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FINNISH RESEARCH PROGRAMME ON ENVIRONMENTAL HEALTH SYTTY NEUROTOXIC EFFECTS OF MICROBIAL TOXINS Project leader:
Kai Savolainen, Finnish Institute of Occupational Health, Topeliuksenkatu 41
a A, Researcher:
Consortium:
Moisture, mould and health. KEY WORDS:
mycotoxin, fumonisin B1, neurotoxicity EXTENDED ABSTRACT 1 Introduction Fusarium moulds are commonly present in buildings with moisture problems and finding of Fusarium indicate that water spills and subsequent water damages may have occurred in these premises. Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides (= F. moniliforme). It is commonly found in corn and its occurrence is also highly likely in buildings with water damage and Fusarium mould growth. FB1 is known to inhibit the normal metabolism of sphingolipids, with subsequent accumulation of ceramide, in cells, as the structure of FB1 is very similar to that of sphingolipids. This inhibition is thought to be the reason why FB1 is toxic to cells. The neuronal effects of FB1 have not been studied very well. Studies with cell cultures show that FB1 inhibits normal growth of neuronal cells and disturbs myelin formation. Experimental animals that were given FB1 had smaller brains than controls and changes in sphingolipid metabolism could be observed in the central nervous system. In glioblastoma cells FB1 causes cell death and apoptosis and inhibits protein synthesis. In vivo FB1 has been associated with animal diseases like leukoencephalomalacia and hemorrhage in the brain of rabbits after systemic administration, and also horses fed with FB1 have suffered from equine leukoencephalomalacia. In humans, FB1 has also been connected with central nervous system effects because FB1 metabolites were found in the urine of patients with chronic idiopathic spastic paraparesis. The aim of this study was to investigate the mechanisms whereby the mycotoxin FB1 affects neuronal and glial cells. 2 Methods Materials. Cell lines used: human SH-SY5Y neuroblastoma cells, human U-118MG glioblastoma cells, mouse GT1-7 hypothalamic cells and rat C6 glioblastoma cells. 2’,7’-Dichlorofluorescein diacetate, monochlorobimane, propidium iodide and digitonin were obtained from Molecular Probes Inc. (Eugene, OR, USA). Caspase-3 substrate and anti-rabbit IgG were from Calbiochem (La Jolla, CA, USA). Dulbecco´s modified Eagle medium, foetal bovine serum, trypsin, penicillin-streptomycin solution and Hank’s balanced salt solution (HBSS; Ca2+- and Mg2+-free) were from Gibco (Paisley, UK). Phosphate buffered saline (PBS; Ca2+- and Mg2+-free) was from Orion diagnostica (Espoo, Finland). Nonidet P40 and proteinase K were from Roche Diagnostics (Mannheim, Germany). Loading Dye Solution was obtained from MBI Fermentas (Hanover, MD, USA). Agarose and ethidium bromide were from Bio Rad (Hercules, CA, USA). p53 protein (DO-7) monoclonal antibody was from Novocastra Laboratories (Newcastle, UK), p53 (Pab 240) monoclonal antibody and MDM2 (H-221) polyclonal antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-mouse IgG was from AmershamPharmacia Biotech (Piscataway, NJ, USA). All other chemicals were obtained from Sigma Co. (St. Louis, MO, USA). Cell culture. All cells were cultured in Dulbecco´s modified Eagle medium (DMEM) supplemented with 10 % inactivated foetal bovine serum (FBS) and 50 U/ml penicillin and 50 µg/ml streptomycin. The cells were incubated in +37 oC in 5% CO2 in an air-ventilated humidified incubator. The cells were grown in 80 cm2 cell culture flasks (Nunc, Denmark) and harvested either by 0,02% EDTA in PBS or by 0,02% EDTA-0,05% trypsin in PBS. Treatment of cells. When treating cells with fumonisin B1, the concentrations 0.01, 0.1, 1, 10, 30 and 100 µM were used. The production of reactive oxygen species, the glutathione levels, cell viability, caspase-3 activity and DNA fragmentation were measured after exposing cells to fumonisin B1 for 0, 0.5, 1, 2, 3, 4, 5, 12, 24, 36, 48, 72 and 144 hours. Some experiments were carried out in the presence or absence of 0.02% diethyl maleate (DEM), which effectively depletes intracellular glutathione. Measurement of intracellular reactive oxygen species. Cells cultured on 48 well plates (Costar, USA) were loaded with 40 µM 2’,7’-dichlorofluorescein diacetate for 20 minutes. After loading, the cells were washed twice with HBSS. In long-term experiments (12 hours or longer), the cells were incubated with FB1 before loading with 2’,7’-dichlorofluorescein diacetate, and in short-term experiments after loading. The formation of the fluorescent compound, dichlorofluorescein, was monitored at an excitation wavelength of 485 nm (bandwidth 15 nm) and an emission wavelength of 535 nm (bandwidth 30 nm) using a Wallac Victor II Multilabel Counter. Measurement of intracellular glutathione levels. When studying changes in intracellular glutathione (GSH) levels the cells were incubated with FB1 on 48-well plates (Costar). The incubations were carried out in cell culture medium in +37 oC if the incubation time was 12 hours or more, and if it was shorter then the incubations were carried out in HBSS at room temperature. After the treatments, the cells were loaded with 40 µM monochlorobimane (MBCL) for 15 minutes in room temperature. The formation of a fluorescent GSH-MBCL-complex was monitored at an excitation wavelength of 355 nm (bandwidth 30 nm) and an emission wavelength of 460 nm (bandwidth 30 nm) using a Wallac Victor II Multilabel Counter. Measurement of cell viability. Cells were grown on 48 well plates (Costar) and incubated with FB1 for 0-144 hours. After the incubation, the cells were washed with HBSS and 50 µM propidium iodide was added. After 20 minutes the fluorescence was measured at an excitation wavelength of 530 nm (bandwidth 12 nm) and an emission wavelength of 590 nm (bandwidth 25 nm) using a Wallac Victor II Multilabel Counter. At the end 160 µM digitonin was added for 20 minutes to permeabilize the cells and to obtain 100 % cell death. The fluorescence at this point was measured using the same wavelengths as previously and the percent viability was counted as follows: V=100-[(F-blank1)/(Fmax-blank2)]x100%. V is percent viability, F is the fluorescence after incubating, Fmax is the fluorescence after permeabilizing of the cells, and blank1 is the baseline fluorescence after adding propidium iodide and blank2 is the baseline fluorescence after the addition of digitonin. Measurement of caspase-3 activity. The cells were cultured on 6-well plates (Costar) and incubated with fumonisin B1. After the incubation the cells were moved to eppendorf tubes and centrifuged. The caspase-3 activity measurement was carried out after incubating cells with a fluorogenic caspase-3 substrate (DEVD-AMC) on black 96-well plates (Nunc) for 60 minutes. The fluorescence was monitored at an excitation wavelength of 355 nm (bandwidth 30 nm) and an emission wavelength of 460 nm (bandwidth 30 nm) using a Wallac Victor II Multilabel Counter. Measurement of DNA-fragmentation (DNA ladder). The cells were cultured on 6-well plates (Costar) and incubated with FB1 for 0-144 hours. After the incubation the cells were suspended into lysis buffer and RNAse A and proteinase K were added. The samples were run on an agarose gel, stained by ethidium bromide. Measurement of DNA damage (Comet assay). The cells were cultured on 6-well plates (Costar) and incubated with FB1 for 0-72 hours. After incubation the cells were collected and counted and added to agarose coated slides. After treatment with lysis buffer the microgel slides were electrophoresed, neutralised, stained with ethidium bromide and visualised by fluorescence microscope. Measurement of p53 and MDM2*). The cells were cultured on petri dishes (Nunc) and incubated with FB1 for 72 hours. After incubation the cells were treated with protease inhibitors and the cytoplasmic and nuclear protein extracts were collected. The expression of p53 and MDM2 proteins was measured by Western blot. *) This study was carried out in collaboration with professor Kirsi Vähäkangas, Katerina Chvalova and Virpi Koponen at the Department of Pharmacology and Toxicology, University of Kuopio. 3 Results and discussion An increased production of reactive oxygen species was seen in SH-SY5Y cells exposed to FB1 for 0,5-5 hours. In U-118MG cells, a decreased production of reactive oxygen species, compared to control cells, was observed after 12-144 hours of exposure, and in C6 cells the same trend was observed after 144 hours of exposure. Intracellular glutathione levels decreased in U-118MG cells exposed to FB1 for 12-144 hours, in GT1-7 and C6 cells after 48-144 hours and in SH-SY5Y cells after 144 hours. Necrotic cell death was observed in all cells treated with FB1 for 144 hours. Caspase-3 activation, which is an indicator if apoptosis, took place in GT1-7 cells, which had been exposed to FB1 for 72 hours. DNA-fragmentation could be seen in U-118MG cells after 144 hours and in C6 cells after 24-144 hours of FB1 exposure. According to our preliminary results obtained by the Comet assay, FB1 does not seem to cause DNA damages in cells. Preliminary studies on the expression of the proteins p53 and MDM2 indicate that FB1 does not clearly affect these proteins. A slight decrease in the expression of MDM2 in GT1-7 cells, treated with FB1 for 72 hours, may occur. The results obtained in this study show that FB1 has clear effects on both neuronal and glial cells. At late time points (144 hours of FB1 exposure) the effects seem to be quite similar in different cell lines. Especially the human U-118MG glioblastoma cell line seems to be sensitive to FB1. The induction of the production of reactive oxygen species in SH-SY5Y cells at early time points compared to the decreased production in U-118MG and C6 cells at late time points can perhaps be explained by alterations in the expression of protecting genes. We will continue studying the mechanisms whereby FB1 affects neuronal and glial cells, by exploring of cytokines and chemokines at mRNA level with a quantitative PCR, Taqman, and expression of pro- and antiapoptotic proteins and transcription factors. 4 Conclusions FB1 does affect the production of reactive oxygen species in SH-SY5Y, U-118MG and C6 cells. FB1 lowers the levels of intracellular glutathione in U-118MG, C6 and GT1-7 cells and decreases cell viability in U-118MG, C6, SH-SY5Y and GT1-7 cells. FB1 exposure also leads to caspase-3 activation in GT1-7 cells and to DNA-fragmentation in U-118MG and C6 cells. The results obtained in the study can be used when assessing the health effects and health risks of mycotoxin producing moulds in buildings. The results can also be useful when evaluating the needs of protection towards mycotoxins and when planning how to avoid mycotoxin exposure.
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