LASU: An efficient and stable phthalocyanine dye with tolerable safety profile for self-disinfecting anti-COVID textiles activated by ambient light.

BACKGROUND: Recent COVID crisis has demonstrated that modern society urgently needs an accessible protection against mass infections, especially viruses, as the new strains are appearing at an ever-increasing pace and cause severe harm to the population and the world economy. METHODS: We have developed an efficient phthalocyanine photosensitizer LASU, that is suitable for dyeing textiles and allows to prepare reusable self-disinfecting fabrics with strong antiviral properties. The safety profile of LASU was evaluated in accredited laboratories by several in vitro assays according to the OECD-guidelines. RESULTS: The textiles impregnated with LASU phthalocyanine showed a significant antiviral photodynamic effect even under moderate indoor and outdoor light. The dye did not show any genotoxic potential in human lymphocyte micronucleus assay. It showed a possible indication for eye irritation in human EpiOcular™ model and was phototoxic when tested in mouse BALB/c 3T3 cell test in the presence and absence of UVA-irradiation. CONCLUSION: Novel phthalocyanine-dyed textiles are suitable for general use as self-disinfecting antiviral barriers and materials in hospitals, households, and public places. The safety profile of LASU is the phototoxic effect which is related to LASU´s mode of action.

The Safety Assessment of Cosmetic Perfumes by Using In Chemico and In Vitro Methods in Combination with GC-MS/MS Analysis.

Animal testing has been prohibited for the safety assessment of cosmetic ingredients or finished products. Thus, alternative non-animal methods, followed by confirmatory clinical studies on human volunteers, should be used as the sole legally acceptable approach within the EU. The safety assessment of cosmetic products requires the involvement of multiple scientific disciplines, including analytical chemistry and biomedicine, as well as in chemico, in vitro and in silico toxicology. Recent data suggest that fragrance components may exert multiple adverse biological effects, e.g. cytotoxicity, skin sensitisation, (photo)genotoxicity, mutagenicity, reprotoxicity and endocrine disruption. Therefore, a pilot study was conducted with selected samples of fragrance-based products, such as deodorant, eau de toilette and eau de parfum, with the aim of integrating results from a number of alternative non-animal methods suitable for the detection of the following toxicological endpoints: cytotoxicity (with 3T3 Balb/c fibroblasts); skin sensitisation potential (in chemico method, DPRA); skin sensitisation potential (LuSens in vitro method, based on human keratinocytes); genotoxicity potential (in vitro Comet assay with 3T3 Balb/c cells); and endocrine disruption (in vitro YES/YAS assay). The presence of twenty-four specific known allergens in the products was determined by using GC-MS/MS. The strategies for estimation of the NOAEL of a mixture of allergens, which were proposed by the Scientific Committee on Consumer Products in their 'Opinion on Tea tree oil' document and by the Norwegian Food Safety Authority in their 'Risk Profile of Tea tree oil' report, were used as models for the NOAEL estimation of the mixtures of allergens that were identified in the individual samples tested in this study.

In Search of Clinical Markers: Indicators of Exposure in Dampness and Mold Hypersensitivity Syndrome (DMHS).

Potential markers were sought to diagnose mold hypersensitivity. Indoor air condensed water and human macrophage THP-1 test were applied to evaluate the buildings. Basophil activation tests (BAT) were conducted and mold-specific immunoglobulins (IgE, IgG, IgA, and IgD) were measured in study subjects' serum and feces. Exposed subjects reported markedly more symptoms from occupational air than controls. Basophils from exposed subjects died/lost activity at 225 times lower concentrations of toxic extracts from the target building than recommended in the common BAT protocol. Fecal IgG and IgD levels against Acrostalagmus luteoalbus and Aspergillus versicolor produced receiver operating curves (ROC) of 0.928 and 0.916, respectively, when plotted against the inflammation marker MRP8/14. Assaying serum immunoglobulin concentrations against the toxic Chaetomium globosum (MTAV35) from another building, a test control, did not differentiate study individuals. However, if liver metabolism produced the same core molecule from other Chaetomium globosum strains, this would explain the increased response in fecal immunoglobulins in the exposed. The altered immunoglobulin values in the samples of exposed when compared to controls revealed the route of mold exposure. The toxicity of indoor air condensed water samples, BAT and serology confirmed the severity of symptoms in the target building's employees, supporting earlier findings of toxicity in this building.

New approach methods for assessing indoor air toxicity.

Indoor air is typically a mixture of many chemicals at low concentrations without any adverse health effects alone, but in mixtures they may cause toxicity and risks to human health. The aim of this study was by using new approach methods to assess the potential toxicity of indoor air condensates. In specific, different in vitro test methods including cyto-and immunotoxicity, skin sensitization and endocrine disruption were applied. In addition to biological effects, the indoor air samples were subjected to targeted analysis of 25 volatile organic compounds (VOCs) and Genapol X-80 (a nonionic emulsifier) suspected to be present in the samples, and to a non-targeted "total chemical scan" to find out whether the chemical composition of the samples is associated with the biological effects. The results confirm that assessing health risks of indoor air by analysing individual chemicals is not an adequate approach: We were not able to detect the VOCs and Genapol X-80 in the indoor air samples, yet, several types of toxicity, namely, cytotoxicity, immunotoxicity, skin sensitization and endocrine disruption were detected. In the non-targeted total chemical scan of the indoor air samples, a larger number of compounds were found in the cytotoxic samples than in the non-cytotoxic samples supporting the biological findings. If only one biological method would be selected for the screening of indoor air quality, THP-1 macrophage/WST-1 assay would best fit for the purpose as it is sensitive and serves as a good representative for different sub-toxic end points, including immunotoxicity, (skin) sensitization and endocrine disruption.

Toxic Indoor Air Is a Potential Risk of Causing Immuno Suppression and Morbidity-A Pilot Study.

We aimed to establish an etiology-based connection between the symptoms experienced by the occupants of a workplace and the presence in the building of toxic dampness microbiota. The occupants (5/6) underwent a medical examination and urine samples (2/6) were analyzed by LC-MS/MS for mycotoxins at two time-points. The magnitude of inhaled water was estimated. Building-derived bacteria and fungi were identified and assessed for toxicity. Separate cytotoxicity tests using human THP-1 macrophages were performed from the office's indoor air water condensates. Office-derived indoor water samples (n = 4/4) were toxic to human THP-1 macrophages. Penicillium, Acremonium sensu lato, Aspergillus ochraceus group and Aspergillus section Aspergillus grew from the building material samples. These colonies were toxic in boar sperm tests (n = 11/32); four were toxic to BHK-21 cells. Mycophenolic acid, which is a potential immunosuppressant, was detected in the initial and follow-up urine samples of (2/2) office workers who did not take immunosuppressive drugs. Their urinary mycotoxin profiles differed from household and unrelated controls. Our study suggests that the presence of mycotoxins in indoor air is linked to the morbidity of the occupants. The cytotoxicity test of the indoor air condensate is a promising tool for risk assessment in moisture-damaged buildings.

Qualitative and Quantitative Analysis of Certain Aspects of the Cytotoxic and Genotoxic Hazard of Hospital Wastewaters by Using a Range of In Vitro Assays.

Health care facilities and hospitals generate significant amounts of wastewater which are released into the sewage system, either after a preliminary treatment or without any further treatment. Hospital wastewater may contain large amounts of hazardous chemicals and pharmaceuticals, some of which cannot be eliminated entirely by wastewater treatment plants. Moreover, hospital effluents may be loaded with a plethora of pathogenic microorganisms or other microbiota and microbiome residues. The need to monitor hospital effluents for their genotoxic hazard is of high importance, as detailed information is scarce. DNA-based information can be acquired directly from samples through the application of various molecular methods, while cell-based biomonitoring assays can provide important information about impaired cellular pathways or mechanisms of toxicity without prior knowledge of the identity of each toxicant. In our study, we evaluated samples of chlorinated hospital wastewater discharged into the sewage system after this disinfection process. The assessment of cytotoxicity, genotoxicity and mutagenicity of the hospital effluents was performed in vitro by using a broad battery of biomonitoring assays that are relevant for human health effects. All the tested hospital wastewater samples could be classified as potentially genotoxic, and it is concluded that the microbiota present in hospital wastewater might contribute to this genotoxic potential.

Development of novel human in vitro vascularized adipose tissue model with functional macrophages.

Inflammation has been proven significant factor in development of type 2 diabetes. So far, most of the adipose tissue related research has been performed in animals, mainly rodent models. The relevance of translation of animal results to humans is questionable. However, in vitro model with relevant human cell source, such as human adipose tissue stromal cells (hASC), can be developed and should be utilized for human adipose tissue research. We developed in vitro models of human adipose tissue utilizing hASC, endothelial cells and monocytes/macrophages. By isolating endothelial cells and macrophages from same adipose tissue as hASC, we were able to provide method for constructing personalized models of adipose tissue. With these models, we studied the effect of macrophages on adipogenesis and protein secretion, with and without vasculature. The models were analyzed for immunocytochemical markers, cell number, triglyceride accumulation and protein secretion. We found that lipid accumulation was greater in adipocytes in the presence of macrophages. Interferon gamma increased this difference between adipocyte culture and Adipocyte-Macrophage co-culture. Protein secretion was affected more by macrophages when vasculature was not present compared to the mild effect when vasculature was present. The vascularized adipose model with macrophages is valuable tool for human adipose tissue research, especially for the personalized medicine approaches; for choosing the right treatments and for studying rare medical conditions.

Association of toxic indoor air with multi-organ symptoms in pupils attending a moisture-damaged school in Finland.

BACKGROUND: There is an on-going debate on how best to test toxic indoor air. Toxicological methods based on condensed water samples and cell culture technique are newly introduced research tools which were tested in this study. METHODS: Pupils (n=47) from a water-damaged and (n=56) healthy schools were interviewed using a questionnaire. Indoor air was collected with a novel condensed water sampling technique and human THP-1 macrophages were exposed to the condensate. The cytotoxicity of cotton wool swab samples was tested using human BJ fibroblasts. Conventional microbiological culture methods were also performed. RESULTS: Gastrointestinal problems (GI) were reported by 51% from the study cohort but only 4% of the control cohort, relative risk RR=14.30. For any neurological or neuropsychological symptoms, the RR was 63.04, muscular-skeletal pain RR=58.28, headache RR=31.00, respiratory symptoms RR=22.64, fatigue RR=21.45, sub febrility RR=15.49, ear infections RR=7.74, skin rash RR=5.96, all being statistically significant (P<0.001). All indoor air (n=7) and cotton wool samples (n=2) taken from the water-damaged classroom or in proximity of the problematic classrooms were toxic in cell culture assays. Low numbers of moisture-damage indicators were recovered from wall, passive air, and swab samples, namely Aspergillus ochraceus species group, Aspergillus, Eurotium species group, Fusarium, Tritirachium, Scopulariopsis genus group and Aspergillus versicolores species group. CONCLUSIONS: Indoor air toxicity and dampness-related microbiota recovered from the classrooms were associated with multi-organ morbidity of the school occupants. These results corroborated our previous reports from two adult cohorts i.e. evidence of causality. These new toxicological methods based on condensed water and cell culturing techniques seem to be superior to conventional microbiological methods in correlating with clinical symptoms.

NANOCI-Nanotechnology Based Cochlear Implant With Gapless Interface to Auditory Neurons.

: Cochlear implants (CI) restore functional hearing in the majority of deaf patients. Despite the tremendous success of these devices, some limitations remain. The bottleneck for optimal electrical stimulation with CI is caused by the anatomical gap between the electrode array and the auditory neurons in the inner ear. As a consequence, current devices are limited through 1) low frequency resolution, hence sub-optimal sound quality and 2), large stimulation currents, hence high energy consumption (responsible for significant battery costs and for impeding the development of fully implantable systems). A recently completed, multinational and interdisciplinary project called NANOCI aimed at overcoming current limitations by creating a gapless interface between auditory nerve fibers and the cochlear implant electrode array. This ambitious goal was achieved in vivo by neurotrophin-induced attraction of neurites through an intracochlear gel-nanomatrix onto a modified nanoCI electrode array located in the scala tympani of deafened guinea pigs. Functionally, the gapless interface led to lower stimulation thresholds and a larger dynamic range in vivo, and to reduced stimulation energy requirement (up to fivefold) in an in vitro model using auditory neurons cultured on multi-electrode arrays. In conclusion, the NANOCI project yielded proof of concept that a gapless interface between auditory neurons and cochlear implant electrode arrays is feasible. These findings may be of relevance for the development of future CI systems with better sound quality and performance and lower energy consumption. The present overview/review paper summarizes the NANOCI project history and highlights achievements of the individual work packages.

Human BJ Fibroblasts is an Alternative to Mouse BALB/c 3T3 Cells in In Vitro Neutral Red Uptake Assay.

The OECD GD 129 BALB/c 3T3 neutral red uptake (NRU) assay is a standardized test method for estimating starting dose for an acute oral systemic toxicity test in rodents. Mouse BALB/c 3T3 fibroblasts are the most commonly used cells in the NRU assay. We have previously transferred and validated BALB/c 3T3 NRU assay in our GLP laboratory. Subsequently, in order to obtain more human-relevant cytotoxicity data, we performed an intralaboratory validation using human BJ fibroblasts in the NRU assay instead of mouse BALB/c 3T3 fibroblasts. Here, we present comparative cytotoxicity data of 26 different test chemicals (pharmaceuticals, industrial chemicals, pesticides and food additives) produced with both BALB/c 3T3 NRU and BJ NRU assays.

The applicability of conventional cytotoxicity assays to predict safety/toxicity of mesoporous silica nanoparticles, silver and gold nanoparticles and multi-walled carbon nanotubes.

Developing new, validated methods for screening of the effects of nanomaterials is a huge and expensive task. It is therefore necessary to try to employ already existing and validated methods, developed for chemicals. In the present study cytotoxicity of gold (Au) and silver (Ag) nanoparticles (NP), two different mesoporous silica nanoparticles (MSNP), and multi-walled carbon nanotubes (MWCNT) were investigated in BALB/c 3T3 fibroblasts, NR8383 macrophages, and U937 monocytes using standard assays, namely WST-1 and NRU. In addition, preliminary attempts were made to investigate ENM-mediated effects on cell motility as a potential end point for NP toxicity. AgNPs were most toxic to BALB/c 3T3 fibroblasts while other ENMs were insignificantly toxic. NR8383 macrophages were most sensitive cells, as in addition to AgNPs, also MWCNTs were toxic to NR8383 cells. AgNP was toxic also to U937 cells, other ENMs had minor effect. Different media resulted in different-sized aggregates of the same ENMs. AgNP inhibited BALB/c motility most, whereas NR8383 motility was inhibited most by MWCNTs. In conclusion, conventional cytotoxicity assays are better suited to rank the order of toxicity of different nanoparticles instead of producing accurate IC50 data. Moreover, using immune cells, especially macrophages together with fibroblasts, would bring more relevant predictions of ENM cytotoxicity as immune cells may discover cytotoxicity that is not captured by BALB/c 3T3 cells alone.

Multilaboratory evaluation of 15 bioassays for (eco)toxicity screening and hazard ranking of engineered nanomaterials: FP7 project NANOVALID.

Within EU FP7 project NANOVALID, the (eco)toxicity of 7 well-characterized engineered nanomaterials (NMs) was evaluated by 15 bioassays in 4 laboratories. The highest tested nominal concentration of NMs was 100 mg/l. The panel of the bioassays yielded the following toxicity order: Ag > ZnO > CuO > TiO2 > MWCNTs > SiO2 > Au. Ag, ZnO and CuO proved very toxic in the majority of assays, assumingly due to dissolution. The latter was supported by the parallel analysis of the toxicity of respective soluble metal salts. The most sensitive tests/species were Daphnia magna (towards Ag NMs, 24-h EC50 = 0.003 mg Ag/l), algae Raphidocelis subcapitata (ZnO and CuO, 72-h EC50 = 0.14 mg Zn/l and 0.7 mg Cu/l, respectively) and murine fibroblasts BALB/3T3 (CuO, 48-h EC50 = 0.7 mg Cu/l). MWCNTs showed toxicity only towards rat alveolar macrophages (EC50 = 15.3 mg/l) assumingly due to high aspect ratio and TiO2 towards R. subcapitata (EC50 = 6.8 mg Ti/l) due to agglomeration of TiO2 and entrapment of algal cells. Finally, we constructed a decision tree to select the bioassays for hazard ranking of NMs. For NM testing, we recommend a multitrophic suite of 4 in vitro (eco)toxicity assays: 48-h D. magna immobilization (OECD202), 72-h R. subcapitata growth inhibition (OECD201), 30-min Vibrio fischeri bioluminescence inhibition (ISO2010) and 48-h murine fibroblast BALB/3T3 neutral red uptake in vitro (OECD129) representing crustaceans, algae, bacteria and mammalian cells, respectively. Notably, our results showed that these assays, standardized for toxicity evaluation of "regular" chemicals, proved efficient also for shortlisting of hazardous NMs. Additional assays are recommended for immunotoxicity evaluation of high aspect ratio NMs (such as MWCNTs).

Toxicity of silver nanoparticle in rat ear and BALB/c 3T3 cell line.

BACKGROUND: Silver nanoparticles (AgNPs) displayed strong activities in anti-bacterial, anti-viral, and anti-fungal studies and was reportedly efficient in treating otitis media .The potential impact of AgNPs on the inner ear was missing. OBJECTIVE: Attempted to evaluate the potential toxicity of AgNPs in the inner ear, middle ear, and external ear canal after transtympanic injection in rats. RESULTS: In in vitro studies, the IC50 for AgNPs in neutral red uptake assay was lower than that in NAD(P)H-dependent cellular oxidoreductase enzyme assay (WST-1) and higher than that in total cellular ATP and nuclear membrane integrity (propidium iodide) assessments. In in vivo experiments, magnetic resonance imaging (MRI) showed that significant changes in the permeability of biological barriers occurred in the middle ear mucosa, the skin of the external ear canal, and the inner ear at 5 h post-transtympanic injection of AgNPs at concentrations ranging from 20 µg/ml to 4000 µg/ml. The alterations in permeability showed a dosage-response relationship, and were reversible. The auditory brainstem response showed that 4000 µg/ml AgNPs induced hearing loss with partial recovery at 7 d, whereas 20 µg/ml caused reversible hearing loss. The functional change in auditory system was in line with the histology results. In general, the BALB/c 3T3 cell line is more than 1000 times more sensitive than the in vivo studies. Impairment of the mitochondrial function was indicated to be the mechanism of toxicity of AgNPs. CONCLUSION: These results suggest that AgNPs caused significant, dose-dependent changes in the permeability of biological barriers in the middle ear mucosa, the skin of the external ear canal, and the inner ear. In general, the BALB/c 3T3 cell line is more than 1000 times more sensitive than the in vivo studies. The rat ear model might be expended to other engineered nanomaterials in nanotoxicology study.

Intra-Laboratory Pre-Validation of a Human Cell Based in vitro Angiogenesis Assay for Testing Angiogenesis Modulators.

The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory pre-validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis, e.g., pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using six reference chemicals, which are widely used pharmaceuticals that inhibit angiogenesis: acetyl salicylic acid, erlotinib, 2-methoxyestradiol, levamisole, thalidomide, and anti-vascular endothelial growth factor. In the intra-laboratory pre-validation, the sensitivity of the assay (upper and lower limits of detection and linearity of response in tubule formation), batch to batch variation in tubule formation between different Master cell bank batches, and precision as well as the reliability of the assay (reproducibility and repeatability) were tested. The pre-set acceptance criteria for the intra-laboratory pre-validation study were met. The relevance of the assay in man was investigated by comparing the effects of reference chemicals and their concentrations to the published human data. The comparison showed a good concordance, which indicates that this human cell based angiogenesis model predicts well the effects in man and has the potential to be used to supplement and/or replace of animal tests.

The combined use of human neural and liver cell lines and mouse hepatocytes improves the predictability of the neurotoxicity of selected drugs.

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).

Modulation of glucose uptake in glial and neuronal cell lines by selected neurological drugs.

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.

Toxicity of selected cationic drugs in retinoblastomal cultures and in cocultures of retinoblastomal and retinal pigment epithelial cell lines.

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.

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 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.

D-Glucose uptake in fish hepatocytes: mediated by transporter in rainbow trout (Oncorhynchus mykiss), but only by diffusion in prespawning lamprey (Lampetra fluviatilis) and in RTH-149 cell line.

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.

Glutamate uptake is inhibited by tamoxifen and toremifene in cultured retinal pigment epithelial cells.

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.