Cytotoxicity Evaluation of Mycotoxins by an MTT-Bioassay

A colorimetric tetrazolium (MTT) cleavage test was modified and established as a bioassay for the cytotoxicity of mycotoxins. Using the human erythroleukemia cell line K562 and porcine white blood cells (lymphocytes and granulocytes) we evaluated the influence of deoxynivalenol, ochratoxin A, and zearalenone on cellular MTT cleavage activity. The yellow MTT is reduced by mitochondrial enzymes of metabolically active cells into a dark blue formazan product, the optical density (OD) of which can be measured by an ELISA reader. After an exposure time of 24hours, concentrations of deoxynivalenol and ochratoxin A as low as 0.4 \gmg/mL were found to inhibit significantly the cleavage activity in K562 cells. Cytotoxicity in lymphocytes and granulocytes was observed at concentrations of 0.8 up to 0.4 \gmg/mL for deoxynivalenol and 3.1 and 0.8 \gmg/mL for ochratoxin A, respectively. Zearalenone concentrations of 25.0 to 12.5 \gm/mL inhibited the mitochondrial cleavage activity of lymphocytes and of K562 cells significantly, whereas in granulocytes none of the concentrations tested was proved to be toxic. Morphological findings on the ultrastractural level showed that toxin incubation (28 hours) resulted in massive cell damage. Similar alterations were observed in about 15% of control cells. This indicates, that the massive cytotoxic effect of the mycotoxin deoxynivalenol is more likely to be an unspecific than a specific one. The modified MTT cleavage assay was found to be a quick (28 hours) and efficient colorimetric test for examining the cytotoxicity of three mycotoxins. The simplicity and speed of the procedure, which allows the simultaneous testing of various parameters and the possibility of objective data analysis could establish this test as an additional bioassay for the evaluation of cytotoxicity of mycotoxins.     

New insights into the mechanisms involved in renal proximal tubular damage induced in vitro by ochratoxin A

The mycotoxin ochratoxin A is a contaminant of human and animal food products. It is a potent nephrotoxin known to damage the proximal tubule. The aim of this work was to investigate the effects of ochratoxin A on a porcine renal proximal tubular epithelial cell line (LLC-PK1), and to identify sensitive endpoints revealing damage at the epithelial barrier level and at the molecular level. Cells exposed for 24 h to 5-10 microM ochratoxin indicated a clear damage to the intactness of the epithelial barrier, as shown by measurements of trans-epithelial resistance and zonula occludens-1 protein expression. At the mitochondrial level we observed alterations of the normal functions, such as an increase of the membrane potential, the formation of straight extensions, and the formation of giant mitochondria. At higher ochratoxin concentrations (50 microM), at which cytotoxicity assays revealed a significant toxicity, alterations of the cytoskeleton organization and induction of apoptosis were evident. In addition, we analyzed the expression of genes by using a cDNA macroarray. Our data indicate that ochratoxin-induced nephrotoxicity can be detected at the barrier and at the mitochondrial level at rather low concentrations, at which conventional cytotoxicity assays are unable to reveal toxic effects.     

Detoxification of Ochratoxin A on Heating under Acidic and Alkaline Conditions

Ochratoxin A was heated under three different moisture conditions, and under acidic and alkaline conditions. Heating to 175°C under the dry conditions produced little change in the molecule and cytotoxicity, detected by TLC and in the effects on the proliferation of HeLa cells. When heated under the moist and watery conditions, a small change in molecule was found by TLC, but cytotoxicity was not reduced. Under the acidic conditions (0.1 N HCl) the decomposition of ochratoxin A was detected by TLC, however the change in cytotoxicity was not observed in this assay system. On the other hand, heating with NaOH (0.1 N) resulted in the decomposition and detoxification of ochratoxin A. The HPTLC analysis showed the formation of some decomposed compounds including L-phenylalanine. This indicates the hydrolysis of ochratoxin A is one of the decomposition reactions induced by alkali at high temperatures.     

The cytotoxic effect of fumonisin B<Subscript>1</Subscript> and ochratoxin A on human and pig lymphocytes using the Methyl Thiazol Tetrazolium (MTT) assay

Lymphocytes cell obtained from healthy human donors and pigs were exposed to fumonisin B₁ (FB₁) and ochratoxin A (OTA), which have been found to be immunosuppressive, carcinogenic and mutagenic, to ascertain their single and combined cytotoxic effects with time and to assess the suitability of animal lymphocytes as test agents in comparison to human cells. The main objectives of this work were to assess the use of animal lymphocytes, particularly pig lymphocytes, for their use in the Methyl Thiazol Tetrazolium (MTT) cytotoxicity test, making them more accessible to animal research-based institutes in comparison to human lymphocytes previously used, and to study the cytotoxic and synergism or antagonistic effects of FB1 and OTA. The MTT assay, which measures cell viability and proliferation based on reduction of MTT to a blue dye, also used the addition of phytohaemagglutinin (PHA) to stimulate the blood cells. The results showed a progressive decrease in lymphocytes viability with time of exposure to the toxins. It was also noted that FB1, as compared to OTA, had a lower cytotoxicity on both human and pig lymphocytes cells. In addition, when the two mycotoxins were combined, a synergistic decrease of cell viability in both human and pig lymphocytes was observed, with pig lymphocytes showing a greater sensitivity. This study has shown that the MTT assay can be used for the determination of cytotoxicity of mycotoxins using animal, and in particular pig, lymphocytes, which eliminates the use of human donors and other cell cultures.     

Cytotoxicity of Nephrotoxic Fungal Toxins to Kidney-derived LLC-PK1 and OK Cell Lines

The nephrotoxic fungal toxins ochratoxin A (OA), ochratoxin B (OB) and citrinin (CIT) are natural contaminants of foods and feeds. While cytotoxicity assays have proven useful for establishing relative toxicity and structure–function relationships within groups of fungal toxins, a drawback of in vitro bioassays is their susceptibility to variation depending on endpoint, target cell, and dosing strategy. These variables were explored for OA, OB, CIT using two continuous kidney cell lines (LLC-PK₁ and OK) and four cytotoxicity assay endpoints. The nephrotoxic antibiotic gentamicin was used as a positive control for cytotoxicity throughout. In general, fungal toxin-induced cytotoxicity was more pronounced in LLC-PK₁ cultures using mitochondrial dehydrogenase inhibition (MTT assay) as the endpoint. Altered dosing strategy, but not seeding density, consistently influenced cytotoxicity: CIT was more toxic to cells when added at the time of seeding, whereas OA was more toxic when added 24 h after cultures were seeded. Toxicity rankings for the fungal toxins were consistent with in vivo studies and were, in order of most to least toxic, OA>OB>CIT. The data indicate that LLC-PK₁ and OK cells compare favorably to existing models in terms of sensitivity to nephrotoxic fungal toxins, but also that relatively minor changes in assay protocols can affect the cytotoxicity of individual toxins and comparative toxicity within a group of toxins.     

Monitoring of residues of ochratoxin A in blood and kidney of chicken using HPLC-FLD

Detoxification of ochratoxin A can be achieved by chemical or enzymatic hydrolyzation, the products of such reactions are ochratoxin α and phenylalanine. Ochratoxin α like ochratoxin A, is a fluorescing molecule, therefore sensitive analysis is possible at very low concentration levels. Methods have been established that make it possible to look for residues of ochratoxin A and its main metabolite ochratoxin α in blood and tissues at very low concentration levels. Plasma is extracted by the use of small amounts of chloroform; the extract is cleaned with water and afterwards evaporated to dryness]. The residue is re-dissolved and analysed by HPLC-FLD. Using this method a limit of detection of 0.5μg/l for both ochratoxin A and ochratoxin α can be reached.     

Increased cytotoxicity of food-borne mycotoxins toward human cell lines in vitro via enhanced cytochrome p450 expression using the MTT bioassay

Eight food-borne mycotoxins epidemiologically implicated in human disease were tested for their cytotoxic effects on human cells previously immortalised and transfected to introduce human cytochrome p450 (CYP 450) genes. Such cells retain many characteristics of normal cell growth and differentiation while simultaneously having the potential of either increasing or decreasing the metabolic activity (cytotoxicity) of the challenging mycotoxins. The MTT assay provided an indication of cytotoxicity. Of the nine CYP450s introduced CYP1A2 was most effective, rendering the cells 540 times more sensitive than the control cells to aflatoxin B1, 28 times more sensitive to aflatoxin G1 and 8-fold more sensitive to ochratoxin A. CYP3A4 resulted in the cells being 211 times more toxic to aflatoxin B1 and 8-fold more toxic to aflatoxin G1 while CYP 2A6, CYP 3A5 and CYP 2E1 also produced observable effects. No increase in metabolic activity was found using cyclopiazonic acid, deoxynivalenol, fumonisin B1, patulin or T-2 toxin. CD5Os were calculated for the mycotoxins against the non-CYP-introduced control cells. There was almost a five order of magnitude difference between the most toxic, T-2 toxin (CD50 0.0057 microgram/ml) and the least toxic, fumonisin B1 (CD50 476.2 micrograms/ml). In vitro biological assays thus provide an excellent system for quantifying the often low CD50s expressed by mycotoxins in foods.     

Ochratoxin A in pig blood: method of analysis and use as a tool for feed studies

A procedure is presented for screening the quality of feed in respect to ochratoxin A contamination based upon the analysis of ochratoxin A in pig blood. Representative samples from large feed lots may be obtained by using pigs as in vivo sample collectors which enrich the toxin and forms homogeneous samples in the blood. The spectrofluorometric procedure for ochratoxin A analysis (K. Hult and S. Gatenbeck, J. Assoc. Off. Anal. Chem. 59:128-129, 1976) has been adapted to pig blood and has been simplified to involve only three extraction steps. A volume of 2.5 ml of blood or plasma is needed, and the detection limit is 2 ng of ochratoxin A per ml. The disappearance of ochratoxin A from pig blood as a function of time has been studied. A feeding experiment with ochratoxin A has been performed, and the time course of the concentration of ochratoxin A in blood has been followed during the experiment.     

Ochratoxin A in pig blood: method of analysis and use as a tool for feed studies.

A procedure is presented for screening the quality of feed in respect to ochratoxin A contamination based upon the analysis of ochratoxin A in pig blood. Representative samples from large feed lots may be obtained by using pigs as in vivo sample collectors which enrich the toxin and forms homogeneous samples in the blood. The spectrofluorometric procedure for ochratoxin A analysis (K. Hult and S. Gatenbeck, J. Assoc. Off. Anal. Chem. 59:128-129, 1976) has been adapted to pig blood and has been simplified to involve only three extraction steps. A volume of 2.5 ml of blood or plasma is needed, and the detection limit is 2 ng of ochratoxin A per ml. The disappearance of ochratoxin A from pig blood as a function of time has been studied. A feeding experiment with ochratoxin A has been performed, and the time course of the concentration of ochratoxin A in blood has been followed during the experiment.     

Metabolism of ochratoxin B and its possible effects upon the metabolism and toxicity of ochratoxin A in rats

A metabolic product was formed from ochratoxin B by rat liver microsomal fractions in the presence of NADPH. It was isolated from the incubation mixture by extraction, thin-layer chromatography, high-pressure liquid chromatography, and crystallization. On the basis of mass and nuclear magnetic resonance spectroscopy, the structure is suggested to be 4-hydroxyochratoxin B. The Km for the formation of 4-hydroxyochratoxin B was determined, and the hydroxylation of ochratoxin A was not altered by the presence of ochratoxin B. Rats were given ochratoxin A or B, or a mixture of both intraperitoneally. The ratios of the three metabolites, ochratoxin A, (4R)-4-hydroxyochratoxin A, and ochratoxin alpha, excreted in the urine did not change in the presence of ochratoxin B. Ochratoxin B was metabolized to 4-hydroxyochratoxin B and ochratoxin beta, but in a different ratio than for the ochratoxin A metabolites. When given intraperitoneally, ochratoxin beta was excreted within 24 h. In rats treated with ochratoxin A alone, the food intake was reduced by 50%, and histologically severe lesions, degeneration, and necrosis were observed in the proximal tubules. When ochratoxin A and B given in combination, the animals were clinically unaffected and histologically there was only slight damage of proximal tubules. These observations indicate that ochratoxin B considerably reduces the toxic effects of ochratoxin A.     

Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes

The mechanism of human peripheral blood monocyte-mediated cytotoxicity for tumor cells was investigated, using the A673 human rhabdomyosarcoma and HT-29 human colon adenocarcinoma lines as target cells. A673 cells were shown to be susceptible to the cytotoxic action of purified recombinant human tumor necrosis factor (TNF). A673 cells were also highly sensitive to the cytotoxic action of peripheral blood monocytes. Clones of A673 cells sensitive and resistant to TNF were isolated and characterized for their sensitivity to monocyte killing. A good correlation was found between the sensitivity of these clones to the cytotoxicity of TNF and their susceptibility to killing by monocytes. A TNF-specific neutralizing monoclonal antibody (MAb) reduced monocyte killing of parental A673 cells and of a TNF-sensitive clone of A673 cells. Inhibition of monocyte killing by this MAb was particularly pronounced at a low effector to target cell ratio. HT-29 cells were relatively resistant to the cytotoxic action of recombinant TNF and to monocyte killing. Treatment of HT-29 cells with recombinant human IFN-gamma increased their susceptibility to both TNF cytotoxicity and monocyte killing. In addition, MAb to TNF inhibited monocyte killing in HT-29 cells sensitized by incubation with IFN-gamma. Our data show that TNF is an important mediator of the cytotoxicity of human monocytes for tumor cells and that IFN-gamma can increase monocyte cytotoxicity by sensitizing target cells to the lytic action of TNF.     

Detection of a specific differentiation antigen of mouse T-cells activated by allogenic transplantation antigens

Xenogeneic antibody was obtained, which displayed cytotoxicity for T lymphocytes in mice belonging to varied inbred strains activated with allogeneic cells. This antibody was not cytotoxic for nonactivated T or B lymphocytes of intact mice. Absorption of antiserum with activated rather than with intact lymphocytes diminished its cytotoxicity. Both activated and nonactivated lymphocytes were found to be equally susceptible to the cytotoxic effect of antilymphocytic or antithymocytic sera. A conclusion is made that murine T lymphocytes activated with allogeneic transplantation antigen carry specific differentiation antigen.     

Metabolism of ochratoxin B and its possible effects upon the metabolism and toxicity of ochratoxin A in rats.

A metabolic product was formed from ochratoxin B by rat liver microsomal fractions in the presence of NADPH. It was isolated from the incubation mixture by extraction, thin-layer chromatography, high-pressure liquid chromatography, and crystallization. On the basis of mass and nuclear magnetic resonance spectroscopy, the structure is suggested to be 4-hydroxyochratoxin B. The Km for the formation of 4-hydroxyochratoxin B was determined, and the hydroxylation of ochratoxin A was not altered by the presence of ochratoxin B. Rats were given ochratoxin A or B, or a mixture of both intraperitoneally. The ratios of the three metabolites, ochratoxin A, (4R)-4-hydroxyochratoxin A, and ochratoxin alpha, excreted in the urine did not change in the presence of ochratoxin B. Ochratoxin B was metabolized to 4-hydroxyochratoxin B and ochratoxin beta, but in a different ratio than for the ochratoxin A metabolites. When given intraperitoneally, ochratoxin beta was excreted within 24 h. In rats treated with ochratoxin A alone, the food intake was reduced by 50%, and histologically severe lesions, degeneration, and necrosis were observed in the proximal tubules. When ochratoxin A and B given in combination, the animals were clinically unaffected and histologically there was only slight damage of proximal tubules. These observations indicate that ochratoxin B considerably reduces the toxic effects of ochratoxin A.     

Production of Ochratoxins in Different Cereal Products by Aspergillus ochraceus

The effects of temperature and length of incubation on ochratoxin A production in various substrates were studied. The optimal temperature for toxin production by Aspergillus ochraceus NRRL-3174 was found to be around 28 C. Very low levels of ochratoxin A are produced in corn, rice, and wheat bran at 4 C. The optimal time for ochratoxin A production depends on the substrate, ranging from 7 to 14 days at 28 C. Ochratoxin B and dihydroisocoumaric acid, i.e., one of the hydrolysis products of ochratoxin A, were produced in rice but at levels considerably lower than ochratoxin A. No ochratoxin C was produced in rice at 28 C. When added to rice cereal or oatmeal, the toxin was found to be very stable over prolonged storage and even to autoclaving for 3 hr.     

Entwicklung eines hochempfindlichen Enzymimmuntests zum Nachweis von Ochratoxin A

Antibodies against ochratoxin A were produced in rabbits after immunization with an ochratoxin A-keyhole limpet hemocyanine conjugate. The immunogen was found to be very efficient, and high antibody titers were detected in the sera of all immunized rabbits. In a competitive enzyme immunoassay using ochratoxin A-horseradish peroxidase as the labelled antigen, low levels of ochratoxin A in buffer solution could be detected. The mean standard curve detection limit and 50% inhibition level of the optimized assay were at 15 pg/ml and 50 pg/ml, respectively. Relative cross-reactivity of ochratoxin B was found to be 2%. With these characteristics, this novel enzyme immunoassay should be useful for the routine detection of ochratoxin A in food and in biological samples at levels well below 1 ng/g.     

Tumor necrosis factor production by human monocytes is a regulated event: induction of TNF-alpha-mediated cellular cytotoxicity by endotoxin

Expression of cellular cytotoxicity by monocytes or macrophages has been conceived as an induced function secondary to collaboration in the immune response or to other agonists. However, a form of spontaneous cellular cytotoxicity by monocytes analyzed with unseparated human peripheral blood mononuclear cells (PBM) has been described by using the 6-hr 51Cr release from actinomycin D (ActD)-treated murine WEHI 164 cells, a target cell refractory to the cytotoxic effects of natural killer and cytolytic T cells. We observe that when cells are isolated under rigorously endotoxin-free conditions, there is no cytotoxicity. Inclusion of serum does not induce cellular cytotoxicity; however, cytotoxic activity is induced by the presence of as little as 1 pg/ml of bacterial lipopolysaccharide (LPS). PBM required 2 hr of preexposure to endotoxin in order to express full cytotoxic activity. We investigated the basis of the cytotoxicity of WEHI 164 cells and the effect of ActD. ActD-treated target cells are highly susceptible to the effects of TNF-alpha and TNF-beta (alpha-lymphotoxin), whereas untreated target cells were resistant. In contrast, ActD does not affect susceptibility to the cytotoxic effects of H2O2, and interleukin 1 is not cytotoxic to the target cells. With the use of a neutralizing monoclonal antibody specific for TNF-alpha, the cytotoxic activity induced by LPS greatly diminished and the amount of TNF-alpha neutralized is similar to that required for equivalent cytotoxicity. We conclude that monocytes present in human PBM are not "spontaneously" cytotoxic for ActD-treated WEHI 164 target cells, but that the reported cytotoxicity results from exposure to a level of endotoxin or endotoxin-like agonists to which the cells are exposed. The cytotoxicity is mediated mostly if not entirely by TNF-alpha, an established product of monocytes/macrophages. With the use of endotoxin-free conditions, PBM can be isolated in a cytotoxically latent state, suitable for analysis of the immunologic regulation of TNF-alpha-mediated monocyte cellular cytotoxicity.     

Influence of light on ochratoxin biosynthesis by <Emphasis Type="Italic">Penicillium</Emphasis>

Light has a profound influence on ochratoxin biosynthesis by Penicillia. When incubated under constant daylight of a certain intensity, ochratoxin A biosynthesis is decreased by about 20–30% compared to incubation under constant darkness. Under day/night oscillation, the ochratoxin A polyketide synthase gene, a key gene of the ochratoxin A biosynthesis pathway, is rhythmically expressed, and moreover, the amount of ochratoxin also oscillates between the amounts produced either during constant darkness or during constant light. This indicates a partial degradation of ochratoxin A (20–30%) under light conditions until a certain lower limit is reached. This behavior is dependent on the light intensity. At 1,600 Lux, only weak effects could be observed; however, at 2,800 Lux, the effects became significant. After growth under constant light conditions, Penicillium produced ochratoxin B at amounts which are 5 times higher than after growth in constant dark or in alternating light/dark conditions. Growth experiments in the dark on medium with increasing amounts of ochratoxin A revealed that externally applied ochratoxin is moderately toxic. However, if the same growth experiments are carried out under light conditions, the growth inhibiting activity of ochratoxin A is greatly increased, indicating that light amplifies the toxic activity of ochratoxin. Because of the oscillation of the concentration of ochratoxin A during night and day incubation, Penicillium seems to have developed an adaptive mechanism to reduce the amount of ochratoxin A during daylight below a toxic level.     

Conversion of ochratoxin C into ochratoxin A in vivo.

The conversion of ochratoxin C to ochratoxin A was studied in rats after oral and intravenous administration. The concentration of ochratoxin A in the blood as a function of time was the same after oral administration of equivalent amounts of either ochratoxin C or ochratoxin A. The maximum ochratoxin A concentrations were measured 60 min after administration. Given intravenously, ochratoxin C was also converted to ochratoxin A. Maximum concentrations were reached after 90 min. It is concluded that ochratoxin C is readily converted to ochratoxin A after both oral and intravenous administration. There is reason to believe that a comparable toxicity of the two toxins is based upon this conversion and that only interference with the biotransformation mechanisms may cause a difference in their toxicity.     

Different phenotypic variants of the mouse B cell tumor A20/2J are selected by antigen- and mitogen-triggered cytotoxicity of L3T4-positive, I-A-restricted T cell clones

The L3T4+, Lyt-2-, cloned BALB/c T cell lines 5.9.24 and 5.8.6 are cytotoxic for the BALB/c B cell tumor line A20/2J. The T cell cytotoxicity against A20/2J cells could be triggered either by the specific antigen ovalbumin (OVA), which is recognized by the T cell clones in association with I-Ad determinants, or by the T cell mitogens Con A and rabbit anti-mouse brain (RaMBr) antiserum. Repeated exposure of A20/2J cells to 5.9.24 and 5.8.6 T cell cytotoxicity selected variant cell lines that had developed resistance to cytotoxicity. The variant lines could be classified into four different variant phenotypes of which three were stably maintained in vitro. The type of variant obtained appeared to be related to the nature of the ligand used to trigger T cell cytotoxicity during selection. Cytotoxicity triggered by the antigen OVA generated type 1 variants that expressed abnormally low levels of I-Ad determinants at the cell surface. Type 1 variants were resistant to OVA-triggered 5.9.24 T cell cytotoxicity, but were fully susceptible to cytotoxicity triggered by Con A or RaMBr antiserum. RaMBr-triggered cytotoxicity generated two unique types of variant cell lines: type 3 variants that were deficient in cell surface Fc receptors and resistant to 5.9.24 cytotoxicity only when triggered by RaMBr antiserum, and type 4 variants that were resistant to cytotoxicity triggered by all three ligands. One type 4 variant, the IC-1 cell line, appeared to be resistant to soluble cytotoxic factors released by 5.9.24 T cells after activation by antigen. All of these variant lines retained sensitivity to cytotoxicity by classic Lyt-2+ cytotoxic T lymphocytes (CTL), a finding that indicates that L3T4a+ T cells and Lyt-2+ CTL use different molecules to attack their target cells. The variant phenotypes were inherited by clones derived from the original cell lines. Because the variants were generated without mutagenesis, they are thought to have been derived by the immunoselection of pre-existing variant cells that arose spontaneously in the parental A20/2J cell line. It is postulated that inheritable variation of A20/2J cells may represent changes that normally occur during B cell differentiation in response to T cell signals. The variant A20/2J cell lines described here provide material for the investigation of B cell surface structures that may regulate T-B cell interactions.