Modulation der zytotoxischen Aktivität von humanen natürlichen Killerzellen durch Ochratoxin A und C

The effect of ochratoxin A (OTA) and C (OTC) on the cytolytic activity of natural killer cells (NK cells) was studied using a fluorescence based bioassay with the cell line NK-92 as effector cell line and K-562 as target cell line. Different OTA and OTC preparations were included in the study. After exposure to very low toxin concentrations (1–10 ng/ml) a slight stimulation of NK cell activity was noted, whereas after addition of higher toxin concentrations (100–1000 ng/ml) NK cell function was suppressed. Preparations containing OTC showed a stronger effect than those containing OTA.     

Adsorption of ochratoxin A and zearalenone by potential probiotic Saccharomyces cerevisiae strains and its relation with cell wall thickness

Aims: To examine Saccharomyces cerevisae strains with previously reported beneficial properties and aflatoxin B₁ binding capacity, for their ability to remove ochratoxin A (OTA) and zearalenone (ZEA) and to study the relation between cell wall thickness and detoxificant ability of yeast strains. Methods and Results: A mycotoxin binding assay at different toxin concentrations and the effect of gastrointestinal conditions on mycotoxin binding were evaluated. Ultrastructural studies of yeast cells were carried out with transmission electronic microscopy. All tested strains were capable of removing OTA and ZEA. Saccharomyces cerevisiae RC012 and RC016 showed the highest OTA removal percentage, whereas RC009 and RC012 strains showed the highest ZEA removal percentages. The cell diameter/cell wall thickness relation showed a correlation between cell wall amount and mycotoxin removal ability. After exposure to gastrointestinal conditions, a significant increase in mycotoxin binding was observed. Conclusions: All tested Saccharomyces cerevisiae strains were able to remove OTA and ZEA, and physical adsorption would be the main mechanism involved in ochratoxin A and ZEA removal. Gastrointestinal conditions would enhance adsorption and not decrease mycotoxin–adsorbent interactions. Significance and Impact of the Study: Live strains with mycotoxin binding ability and beneficial properties are potential probiotics that could be included in animal feed. Previous and present results suggest that the RC008 and RC016 strains are very promising candidates for functional feed product development.     

Effects of a long-term exposure with OTA or OTC on functions of a human monocytic cell line

An in-vitro model was developed to study the effects of a long-term exposure with a low concentration of ochratoxin A (OTA) or ochratoxin C (OTC) on a human monocytic cell line (THP-1). Cells were propagated in 24-well cell culture plates for 15 days. OTA and OTC preparations, respectively, at a concentration of 1 ng/ml were included in the cell culture medium during the whole cultivation period. At the end of the exposure time, parameters of cell viability and cell function were examined. After 15 days of exposure to ochratoxins, viability and function of the THP-1 cell line were modulated. Mitochondrial activity and the production of IL-6 were increased by all mycotoxin preparations. Cell membrane integrity was disturbed, proliferation and the production of TNF-α and IL-8 were inhibited. These parameters were most severely affected by mycotoxin preparations containing OTC. Our results show that long term exposure to OTA and especially OTC in low concentrations can cause subtle alterations of cell viability and function which may have remarkable consequences for human and animal health. In this context it seems to be necessary to study the contamination of food and feed stuffs with OTC more intensively. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Allium cepa as a biomonitor of ochratoxin A toxicity and genotoxicity

Ochratoxin A (OTA) is a toxin produced by Aspergillus and Penicillum moulds. Since OTA has not yet been evaluated in plant systems, this paper focused on describing the controversial effect OTA in an Allium root test model, which has known sensitivity to genotoxins and could be useful in toxin screening. Analyses of root growth and the root meristematic zone in response to OTA treatment were undertaken. The results show OTA toxicity to root growth at a concentration of 10 ug·ml^?1 associated with inhibition of proliferation activity. Cytological changes observed in the Allium chromosome aberrations assay, at a concentration of 5.0 ug·ml^?1, showed that OTA was able to induce genotoxicity at the chromosome level. These results indicate that plants cells (Allium cepa) are very sensitive to the mycotoxin OTA, as observed at the highest concentration. Under these conditions, OTA produced toxicity and cytogenetic injury. Evidence in vitro and in vivo indicates that OTA can induce damage at the DNA level.     

Beeinflussung der Zytokinsekretion von Maus-Thymom-Zellen (EL-4) durch Ochratoxin A

The murine thymoma cell line EL-4 was used as an in vitro T-cell model to assess the immunomodulating effect of pure Ochratoxin A (OTA) and an Aspergillus ochraceus raw toxin preparation. Cytokine production (IL-2, IL-4, IL-5, IL-6) and cell viability of PMA-stimulated EL-4 cells were investigated in the presence of OTA. The cytotoxic effect of the raw toxin could be observed at lower concentrations than pure OTA. The IC50 values were 3 µg/ml and 11 µg/ml, respectively. Increasing concentrations of both OTA preparations caused an inhibition of cytokine production, but the inhibition effect of the raw toxin was stronger than of pure OTA. This is supposed to be the effect of further up to now not characterized substances in the raw toxin. Differences in the susceptibility of the mechanisms of production and regulation of each cytokine are indicated by the different concentrations for inhibition effects. Both toxin preparations showed also stimulating effects on some cytokines (IL-2 and IL-6) while others (IL-4 and IL-5) were depressed at these OTA concentrations. This indicates the immunomodulating properties of the toxin.     

Characterization of ochratoxin A-induced apoptosis in primary rat hepatocytes

The main target organ of the mycotoxin ochratoxin A (OTA) in mammals is the kidney but OTA has also been shown to be hepatotoxic in rats and to induce tumors in mouse liver. Even at very low concentrations, OTA causes perturbations of cellular signaling pathways as well as enhanced apoptosis. OTA has been extensively studied in kidney cell systems. Since this substance also affects liver health, we focused our work on apoptosis-related events induced by OTA in primary rat hepatocytes. We performed pathway-specific polymerase chain reaction arrays to assess the expression of genes involved in apoptosis. Treatment with 1 μM OTA for 24 h caused marked changes in apoptosis-related gene expression. Genes as apaf1, bad, caspase 7, polb (DNA polymerase beta, performs base excision repair), and p53, which are marker genes for DNA damage, were upregulated. FAS and faslg were also markedly induced by treatment with OTA. Treatment of hepatocytes with OTA led to a concentration-dependent inhibition of protein biosynthesis. Apoptosis-inducing factor was released from mitochondria following OTA treatment; the mycotoxin induced the activity of caspases 8, 9, and 3/7 and caused chromatin condensation and fragmentation. Caspase inhibition led to a significant but not complete reduction of OTA-induced apoptosis. Our data suggest that not only OTA leads to p53-dependent apoptosis in rat hepatocytes but it also hints to other mechanisms, independent of caspase activation or protein biosynthesis, being involved.     

The nephrotoxin ochratoxin A induces apoptosis in cultured human proximal tubule cells

To test the apoptotic potential of the nephrotoxic mycotoxin ochratoxin A (OTA), we exposed human proximal tubule-derived cells (IHKE cells) for various times to OTA concentrations close to those occurring during dietary exposure (from 2 to 100 nmol/L) and investigated caspase 3 activation, chromatin condensation, and DNA fragmentation. OTA induced a time- and concentration-dependent activation of caspase 3: concentrations as low as 5 nmol/L OTA caused a slight but significant increase in caspase 3 activity after 7 days of OTA exposure. Exposure to 10 nmol/L OTA for 72 or 24 h led to a significantly increased activity of caspase 3 in human proximal tubule-derived cells. Radical scavengers such as N-acetylcysteine had no effect on OTA-induced caspase 3 activation. Chelation of intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethylester) (BAPTA-AM) also showed no effect. Exposure to 30 nmol/L or more OTA led to DNA fragmentation and chromatin condensation in IHKE cells. Cultured renal epithelial MDCK-C7 and MDCK-C11 or OK cells also showed increased caspase 3 activity after OTA exposure. We conclude that exposure to low OTA concentrations can lead to direct or indirect caspase 3 activation and subsequently to apoptosis in cultured human proximal tubule cells and in other renal epithelial cell lines of different origins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Modulation of ochratoxin A induced DNA-damage in urothelial cell cultures

Despite good evidence for a genotoxic potential of ochratoxin A (OTA), the mechanism of OTA-induced genotoxicity (direct or indirect?) is still unclear. This calls for a further characterization of OTA-related DNA damage, and investigations of factors that may modulate dose-effect relationships in cells. Since bladder epithelium is a target tissue for the toxicity of OTA, its effects were studied in cultures of human bladder carcinoma (H5637) cells. Cytotoxicity of OTA, assessed by Neutral red (NR) uptake or Alamar-Blue assay, is concentration- and time-dependent: Upon 24 h treatment of 5637 cells, NR uptake is reduced by 50% with OTA concentrations of ≥0.2 microM, but not with 3 h treatment of the cells. Since cytotoxicity of OTA was not affected by addition of xenobiotic metabolizing enzymes (S-9 mix), it appears to be unrelated to biotransformation of the mycotoxin. Also, addition of S-9 mix did not significantly affect the genotoxicity of OTA as studied by alkaline single cell gel electrophoresis (Comet assay). DNA damage was detectable after 3 h treatment of cells at OTA concentrations between 0.1 and 1 microM, and increased further at higher concentrations. The magnitude of OTA-induced DNA damage did not increase with longer treatment times (18, 24 h), probably due to repair processes in the cells. Repair of OTA-induced lesions is quite efficient in kidney (Arch Toxicol 2002, 75, 734–741) and in porcine bladder cells (Föllmann and Lebrun, 2005, Mycotoxin Research, this volume). Interestingly, the genotoxicity of OTA is modulated by the pH of the culture medium, with higher damage at pH 5 compared to pH 7.5. In line with this, uptake studies with tritiated OTA show a higher cellular accumulation of the mycotoxin at pH 5 than in buffer of pH 7.5. Thus, bladder cells exposed to OTA in slightly acidic urine (which facilitates reabsorption) may be at higher risk.     

Toxicokinetics and toxicodynamics of ochratoxin A, an update

Ochratoxin A (OTA) is a mycotoxin produced by fungi of two genera: Penicillium and Aspergillus. OTA has been shown to be nephrotoxic, hepatotoxic, teratogenic and immunotoxic to several species of animals and to cause kidney and liver tumours in mice and rats. Because of differences in the physiology of animal species, wide variations are seen in the toxicokinetic patterns of absorption, distribution and elimination of the toxin. Biotransformation of OTA has not been entirely elucidated. At present, data regarding OTA metabolism are controversial. Several metabolites have been characterized in vitro and/or in vivo, whereas other metabolites remain to be characterized. Several major mechanisms have been shown as involved in the toxicity of OTA: inhibition of protein synthesis, promotion of membrane peroxidation, disruption of calcium homeostasis, inhibition of mitochondrial respiration and DNA damage. The contribution of metabolites in OTA genotoxicity and carcinogenicity is still unclear. The genotoxic status of OTA is still controversial because contradictory results were obtained in various microbial and mammalian tests, notably regarding the formation of DNA adducts. More recent studies are focused on the OTA ability to disturb cellular signalling and regulation, to modulate physiological signals and thereby to influence cells viability and proliferation. The present paper offers an update on these different issues. In addition since humans and animals are likely to be simultaneously exposed to several mycotoxins, especially through their diet, the little information available on the combined effects of OTA and other mycotoxins has also been reviewed.     

The effects of the Penicillium mycotoxins citrinin, cyclopiazonic acid, ochratoxin A, patulin, penicillic acid, and roquefortine C on in vitro proliferation of porcine lymphocytes

The in vitro effect of each of the Penicillium mycotoxins citrinin (CIT), cyclopiazonic acid (CPA), ochratoxin A (OTA), patulin (PAT), penicillic acid (PIA) and roquefortine C (RQC) on mitogen induced lymphocyte proliferation was determined using purified lymphocytes from 6 piglets. Dose response curves for each mycotoxin were generated and the concentrations producing 50% inhibition of cell proliferation (IC₅₀) were estimated. OTA and PAT were the most potent toxins with IC₅₀ of 1.3 and 1.2 μmol/l, respectively (0.52 and 0.18 mg/l, respectively). Based on molar concentrations, OTA was 15, 30, 40, and 65 times more potent as an inhibitor than PIA, CIT, CPA and RQC, respectively.     

Epithelial-fibroblast cross talk aggravates the impact of the nephrotoxin ochratoxin A

BackgroundChronic nephropathies result from different pathogenic agents, including nutritional factors triggering vicious pathophysiological cycles. Ochratoxin A (OTA) is a globally occurring nephrotoxic mycotoxin detectable in a variety of foodstuff and suspected to cause tubulointerstitial damage. The underlying mechanisms are not sufficiently understood, compromising risk assessment. Because crosstalk of proximal tubule cells with fibroblasts is crucial for tubulointerstitial damage, we investigated the effects of OTA in co-culture of these two cell types.MethodsRat renal proximal tubule cells (NRK-52E) and renal fibroblasts (NRK-49F) were exposed to nanomolar OTA concentrations under mono- and/or co-culture conditions for up to 48 h. We determined the impact on inflammation-, EMT- and fibrosis-associated proteins as well as microRNAs by western blot or qPCR, respectively. Alterations in cell morphology were quantitatively assessed. The roles of miRs, COX-2 and ERK1/2 in OTA-induced effects were investigated by specific inhibition.FindingsOnly under co-culture condition, OTA caused an increase of vimentin, fibronectin and miR-21 and a decrease of collagen III, E-cadherin, COX-2 and WISP1 mRNA abundance in NRK-52E cells. In NRK-49F cells, OTA induced an increase of N-cadherin, COX-2, WISP1 in co-culture only. The OTA-induced increase of fibronectin in NRK-52E cells was prevented by simultaneous inhibition of miR-21 and -200a, COX-2 or ERK1/2. The OTA-induced increase of COX-2 in NRK-49F cells was prevented by inhibition of miR-21 and -200a or ERK1/2.InterpretationOur results show that the complete nephropathic potential of nanomolar OTA, leading to EMT, is unveiled when cellular crosstalk is possible. In monoculture, the nephropathic potential is underestimated.Research in contextChronic nephropathies are a severe health burden and the result of different pathogenic mechanisms, including nutritional factors that trigger vicious pathophysiological cycles. Ochratoxin A (OTA) is a ubiquitous, globally occurring nephrotoxic mycotoxin detectable in a variety of foodstuff and suspected to cause tubulointerstitial damage. Because underlying pathomechanisms are unclear, risk assessment is problematic. Crosstalk of proximal tubule cells (the main target of OTA) with fibroblasts is crucial for the development of tubulointerstitial damage. We show that during co-culture of proximal tubule cells and fibroblasts, OTA-induced effects (e.g. epithelial-mesenchymal transition (EMT)) change significantly as compared to monoculture. Our results show that the complete nephropathic potential of OTA is unveiled when cellular crosstalk is possible. In monoculture, the nephropathic potential of OTA is underestimated.     

Differences in neurotoxic effects of ochratoxin A, ochracin and ochratoxin-alpha in vitro.

The mycotoxin ochratoxin A (OTA) is a chlorinated dihydroisocoumarin derivative connected through an amide-bond to L-phenylalanine. In a previous study we could show that competition with L-phenylalanine-dependent processes does not play a role in OTA neurotoxicity. To test whether the isocoumarin part is responsible for the neurotoxic effects, we determined in the present study the effects of the hydrolysis product of OTA, ochratoxin-alpha (OTalpha), and of ochracin on embryonic chick brain cell cultures. In addition, we investigated the interaction between OTA and ochracin regarding the neurotoxic effects. We report here that OTalpha did not affect brain cell cultures at concentrations up to 15 microM. With the exception of a small (20%) but significant reduction in cell culture, cellular protein at concentrations above 0.3 microM, in our cell cultures' cell function, as defined by neutral red uptake and MTT-dehydrogenase activity, was only reduced by high OTalpha concentrations (1 mM). Addition of 0.1 microM OTA increased ochracin cytotoxicity as defined by latter parameters. No effects on cell culture NF68kD content could be detected. The results are discussed with regard to the existence of an OTA target interaction binding site.     

Combinatory effects of citrinin and ochratoxin A in immortalized human proximal tubule cells

Ochratoxin A (OTA) and citrinin (CIT) are two mycotoxins often occurring together in grains and cereals. Although both are nephrotoxic and can induce apoptosis, combination effects have not been examined up to now. Therefore, the aim of this study was to take a close look at the interactions of citrinin and OTA in cultured human proximal tubule-derived cells (IHKE cells). The cytotoxicity of both mycotoxins was studied, measuring the metabolic activity and the cell number. Furthermore, caspase 3-activation as a marker for apoptosis was examined for both mycotoxin alone and in combination. The results show that citrinin had an antagonistic effect on ochratoxin A induced caspase 3-activation in concentrations of 2.5 and 5 μmol/l. Higher concentrations (7.5 and 15 μmol/l) lead to additive effects, lower citrinin concentrations (0.25 and 1 μmol/l) did not show any effect at all. The observed decrease in caspase 3-activity was specific for the combination with OTA, since the combination of citrinin with cisplatin did not show any effect. Citrinin did not influence of the OTA-induced apoptosis when added two hours after applying ochratoxin A. Also the combination of both toxins decreased the uptake of OTA into the cells which might be an explanation for the antagonistic effect of citrinin in certain concentrations. However, the transport into cells can not be the only explanation. so further examinations are necessary. Presented at the 27^th Mykotoxin-Workshop. Dortmund, Germany, June 13–15, 2005.     

The effect of high and low doses of ochratoxin A in feed on growing chicken

Effects of high and low dose of ochratoxin A (OTA) as pure toxin supplemented to feed were investigated on the performance of growing chicken. Two groups were fed with different doses of chemical pure OTA 0.5 ppm and 5 ppm in feed and the effects of toxin on body and organ weights were studied and compared with control group. No effects were observed by feeding 0.5 ppm OTA, whereas 5 ppm had a negative effect on the daily body mass gain. OTA in feed had a negative effect on the daily body mass gain. In contrary nephrotoxic effects could be observed by feeding the naturally OTA contaminated feed with only 0.2 ppm of OTA produced byPenicillium verrucosum.     

Simultaneous analysis of ochratoxin A and its major metabolite ochratoxin alpha in plasma and urine for an advanced biomonitoring of the mycotoxin

Ochratoxin A (OTA) is a frequent mycotoxin contaminant found worldwide in foods and feedstuffs. Biomonitoring has been used to assess internal OTA exposure resulting from dietary intake and from other sources. Mycotoxin levels in blood and/or urine provide good estimates of past and recent exposure since OTA binds to serum proteins and is also partly excreted via the kidney. But, measuring OTA alone does not reflect its biotransformation. In light of scarce data on its metabolites in humans, it was the aim of this study to develop a method that allows analysis of OTA and its detoxication product ochratoxin alpha (OTα) in urine and in blood plasma. The method involves enzymatic hydrolysis of conjugates, liquid–liquid extraction, and analysis of sample extracts by liquid chromatography with fluorescence detection. Application of the validated method in a pilot study with 13 volunteers revealed the presence of OTA and OTα in all samples (limit of quantification: 0.05 ng/mL in urine, and 0.1 ng/mL in plasma). In line with negative findings of others, an OTA glucuronide was not detected, neither in urine nor in plasma. By contrast, conjugates of OTα (glucuronide and/or sulfate) are major products in these samples. This was confirmed by mass spectrometry detection. As OTα represents a large fraction of ingested mycotoxin, we propose to include analyses of this metabolite in future biomonitoring studies, also in light of the observed variations for urine OTα-levels that suggest different interindividual abilities for OTA-detoxification in humans.     

The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells

Balkan endemic nephropathy (BEN), a disease characterized by progressive renal fibrosis in human patients, has been associated with exposure to ochratoxin A (OTA). This mycotoxin is a frequent contaminant of human and animal food products, and is toxic to all animal species tested. OTA predominantly affects the kidney and is known to accumulate in the proximal tubule (PT). The induction of oxidative stress is implicated in the toxicity of this mycotoxin.In the present study, primary rat PT cells and LLC-PK(1) cells, which express characteristics of the PT, were used to investigate the OTA-mediated oxidative stress response. OTA exposure of these cells resulted in a concentration-dependent elevation of reactive oxygen species (ROS) levels, depletion of cellular glutathione (GSH) levels and an increase in the formation of 8-oxoguanine.The OTA-induced ROS response was significantly reduced following treatment with alpha-tocopherol (TOCO). However, this chain-braking anti-oxidant did not reduce the cytotoxicity of OTA and was unable to prevent the depletion of total GSH levels in OTA-exposed cells. In contrast, pre-incubation of the cell with N-acetyl-L-cysteine (NAC) completely prevented the OTA-induced increase in ROS levels as well as the formation of 8-oxoguanine and completely protected against the cytotoxicity of OTA. In addition, NAC treatment also limited the GSH depletion in OTA-exposed PT- and LLC-PK(1) cells.From these data, we conclude that oxidative stress contributes to the tubular toxicity of OTA. Subsequently, cellular GSH levels play a pivotal role in limiting the short-term toxicity of this mycotoxin in renal tubular cells.     

Biomonitoring of ochratoxin A in grain workers

Handling agricultural commodities such as grain can result in an inhalation of mycotoxin-containing dusts. Ochratoxin A (OTA) is particularly well suited for biomonitoring studies due to its long half-life in blood, and served as a marker toxin to investigate whether or not exposure to dusts in occupational contexts may result in elevated OTA blood serum levels. OTA analysis was performed for blood samples (n=61) obtained from a cohort of male workers employed at granaries of several grain handling companies in Germany. OTA was analyzed in plasma extracts by HPLC with fluorimetric detection; calibration curves were run for each batch of samples collected between July 2005 and March 2006, and the level of detection was 0.05 ng/ml plasma. The OTA plasma levels of the 61 grain workers ranged between 0.07 ng/ml and 0.75 ng/ml. The mean (0.28±0.13 ng/ml) and median (0.26 ng/ml) OTA value for this cohort was similar to average values previously reported for the German population. Our results gave no indication that OTA in excess of those originating from typical dietary sources was ingested by these workers. Although measurable OTA concentrations have been found in dust samples collected at the corresponding workplaces (Mayeret al, this issue), the biomonitoring data do not provide evidence for a significant inhalatory burden of OTA in grain workers. Since deoxynivalenol and zearalenone were also detected in the dust samples in concentrations much higher than that of OTA, additional research should try to assess the potential relevance of an inhalation exposure to these mycotoxins. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Guttation droplets of <Emphasis Type="Italic">Penicillium nordicum</Emphasis> and <Emphasis Type="Italic">Penicillium verrucosum</Emphasis> contain high concentrations of the mycotoxins ochratoxin A and B

Eight of eleven ochratoxigenic isolates of Penicillium nordicum and Penicillium verrucosum produced guttation droplets when grown on Czapek yeast extract (CYA) agar for 10–14 days at 25°C. Parallel cultivation of one strain each of P. nordicum and P. verrucosum on malt extract agar demonstrated that higher volumes of exudate are produced on this agar. However, HPLC analyses revealed higher concentrations of ochratoxin A (OTA) and B (OTB) in droplets originating from cultures on CYA. For quantitative determination of the mycotoxin contents, triplicates of three isolates each of P. nordicum and P. verrucosum were grown as single spot cultures on CYA for up to 14 days at 25°C. Guttation droplets were carefully collected between day 11 and 14 with a microliter syringe from each culture. Extracts from exudates and corresponding mycelia as well as fungal free agar were analyzed by HPLC for the occurrence of ochratoxin A (OTA) and B (OTB). Mean concentrations ranging between 92.7–8667.0 ng OTA and 159.7–2943.3 ng OTB per ml were detected in the guttation fluids. Considerably lower toxin levels were found in corresponding samples of the underlying mycelia (9.0–819.3 ng OTA and 4.5–409.7 ng OTB/g) and fungal free agar (15.3–417.0 ng OTA and 12.7–151.3 ng OTB/g). This is the first report which shows that high amounts of mycotoxins could be excreted from toxigenic Penicillium isolates into guttation droplets.     

Combined effects of selected Penicillium mycotoxins on in vitro proliferation of porcine lymphocytes

The in vitro effect of combinations of the Penicillium mycotoxins citrinin (CIT), cyclopiazonic acid (CPA), ochratoxin A (OTA), patulin (PAT), penicillic acid (PIA) and roquefortine C (RQC) on mitogen induced lymphocyte proliferation was determined using purified lymphocytes from six piglets. Dose–response curves for each mycotoxin and mycotoxin combinations were generated. The combined effects of toxin pairs based on IC₂₀ were illustrated in isobole diagrams and statistically calculated. OTA and CIT elicited a synergistic effect. Four toxin pairs elicited additive effects, four pairs less–than–additive effects and six pairs independent effects. Thus, the majority of toxin pairs tested produced lower combined effects than an additive effect. The results indicate that the sum effect of all toxins is less than that from the summation of concentrations of the individual compounds, adjusted for differences in potencies.