407 cells and 3.6 microM for pig retinal pigment epithelial cells) and toremifene (EC(50) 6.2 microM and 3.1 microM respectively) than to chloroquine (EC(50) 77.2 microM for D407 cells). The inhibition of rod outer segment phagocytosis in both cell cultures started at lower dose levels of test compounds than the cytotoxicity indicated by the WST-1-test. The experiments were carried out both in serum-free medium and serum-containing medium. Serum seemed to be a critical factor in the medium and caused difficulties in the interpretation of the results.

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 cells carry out phagocytosis and digestion of material shed from the photoreceptor outer segments. In this process, the integrity of lysosomal enzymes is of major importance. In the present study the effects of tamoxifen, toremifene and chloroquine on the activity of two lysosomal enzymes (cathepsin D and N-acetyl-beta-D-glucosaminidase) in the retinal pigment epithelial cells were studied. Retinal pigment epithelial cells from pig eyes were cultured for two weeks in Dulbecco's Modified Eagle Medium, after which the cells were exposed to 1-40 microM concentrations of tamoxifen citrate, toremifene citrate and chloroquine diphosphate. To eliminate possible medium-borne oestrogenic mechanisms, the test was repeated using phenol red-free medium with charcoal-stripped fetal calf serum. The exposure time was one week, after which the lysosomal enzymes cathepsin D and N-acetyl-beta-glucosaminidase were determined. Cellular injuries were assessed by quantifying the leakage of lactate dehydrogenase into the culture medium. Cathepsin D and N-acetyl-beta-D-glucosaminidase showed different sensitivities to tamoxifen, toremifene and chloroquine. The main lysosomal protease cathepsin D was more sensitive than N-acetyl-beta-D-glucosaminidase to the effects of tamoxifen and toremifene, possibly due to their antioestrogenic properties. The phenol red-free medium with charcoal-stripped serum seemed to make the drugs more effective than the reference medium. Chloroquine had only a minor effect on the lysosomal protease cathepsin D, but a clearer effect could be seen on N-acetyl-beta-glucosaminidase.

The anticancer drug tamoxifen is widely used in breast cancer therapy. Tamoxifen has been reported to cause ocular toxicity and impairment of vision in epidemiological studies. To study the possible role of an excitotoxic mechanism in the ocular toxicity of tamoxifen, we investigated the effect of tamoxifen on retinal pigment epithelium (RPE) glutamate uptake in vitro. RPE, a layer of cells between photoreceptors and choroidal capillaries, contributes to the regulation of the concentration of the major excitatory amino acid, glutamate, in the sub-retinal space. Dysfunction in RPE glutamate uptake can lead to accumulation of extracellular glutamate and can cause various excitotoxic effects in the retina. The study was conducted by using cultured pig RPE cells. Six different tamoxifen citrate concentrations, ranging from lμM to 100μM, and [3H]-L-glutamate were added to the culture medium. To specify the glutamate uptake, 1mM dinitrophenol was added and a Na+-free culture was used. Due to the anti-oestrogenic character of tamoxifen, the possible effect of β-oestradiol on the glutamate uptake of RPE was also examined. The results show that glutamate uptake by RPE cells was reduced in the presence of tamoxifen, and that the reduction was dose-dependent. These results suggest that tamoxifen exposure could lead to the extracellular accumulation of glutamate. Disturbances in glutamate uptake can cause eye toxicity via an excitotoxic mechanism. The glutamate uptake of RPE cells was reduced under Na+-free conditions and was also reduced in the presence of dinitrophenol.

There is an urgent need to develop predictive test methods better than animal experiments for assessing the safety of chemical substances to man. According to today's vision this is achieved by using human cell based tissue and organ models. In the new testing strategy the toxic effects are assessed by the changes in the critical parameters of the cellular biochemical routes (AOP, adverse toxic outcome pathway-principle) in the target tissues. In vitro-tests are rapid and effective, and with them automation can be applied. The change in the testing paradigm is supported by all stakeholders: scientists, regulators and people concerned on animal welfare.

— There are several hundred industrial chemicals with neurotoxic potential. The neurotoxic risks of most of these chemicals are unknown. Additional methods are needed to assess the risks more effectively and to elucidate the mechanisms of neurotoxicity more accurately than is possible with the conventional methods. This paper deals with general tasks concerning the use of in vitro models in the evaluation of neurotoxic risks. It is based on our previous studies with various in vitro models and on recent literature. The induction of glial fibrillary acidic protein in astrocyte cultures after treatment with known neurotoxicants (mercury compounds and aluminium) is discussed in more detail as an important response which can be detected in vitro. When used appropriately with in vivo tests and with previous toxicological data, in vitro neurotoxicity testing considerably improves risk assessment. The incorporation of in vitro tests into the early stages of risk evaluation can reduce the number of animals used in routine toxicity testing, by identifying chemicals with high neurotoxic potential.

The retinal pigment epithelium (RPE) removes the outer segments of photoreceptor cells by phagocytosis. We studied the effects of tamoxifen and chloroquine on the activity of the lysosomal enzymes N-acetyl-beta-glucosaminidase and cathepsin D in RPE in vitro to evaluate the possible eye toxicity caused by these drugs. The results show decreases in the activities of lysosomal enzymes after drug exposure. The enzymes tested seemed to be more sensitive to tamoxifen than to chloroquine. A profound decrease in the activities of the lysosomal enzymes only started at concentrations above therapeutic dose levels.

The most common acute neurotoxic effect of organic solvents is their central nervous system (CNS) depressant effect. The molecular mechanism underlying this effect is not known. The purpose of our studies has been to evaluate the adverse effects of organic solvents on the CNS in vitro. Synaptosomal membranes, whole brain reaggregate and astrocyte cultures were studied. Our results suggest that cell membrane integral proteins are targets for solvent impact, but that there are differences among various cell types. In addition to lipophilicity, the structure of solvent molecules seems to be important when considering CNS toxicity. Organic solvents increase the fluidity of the membranes, which may disturb the lipid-protein interaction and the optimal functioning of the enzyme. However, direct effects of solvents on proteins cannot be excluded. In vitro cell models can be used in methods designed to predict acute neurotoxic effects of foreign compounds, and in studies of neurotoxic mechanisms.

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.

The present paper deals with the general anaesthetic effect of organic solvents. The emphasis is on cell membranes as models for predicting the adverse reactions of the human nervous system to organic solvents, and for studying the mechanisms of their anaesthetic action. Human and rat erythrocyte and platelet membranes, rat synaptosomal membranes and cultured neural cell membranes were exposed in vitro to various concentrations (3–30mM) of benzene, toluene, o-xylene and styrene in the incubation mixtures. After exposure to a solvent for 30 minutes, the membrane integral protein integrity was determined by measuring ATPase and acetylcholinesterase activities. All measurements were performed in triplicate from 3 or 4 independent experiments with each exposure of solvent and each membrane isolation. The enzyme inhibition was dose-dependent, and differed after solvent treatment in all membrane preparations tested in increasing order, as follows: benzene, toluene, styrene, o-xylene. The results support the protein theories on the mechanism of the anaesthetic action of various industrial organic solvents. The integral membrane proteins contain hydrophobic pockets which may bind anaesthetics, e.g. solvents which change the properties of integral enzymes. The effects of solvents in peripheral models are quite similar to those in neural membranes in vitro. Therefore, these membrane models can serve as test models when the narcotic potencies of the solvents are being evaluated, or at least for screening purposes and for studies of the mechanisms of solvent neurotoxicity.

International audience; The combination of an in vitro BBB model (4d/24w) with a neuronal cell line (SH-SY5Y) provides a convenient approach to explore the importance of BBB permeability in neurotoxicity assessment of compounds. The toxicity of 16 compounds on SH-SY5Y cells was evaluated after 24 h incubation with each compound and compared to their toxicity on SH-SY5Y after passage through the BBB model. Nine out of 16 compounds were found toxic after direct exposure at 100 μM while only three still induced toxicity on SH-SY5Y cells after BBB transport. The BBB permeability values of each compound revealed that in the case of compounds that did not induce toxicity, the amount that crossed the BBB was not enough to exert a toxic effect on the neuronal cells. Since disrupting the BBB may also cause unwanted effect on brain cells, the BBB toxicity of these compounds have been assessed. Our results prompted the importance of BBB permeability assessment in neurotoxicity evaluation, as it allows a better estimation of the actual concentration at the target site.

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.

In this in vitro study, changes in the activity of the neural membrane integral protein, ATPase, were recorded after the exposure of isolated synaptosomes to different concentrations of aluminium and lead. Both total ATPase activity and Mg2+-ATPase activity were studied. A specific mouse strain, heterozygous for a glial cell line-derived neurotrophic factor (GDNF), and the corresponding wild-type mouse cerebral tissue, were used for the synaptosome isolations. The ATPase activities of the GDNF+/– mouse synaptosomes were compared with those of wild-type synaptosomes. The decrease in total ATPase activity was similar in both types of synaptosomes, but after exposure to aluminium, the decrease of Mg2+-ATPase activity in the GDNF+/– synaptosomes was smaller than that in the wild-type synaptosomes. After exposure to lead, the protective effect of GDNF was not so clear. The synaptosomal effects of lead were already found at concentrations lower than those where cell toxicity appeared in SHSY5Y cell cultures. Thus, synaptosomal ATPase activity was considered to be a sensitive marker for the detection of lead-induced neurotoxicity.

The purpose of this paper is to review the current state of development of advanced in vitro blood–brain barrier (BBB) models. The BBB is a special capillary bed that separates the blood from the central nervous system (CNS) parenchyma. Astrocytes maintain the integrity of the BBB, and, without astrocytic contacts, isolated brain capillary endothelial cells in culture lose their barrier characteristics. Therefore, when developing in vitro BBB models, it is important to add astrocytic factors into the culture system. Recently, novel filter techniques and co-culture methods have made it possible to develop models which resemble the in vivo functions of the BBB in an effective way. With a BBB model, kinetic factors can be added into the in vitro batteries used for evaluating the neurotoxic potential of chemicals. The in vitro BBB model also represents a useful tool for the in vitro prediction of the BBB permeability of drugs, and offers the possibility to scan a large number of drugs for their potential to enter the CNS. Cultured monolayers of brain endothelial cell lines or selected epithelial cell lines, combined with astrocyte and neuron cultures, form a novel three-dimensional technique for the screening of neurotoxic compounds.

Blood-brain barrier in vitro models and their application in toxicology. The report and recommendations of ECVAM Workshop 49.

Glucose is the main energy source of brain cells. The transport of glucose across the cell membrane is the first step of its utilization. Any modification in glucose uptake capacity may cause deleterious effects on neural cell functions. In the present study, 3-O-methyl-D-glucose (3-OMG) uptake and its modulation by selected neurological drugs (amitriptyline, selegiline, carbamazepine and phenytoin) were studied in differentiated (with retinoic acid and 12-O-tetradecanoyl phorbol 13-acetate) and undifferentiated neuroblastoma SH-SY5Y and astrocytoma U-373 MG cell lines, using tracer methods. The expression of glucose transporters was studied by immunocytochemistry. SH-SY5Y and U-373 MG cells showed differences both in their glucose uptake properties and in the modulation of glucose uptake by the drugs, which might reflect different specialization of neuronal and glial cells in vivo. While selegiline and amitriptyline had a minor and variable effect on 3-OMG uptake in all cell cultures, the anticonvulsants carbamazepine and phenytoin increased 3-OMG uptake in U-373 MG cells, but decreased that in SH-SY5Y cells. Differentiated SH-SY5Y cells were more sensitive to the effects of the anticonvulsants than undifferentiated SH-SY5Y cells. The results suggest that, the cell lines are promising neural models for the evaluation of drug side effects due to disturbances in glucose uptake.

Mercury and aluminum are considered to be neurotoxic metals, and they are often connected with the onset of neurodegenerative diseases. In this study, mercuric mercury, methylmercury and aluminum were studied in three different cell lines of neural origin. To evaluate the effects, mitochondrial cytotoxicity and apoptosis induced by the metals were measured after various incubation times. SH-SY5Y neuroblastoma, U 373MG glioblastoma, and RPE D407 retinal pigment epithelial cells were subcultured to appropriate cell culture plates and 0.01-1,000 microM concentrations of methylmercury, mercuric and aluminum chloride were added into the growth medium. In the assay measuring the mitochondrial dehydrogenase activity, WST-1, the cultures were exposed for 15 min, 24 or 48 h before measurement. Cells were allowed to recover from the exposure in part of the study. Apoptosis induced by the metals was measured after 6-, 24- and 48-h exposure times with the determination of activated caspase 3 enzyme. Mitochondrial assays showed a clear dose-response and exposure time-response to the metals. The most toxic was methylmercury (EC50 ~0.8 microM, 48 h), and the most sensitive cell line was the neuroblastoma cell line SH-SY5Y. Furthermore, there was marked mitochondrial activation, especially in connection with aluminum and methylmercury at low concentrations. This activation may be important during the initiation of cellular processes. All the metals tested induced apoptosis, but with a different time-course and cell-line specificity. In microscopic photographs, glioblastoma cells formed fibrillary tangles, and neuroblastoma cells settled along the fibrilles in cocultures of glial and neuronal cell lines during aluminum exposure. The study emphasized the toxicity of methylmercury to neural cells and showed that aluminum alters various cellular activities.

The antioestrogens, tamoxifen and its more recent homologue toremifene, are used in the therapy of breast cancer. Tamoxifen has been reported to cause retinal changes as side effects. Both compounds inhibited glutamate uptake in retinal pigment epithelial cells, and the present study was conducted to clarify the mechanism of this inhibition. Retinal pigment epithelial cells are part of the blood-retina barrier, and their glutamate transporters are essential for retinal glutamate homeostasis. Glutamate uptake was investigated in human retinal pigment epithelial cell line D407 and in cultured pig retinal pigment epithelial cells using L-[3H]glutamate as a tracer. The cells were exposed to 7.5 microM tamoxifen and toremifene. beta-Hydroxyaspartate, a transportable inhibitor of glutamate transport, was used as a reference compound. In kinetic analyses, beta-hydroxyaspartate increased the Km constant for glutamate transport. Tamoxifen and toremifene exhibited the same effect, which indicates that inhibition evoked by them is also competitive in nature. Both drugs were more effective in the human retinal pigment epithelial cell line than in the pig retinal pigment epithelial cells. The results show for the first time that the antioestrogens tamoxifen and toremifene could possibly hamper glutamate transport by replacing glutamate as the substrate.

The transport of D-glucose into rainbow trout (Oncorhynchus mykiss) and river lamprey (Lampetra fluviatilis) hepatocytes, as well as into rainbow trout hepatoblastoma cell line RTH-149 was studied using tracer methods. The half-time for D-glucose equilibration was 15 s for rainbow trout. The half-times for the non-metabolizable D-glucose analog, 3-O-methyl-D-glucose equilibration were 8 s, 37 s and 38 s for rainbow trout, lamprey and RTH-149 cells, respectively. The 3-O-methyl-D-glucose was taken up by rainbow trout hepatocytes by facilitated diffusion in addition to simple diffusion. The uptake showed saturation kinetics with the K(m) of 37 mM and V(max) of 62 mmol kg(-1) cells min(-1). The uptake was sensitive to phloretin and cytochalasin B, but not affected by ouabain. The 3-O-methyl-D-glucose uptake by lamprey hepatocytes and RTH-149 cells showed no indication of saturation up to 160 mM, and was not affected by phloretin, cytochalasin B or ouabain, which suggests the mode of transport to be by passive diffusion. However, immunocytochemical stainings revealed the existence of mammalian type GLUT1 and GLUT2 transporters in all cells studied. The lack of a functioning carrier-mediated glucose uptake in lamprey hepatocytes might be due to its physiological state (prespawning starvation). The minor 3-O-methyl-D-glucose uptake into RTH-149 cells compared to freshly isolated rainbow trout hepatocytes might reflect low metabolic activity of the cell lines. Under the conditions applied the RTH-149 cell line is no suitable in vitro model for glucose transport in fish cells.

Pyrethroids are important insecticides used largely because of their high activity as an insecticide and their low mammalian toxicity. Some studies have demonstrated that these products, especially compounds with an α-cyano group, show neurotoxic effects on the mammalian central nervous system (CNS). In this study, we investigate with different methods the cell toxic effects of commercial, chemically different pyrethroid compounds on neuronal cell line SH-SY5Y. Natural pyrethrin and permethrin (both with no α-cyano group) and cypermethrin (with an α-cyano group), were studied. For toxicity determinations, SH-SY5Y neuroblastoma cells were exposed to pyrethroids at 0.1-100μM concentrations for 1 day. The cell toxicity was evaluated by determining the total ATP with a luminescence method, the mitochondrial metabolic activity (WST-test) with a photometric method, and the morphological changes of the cell cultures with microscopic digital photographing at different dose levels of compounds. The results obtained with WST-1 method and with the measurement of total ATP were different. ATP measurement seemed to show cytotoxicity at lower concentrations than WST-1 method. There was induction of enzyme activities with WST-1 test with all pyrethroid compounds studied at low concentrations. With the ATP assay, exposure to 0.1-100μM of natural pyrethrin, as well as of permethrin and cypermethrin showed dose-dependent cytotoxicity. The most toxic pyrethroid was cypermethrin followed by permethrin and natural pyrethrin. Our study confirms that the cell toxicity was dependent on the chemical structure of pyrethroids and pyrethroids without an α-cyano group show the weakest physiological effect. Microscopic photographs of exposed cell cultures correlated to the toxic effects revealed by the metabolic tests.

This study was undertaken to investigate the use of the in vitro test WST-1, an assay of cell proliferation and viability, for a preliminary safety evaluation of topical ophthalmic preparations. The cytotoxicity of two surfactants, benzalkonium chloride (BAC) and polyoxyethylene-20-stearyl ether (Brij78, PSE) was independently investigated in four laboratories in the EU by using an immortalized human corneal epithelial (HCE) cell line. The HCE cells were exposed to BAC and PSE for 5 min, 15 min, and 1 hour, and the results of the HCE-WST-1 tests were collected and compared. After one-hour exposure, the EC(50) values in BAC-treated cells in the presence of serum ranged between 0.0650 +/- 0.0284 (mean +/- SD) mM, and those in the absence of serum 0.0296 +/- 0.0081 mM. The corresponding values for PSE were 0.0581 +/-.0300 mM and 0.0228 +/-.0063 mM. There were variations in the results between different laboratories, with coefficients of variation ranging from 31 to 121%, mean 58%. The use of one-hour exposure time is to be preferred, and the elimination of serum in the culture medium is recommended to avoid both underestimation of toxic effects and variability of the test results. J Ocul Pharmacol Ther

The systemic drugs chloroquine and tamoxifen have caused retinal defects in human eye. The aim of our study was to investigate the effects of the amphiphilic drug tamoxifen, of its homologue toremifene, and of chloroquine on the glutamate uptake in retinal pigment epithelial (RPE) cells. Cultured human RPE cell line D407 and pig RPE cells were used in the study. Glutamate uptake was characterised and the glutamate transporters of pig RPE cells and the human RPE cell line D407 were compared to each other. The uptake of glutamate was studied using L-[3H]glutamate as a tracer. The radioactivity in the solubilised RPE was measured with a liquid scintillation counter. In the uptake experiments, the cells were exposed to the test drugs, to the selected glutamate receptor antagonists, and to the glutamate transporter inhibitors. Both RPE cell types exhibited a high-affinity transport system for glutamate. The glutamate transporter in RPE exhibited features characteristic of the uptake systems of neurotransmitters. The transport was Na+-dependent, and L- and D-aspartate were transported into the cell by the same transporter. Chloroquine had no effect on glutamate uptake, but tamoxifen and toremifene decreased the glutamate uptake of RPE cells dose-dependently both in pig RPE cells and in human RPE cell line. The IC50 values of tamoxifen and toremifene were lower for pig RPE cells, compared to the human RPE cell line D407. The glutamate uptake was a sensitive target for the effects of tamoxifen and toremifene, and disturbances in this function could be considered as one of the possible mechanisms of retinal defects.

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 mumol/l, and in pig RPE cells between 10.1 and 12.2 mumol/l, depending on the cell density used. The corresponding values for toremifene were 7.4 to 11.1 mumol/l in D407 cells and 10.0 to 11.6 mumol/l in pig RPE cells. In chloroquine-treated cells, the EC50 values were 110.0 mumol/l for D407 cells and 58.4 mumol/l for pig RPE cells. Gentamicin and ganciclovir did not shock 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 or 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 tyupes. 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. (C) 2002 Published by Elsevier Science Ltd. Toxicology in Vitro

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 μ m ) 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 μ m 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 μ m 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.