In addition to its nephrotoxic effects, hepato-, terato- and immunotoxic activities of OTA have also been reported [18, 24]. accompanied by a loss of mitochondria membrane potential. Overall, present data indicated that OTA is neurotoxic at relatively low concentrations. OTA induced neurotoxicity seems to be, at least party, mediated by apoptosis. OTA may contribute to the pathogenesis of neurodegenerative diseases (e.g. Alzheimers and Parkinsons disease) in which apoptotic processes are centrally involved. and Their widespread occurrence and the persistence of OTA in the food chain may contribute to a significant OTA exposure to humans [25, 26]. In fact OTA has been frequently found in the human blood [20]. The kidney is the main target tissue of OTA toxicity [10, 19]. In addition to its nephrotoxic effects, hepato-, terato- and immunotoxic activities of OTA have also been reported [18, 24]. OTA has been classified as a putative human carcinogen by the International Agency for Research on Cancer (IARC [14]). Open in a separate window Fig.?1 Chemical structure of ochratoxin A Furthermore, recent findings indicate that OTA may, to some extent, also affect the neuronal system [2, 27]. Studies in rodents suggest that OTA crosses the blood brain barrier and accumulates in most parts of the brain as a function of time and concentration [2, 27, 28]. After administration of a single OTA dose to mice (3.5?mg/kg body weight; i.p.), highest OTA concentrations were measured in the cerebellum (1.7?ng/mg) and the pons (0.7?ng/mg) followed by the cerebral cortex (0.3?ng/mg) [28]. Interestingly, the sites of OTA accumulation and tissue susceptibility towards OTA induced toxicity in the brain did not always correlate. However, the hippocampus, a primary site of neurodegeneration in Alzheimers disease, turned out to exhibit relatively high OTA levels with concurrently pronounced OTA neurotoxicity [3]. In this context, Sava and coworkers [28] hypothesized that low level exposure of OTA may exert delayed neurotoxic effects which may in turn contribute to the development of neurodegenerative disorders. Interestingly, OTA has also been shown to cause widespread oxidative stress as measured by an increase in lipid peroxidation and DNA damages in mice brain [27], features that are associated with a number of neurodegenerative disorders including Parkinsons Arterolane and Alzheimers disease. However, the underlying molecular mechanisms for OTA neurotoxicity are not fully understood. The present study in cultured neurons aimed at investigating whether OTA induced neurotoxicity may be mediated by apoptosis. Materials and methods Cell culture SH-SY5Y neuroblastoma cells were routinely cultured in RPMI 1640 medium supplemented with 5% fetal bovine serum, 5?mmol/L glutamine, 1?mmol/L MEM sodium pyruvate, 1% MEM non-essential amino acids, 1% MEM vitamins, with 100?IU/mL penicillin and 100?g/mL streptomycin (all from PAA, Pasching, Austria) under standard conditions (37C, humidified 5% CO2 atmosphere). Cells were subcultured every 4C5?days after reaching 80% confluence and seeded at an initial density of 6.3??104?cells/cm2 in 24-well and 6-well plates for further experiments. Primary rat cortical neuronal cell tradition was carried out as previously explained with small modifications [9, 32]. Briefly, neonatal Wistar rats were sacrificed; cortices were separated on snow and slice into pieces previous digestion in 0.25% trypsin for 15?min at 37C. Cortical cells lysate was then passaged through a series of fire-polished pipettes to get a single cell suspension. After centrifuging, cells were resuspended in Neurobasal medium supplemented with 2% B27 (Invitrogen, Carlsbad, CA, USA), 100?IU/mL penicillin, 100?g/mL streptomycin and seeded at 1??105?cells/cm2 in poly-d-lysine (Sigma, Deisenhofen, Germany) coated plates. Tradition medium was refreshed every 2C3?days and cells were kept under standard conditions?7?days before further treatment. Ochratoxin A (from Sigma) was dissolved in methanol (5?mmol/L stock solution) and further diluted in culture medium before use. Cells were treated with 0.1, 0.25, 0.5, 1.0 and 2.5?mol/L of OTA in serum or B27 free medium while described above prior to the neutral red assay or cell collection. Control cells were supplemented with 0.1% methanol as a vehicle control. In the caspase inhibitor experiments, Z-VAD-fmk and Z-DEVD-fmk (R&D system Inc., MN, USA) were dissolved in phosphate buffered saline (PBS) at 20?mmol/L and were further diluted by corresponding serum or B27 free medium to working concentrations. Cells were pre-incubated with Z-VAD-fmk Arterolane or Z-DEVD-fmk for 24?h followed by a 24?h exposure with OTA without Z-VAD-fmk or Z-DEVD-fmk. After that, cell viability was determined by the neutral reddish assay. For DNA laddering, cells were treated with 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0?mol/L of OTA for 48?h in serum or B27-free medium before collection. Neutral reddish assay Cell viability was assessed by the neutral reddish assay as explained previously [7]. Briefly, cells, after treatment with OTA, were washed with PBS. New medium supplemented with 50?g/ml neutral red was added and incubated for 1.5?h. Subsequently, neutral red medium was removed and the integrated.JC-1 is a m specific probe which indicates loss of m by changing fluorescence emission from red to green. potential. Overall, present data indicated that OTA is definitely neurotoxic at relatively low concentrations. OTA induced neurotoxicity seems to be, at least party, mediated by apoptosis. OTA may contribute to the pathogenesis of neurodegenerative diseases (e.g. Alzheimers and Parkinsons disease) in which apoptotic processes are centrally involved. and Their common occurrence and the persistence of Arterolane OTA in the food chain may contribute to a significant OTA exposure to humans [25, 26]. In fact OTA has been frequently found in the human being blood [20]. The kidney is the main target cells of OTA toxicity [10, 19]. In addition to its nephrotoxic effects, hepato-, terato- and immunotoxic activities of OTA have also been reported [18, 24]. OTA has been classified like a putative human being carcinogen from the International Agency for Study on Malignancy (IARC [14]). Open in a separate windows Fig.?1 Chemical structure of ochratoxin A Furthermore, recent findings indicate that OTA may, to some extent, also affect the neuronal system [2, 27]. Studies in rodents suggest that OTA crosses the blood brain barrier and accumulates in most parts of the brain like a function of time and concentration [2, 27, 28]. After administration of a single OTA dose to mice (3.5?mg/kg body weight; i.p.), highest OTA concentrations were measured in the cerebellum (1.7?ng/mg) and the pons (0.7?ng/mg) followed by the cerebral cortex (0.3?ng/mg) [28]. Interestingly, the sites of OTA build up and cells susceptibility towards OTA induced toxicity in the brain did not usually correlate. However, the hippocampus, a primary site of neurodegeneration in Alzheimers disease, turned out to exhibit relatively high OTA levels with concurrently pronounced OTA neurotoxicity [3]. With this context, Sava and coworkers [28] hypothesized that low level exposure of OTA may exert delayed neurotoxic effects which may in turn contribute to the development of neurodegenerative disorders. Interestingly, OTA has also been shown to cause common oxidative stress as measured by an increase in lipid peroxidation and DNA damages in mice mind [27], features that are associated with a number of neurodegenerative disorders including Parkinsons and Alzheimers disease. However, the underlying molecular mechanisms for OTA neurotoxicity are not fully understood. The present Rabbit Polyclonal to PKA-R2beta study in cultured neurons aimed at investigating whether OTA induced neurotoxicity may be mediated by apoptosis. Materials and methods Cell tradition SH-SY5Y neuroblastoma cells were regularly cultured in RPMI 1640 medium supplemented with 5% fetal bovine serum, 5?mmol/L glutamine, 1?mmol/L MEM sodium pyruvate, 1% MEM non-essential amino acids, 1% MEM vitamins, with 100?IU/mL penicillin and 100?g/mL streptomycin (all from PAA, Pasching, Austria) less than standard conditions (37C, humidified 5% CO2 atmosphere). Cells were subcultured every 4C5?days after reaching 80% confluence and seeded at an initial denseness of 6.3??104?cells/cm2 in 24-well and 6-well plates for further experiments. Main rat cortical neuronal cell tradition was carried out as previously explained with minor modifications [9, 32]. Briefly, neonatal Wistar rats were sacrificed; cortices were separated on snow and slice into pieces previous digestion in 0.25% trypsin for 15?min at 37C. Cortical cells lysate was then passaged through a series of fire-polished pipettes to get a single cell suspension. After centrifuging, cells were resuspended in Neurobasal medium supplemented with 2% B27 (Invitrogen, Carlsbad, CA, USA), 100?IU/mL penicillin, 100?g/mL streptomycin and seeded at 1??105?cells/cm2 in poly-d-lysine (Sigma, Deisenhofen, Germany) coated plates. Tradition medium was refreshed every 2C3?days and cells were kept under standard conditions?7?days before further treatment. Ochratoxin A (from Sigma) was dissolved in methanol (5?mmol/L stock solution) and further diluted in culture medium before use. Cells were treated with 0.1, 0.25, 0.5, 1.0 and 2.5?mol/L of OTA in serum or B27 free medium while described above prior to the neutral red assay or cell collection. Control cells were supplemented with 0.1% methanol as a vehicle control. In the caspase inhibitor experiments, Z-VAD-fmk and Z-DEVD-fmk (R&D system Inc., MN, USA) were dissolved in phosphate buffered saline (PBS) at 20?mmol/L and were further diluted by corresponding serum or B27 free medium to working concentrations. Cells were pre-incubated with Z-VAD-fmk or Z-DEVD-fmk for 24?h followed by a 24?h exposure with OTA without Z-VAD-fmk or Z-DEVD-fmk. After that, cell viability was determined by the neutral reddish assay. For DNA laddering, cells were treated with.