Bioavailability of the Fusarium toxin deoxynivalenol (DON) from naturally contaminated wheat for the pig

Experiments were carried out with 16 castrated male pigs (41.5 +/- 2.0 kg) to examine the toxicokinetics of deoxynivalenol (DON) from naturally contaminated wheat (16.6 mg DON/kg) after chronic exposure or one single oral dose (acute). The systemic absorption (bioavailability) of DON was estimated based on the area under the curves (AUC) after oral (chronic or acute) and intravenous application of pure DON (53 microg/kg live weight). Additionally, a balance study was conducted to quantitatively trace the DON metabolism. After intravenous (IV) DON application (n = 5), serum DON concentrations decreased biphasically with terminal elimination half-lives (t(1/2)beta) of between 4.2 and 33.6h. DON was rapidly absorbed following oral exposure and reached maximal plasma concentrations (C(max)) of 21.79 and 15.21 ng DON/ml serum after (t(max)) 88.4 and 99.1 min in the chronic (n = 5) and acute (n = 6) fed group, respectively. Thereafter serum DON levels declined slowly with an elimination half-life (t(1/2)beta) of 6.28 and 5.32 h for both oral groups. The mean bioavailability (F) of DON was 89% for the chronic group and 54% for the acute oral group. DON was highly distributed in all groups, with an apparent volume of distribution (V(d)) higher than the total body water. Glucuronide conjugation of deoxynivalenol was found in serum samples after oral exposure, but not after intravenous application. Dietary DON caused a significant increase in DON concentrations of urine and faeces, whereby the metabolite de-epoxy-DON was found only in the trials with a pre-period of longer than 4 weeks. The total recovery was 66.6 +/- 39.0% and 54.0 +/- 9.7% for the control and the chronic DON groups, respectively, with urine being the main excretory route. In conclusion, orally administered DON was quickly absorbed to an extent of over 50%, highly distributed and only poorly metabolized. Twenty-four hours following oral dosing, DON could not be detected in the serum, except in one chronically fed pig at the level of the detection limit.     

Effect of the Fusarium toxins,zearalenone and deoxynivalenol,on the mouse brain

The aim of this study was to find effects of Fusarium toxins on brain injury in mice. We evaluated the individual and combined effect of the Fusarium toxins zearalenone and deoxynivalenol on the mouse brain. We examined brain weight, protein, antioxidant indicators, and apoptosis. After 3 and 5 days of treatment, increased levels of nitric oxide, total nitric oxide synthase, hydroxyl radical scavenging, and malondialdehyde were observed in the treatment groups. This was accompanied by reduced levels of brain protein, superoxide dismutase (apart from the low-dose zearalenone groups), glutathione, glutathione peroxidase activity, and percentage of apoptotic cells. By day 12, most of these indicators had returned to control group levels. The effects of zearalenone and deoxynivalenol were dose-dependent, and were synergistic in combination. Our results suggest that brain function is affected by zearalenone and deoxynivalenol.     

Effects of the Fusarium toxin deoxynivalenol on tissue protein synthesis in pigs

The Fusarium toxin deoxynivalenol is known to be a potent protein synthesis inhibitor according to research in different in vitro and in vivo systems. However, the significance of this toxic feature was not yet evaluated for the pig. Therefore, an experiment was carried out to measure porcine tissue protein synthesis employing the so-called flooding dose technique using [(2)H(5)]-phenylalanine as tracer. A total of 25 male castrated pigs (initial weight of 23kg) were used. Seventeen pigs were fed the control diet (control) and eight the Fusarium toxin-contaminated diet (chronic oral deoxynivalenol (DON)) for approximately 4wks. Pigs recruited from the control group were treated with acute oral (n=7) and intravenous (i.v.) DON (n=5), and were exposed to the toxin once on the day of protein synthesis measurement. The DON-dose given prior to the beginning of protein synthesis measurement amounted to 2, 77, 83 and 53microg DONkg(-1) live weight for the control group, chronic oral DON-group, acute oral DON-group and acute i.v. DON-group, respectively. Protein synthesis expressed as fractional synthesis rate (FSR) was significantly reduced in kidneys, spleen and ileum of DON-exposed pigs with effects being less pronounced in the acute oral DON-group. FSR of liver, skeletal and heart muscle, mesenteric lymph nodes, duodenum, jejunum, jejunal mucosa cells, pancreas and lung were not affected by DON.     

Effects of the Fusarium mycotoxins zearalenone and deoxynivalenol on the mitochondrial methylthiazol tetrazolium-cleavage activity of monolayer cells

The application of a modified colorimetric assay for the evaluation of mycotoxin-derived cytotoxicity is reported. Using four mammalian monolayer cell types (swine kidney, African green monkey kidney, Madin Darbin canine kidney and bovine embryonic lung cells), the influence of the mycotoxins zearalenone and deoxynivalenol on the cellular methylthiazoltetrazolium (MTT)-cleavage activity was evaluated after different toxin exposure times. The yellow tetrazolium salt is converted by mitochondrial enzymes of metabolically active cells into purple formazan products. These crystals were dissolved in dimethylsulphoxide to obtain a homogeneous solution, the optical density of which was suitable for precise spectrophotometric measurement by a plate reader. Zearalenone proved to be less cytotoxic than deoxynivalenol and even enhanced the cleavage activity of the cell types tested. At concentrations of 10 and 100 μg/ml, deoxynivalenol showed a marked suppressive effect on the activity of all cell types, depending on the length of the incubation time. The results indicate that monolayers are useful targets in the MTT assay for investigating the action of mycotoxins and for performing comparative studies in mammalian cells.     

Systemic and local effects of the Fusarium toxin deoxynivalenol (DON) are not alleviated by dietary supplementation of humic substances (HS)

The aim of this study was to examine the effects of a control diet (CON) or a Fusarium toxin contaminated diet (FUS) with and without HS (CON-HS and FUS-HS, respectively) on pigs during a 10-week growth trial starting at 35.1±3.2 kg live weight (n=12/group). Moreover, 2 additional choice feeding groups were included to test the ability of the pigs to differentiate between the CON and FUS diet. Feeding the FUS diets (～3 mg DON/kg) did not depress feed intake irrespective of HS addition. However, the pigs of the choice feeding groups recognised the FUS diets and acquired an ability to avoid these diets. DON residues were detected exclusively in the blood of pigs exposed to the FUS diets (7-21 ng/mL) but their levels were not affected by HS, suggesting their inefficiency in preventing DON absorption. While zonula occludens-1 protein expression and villus height in jejunum and ileum were not compromised by FUS feeding, the jejunal crypts were significantly deepened at 31% compared to the CON group. These changes had no consequences for nutrient digestibility or LPS levels in systemic blood (0.02-0.08 EU/mL). As portal LPS levels were not measured, FUS effects on intestinal LPS translocation cannot be excluded.     

Toxicokinetic study and absolute oral bioavailability of deoxynivalenol,T-2 toxin and zearalenone in broiler chickens

Mycotoxins lead to economic losses in animal production. A way to counteract mycotoxicosis is the use of detoxifiers. The European Food Safety Authority stated that the efficacy of detoxifiers should be investigated based on toxicokinetic studies. Little information is available on the absolute oral bioavailability and the toxicokinetic parameters of deoxynivalenol, T-2 and zearalenone in broilers. Toxins were administered intravenously and orally in a two-way cross-over design. For deoxynivalenol a bolus of 0.75 mg/kg BW was administered, for T-2 toxin 0.02 mg/kg BW and for zearalenone 0.3 mg/kg BW. Blood was collected at several time points. Plasma levels of the mycotoxins and their metabolite(s) were quantified using LC–MS/MS methods and toxicokinetic parameters were analyzed. Deoxynivalenol has a low absolute oral bioavailability (19.3%). For zearalenone and T-2 no plasma levels above the limit of quantification were observed after an oral bolus. Volumes of distribution were recorded, i.e. 4.99, 0.14 and 22.26 L/kg for deoxynivalenol, T-2 toxin and zearalenone, respectively. Total body clearance was 0.12, 0.03 and 0.48 L/min kg for deoxynivalenol, T-2 toxin and zearalenone, respectively. After IV administration, T-2 toxin had the shortest elimination half-life (3.9 min), followed by deoxynivalenol (27.9 min) and zearalenone (31.8 min).     

On the transfer of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) from sows to their fetuses during days 35-70 of gestation

Eleven pregnant sows with a body weight between 153 and 197 kg were fed a control diet (CON, 0.15 mg DON and 0.0035 mg ZON/kg diet) or a diet containing 15% of Fusarium toxin contaminated triticale (MYCO, 4.42 mg DON and 0.048 mg ZON/kg diet) in the period of day 35 and 70 of gestation. The indirect effect of feed intake was separated from the direct effects of the Fusarium toxins by the restricted feeding regimen where all sows were fed the same amount of feed (2000 g/d) over the whole study. At the end of experiment, fetuses were delivered by Caesarian section and samples of serum, bile, urine, liver, kidney and spleen of euthanatized sows and fetuses were taken to analyze the concentrations of DON, ZON and their metabolites. Feeding the Fusarium toxin contaminated diet to pregnant sows caused neither adverse effects on performance, organ weights and maintenance of pregnancy of sows nor on fetus weight and length. Furthermore, no teratogenic or embryolethal effects could be observed in the MYCO group. Hematological and clinical-chemical parameters of sows and fetuses were not affected by feeding, with the exception of significantly lower GLDH (glutamate dehydrogenase) serum activities in MYCO sows. The carry over of DON and ZON from the diet to the sow or fetus tissues was calculated by the diet ratio (sum of concentrations of all metabolites in the physiological specimen divided by the dietary toxin concentration), while the fetus ratio was evaluated by the sum of concentrations of all metabolites in the physiological specimen of the fetus divided by that of the sows. DON and deepoxy-DON were found in urine, bile, serum, liver, kidney and spleen of sows of the MYCO group, but not in the bile of fetuses (spleen not analyzed). ZON and its metabolite alpha-zearalenol (alpha-ZOL) were detected in urine and bile of sows, while all specimens of fetuses as well as serum and liver of sows were negative for ZON metabolites. The maximum diet ratios for urine and bile in sows of the MYCO group were 0.84 and 0.05 for DON metabolites and 1.2 and 3.8 for ZON metabolites, underscoring the differences in metabolism and excretion of both toxins. The maximum diet ratio of DON and deepoxy-DON into liver, kidney and spleen of MYCO sows were 0.003, 0.007 and 0.003, respectively. The maximum fetus ratio of DON and deepoxy-DON into urine, bile, serum, liver and kidney of fetuses were 0.006, 0, 0.5, 0.88, and 0.33, while the maximum placental ratio (sum of toxin concentrations in the physiological specimen of the fetus divided by the toxin serum concentration of the sow) were 0.64, 0, 0.50, 0.70 and 0.52, respectively. Therefore, it can be concluded that the developing fetus is exposed to DON between the gestation days 35 and 70 when the sows are fed a Fusarium toxin contaminated diet. ZON concentration in the MYCO diet was too low to get reliable results for fetus or placental ratios.     

On the transfer of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) from the sow to the full-term piglet during the last third of gestation.

Pregnant sows were fed either a control diet (CON, n=8, 0.21 mg DON and 0.004 mg ZON/kg diet) or a diet containing 40% of a Fusarium toxin contaminated wheat (MYCO, n=7, 9.57 mg DON and 0.358 mg ZON/kg diet) from day 75 to 110 of gestation. Piglets were delivered by Caesarean section at the end. Spleen weights of piglets from the MYCO group were significantly lower. Hemoglobin concentration and hematocrit were also significantly decreased in these piglets, although this effect was more obvious in female than in male piglets. The transfer of DON and ZON was evaluated by the diet ratio (sum of concentrations of all metabolites in the physiological specimen divided by the dietary toxin concentration) and the piglet ratio (sum of concentrations of all metabolites in the physiological specimen of the piglet divided by that of the sows). The diet ratio for the liver (sows only) amounted to 0.001 (DON+de-epoxy-DON) and 0.016 (ZON and metabolites). The diet ratios of DON in bile reached up to 0.041 and 0.003 for sows and piglets, respectively, and those for ZON up to 2.896 and 0.128. The piglet ratios in bile varied up to 0.309 and 0.518 for DON and ZON, respectively, whereas nearly similar DON concentrations were found in serum of piglets and sows (median piglet ratio of 0.750). The results of the study suggest that the developing fetus is exposed to DON, ZON and their metabolites when the sows are fed a Fusarium toxin contaminated diet.     

Detection of zearalenone and its metabolites in naturally contaminated follicular fluids by using LC/MS/MS and in vitro effects of zearalenone on oocyte maturation in cattle

Zearalenone (Zen) and its metabolites are estrogenic and may be important factors involved in reproductive disorders in domestic animals. We aimed to (1) simultaneously detect Zen and its metabolites in bovine follicular fluids (FFs) by liquid chromatography-tandem mass spectrometry and (2) examine the in vitro effects of Zen on bovine oocytes. Zen and its metabolites were detected in 6 of 32 normal follicles and 7 of 20 cystic follicles. Bovine oocytes were cultured in a maturation media containing various Zen concentrations (0 [control], 1, 10, 100, and 1000microg/L), fertilized, and cultured further. Maturation rates decreased dose-dependently. Further, maturation of 62 (50%) of 124 oocytes examined in the 1000-microg/L group was arrested in metaphase I, without affecting the fertilization rate. Blastocyst-formation rates did not significantly differ among the groups. Zen and its metabolites were detectable in bovine FFs. High Zen concentration may adversely affect meiotic competence but not the fertilization and development rates.     

Effect of the Fusarium toxin deoxynivalenol (DON) on IgA, IgM and IgG concentrations and proliferation of porcine blood lymphocytes.

An important effect of the trichothecene mycotoxins is the impairment of the immune function, but immunotoxicity studies have mainly been conducted on the mouse model. In the present study, the effect of deoxynivalenol (DON) on the proliferation of ConA stimulated porcine peripheral blood lymphocytes (PBL) was assessed in vitro after adding of 70-560 ng DON per ml medium, and in vivo after chronic and acute (one single dose) dietary DON exposure (5.7 mg/kg). Immunoglobulin (IgA, IgG, IgM) concentrations were measured by ELISA in supernatants and serum of pigs. The proliferation rate was estimated with two different assays (BrdU incorporation and MTT cleavage). In vitro the ConA stimulated proliferation was inhibited to 50% (IC50) at 200 and 309 ng DON/ml for the BrdU and MTT assay, respectively, indicating a higher sensitivity of DNA synthesis to DON. Immunoglobulin concentrations in the supernatant after in vitro proliferation of PBL with increasing DON concentrations for 72 h were significantly decreased, with IC50 values of 120.6, 84.1 and 71.7 ng DON/ml for IgA, IgM and IgG, respectively. In vivo significant inhibition of lymphocyte proliferation was observed only in the DON acute group using the MTT assay, but values tended to be decreased in the BrdU assay and after chronic DON exposure. Immunoglobulins (IgA, IgM and IgG) in the supernatant of cultured lymphocytes were not significantly affected after dietary DON exposure. Serum IgA of pigs showed no significant differences between the groups, whereas IgM and IgG were significant increased in the DON acute group.     

Suppression of immune response in the B6C3F1 mouse after dietary exposure to the Fusarium mycotoxins deoxynivalenol (vomitoxin) and zearalenone

The effect that dietary exposure to the naturally-occurring Fusarium graminearum toxins deoxynivalenol (DON) and zearalenone (ZEA) may have on immune function was assessed in the B6C3F1 mouse. Dietary DON depressed the plaque-forming response to sheep red blood cells, the delayed hypersensitivity response to keyhole limpet haemocyanin and the ability to resist Listeria monocytogenes. Listerial resistance was similarly decreased in control mice fed restricted diets comparable to the dietary restriction caused by DON-induced feed refusal, whereas equivalent food restriction did not decrease the plaque or delayed hypersensitivity responses. ZEA ingestion decreased resistance to L. monocytogenes but did not affect splenic plaque-forming or delayed hypersensitivity responses. Resistance to Listeria was reduced to a greater extent by co-administration of DON and ZEA than by DON alone, whereas the ability of DON to inhibit the delayed hypersensitivity response was significantly lessened in the presence of ZEA. While effects on resistance to Listeria and delayed hypersensitivity were detectable in mice ingesting the mycotoxins for 2-3 wk, these effects disappeared upon extension of the feeding period to 8 wk. In contrast, some effect on the plaque-forming response was detectable with both the 2- and the 8-wk period of mycotoxin ingestion. Immunosuppression can thus result from ingestion of F. graminearum-infected agricultural staples, the suppression being attributable to interactions between direct immunotoxic effects of DON and ZEA and nutritional effects associated with DON-induced food refusal.     

The effect of Fusarium mycotoxins deoxynivalenol,fumonisin, and moniliformin from contaminated moldy grains on aquaculture fish

Fusarium spp. are types of fungi that invade agriculturally important grains, such as corn and wheat, where they may produce mycotoxins that are harmful to the productivity and health of food animals, such as swine, poultry, and aquacultural fish. Because corn and wheat are used for other industrial purposes, such as ethanol and flour production, by-products of these processes are available to supplement or replace expensive grains in fish feed formulations. Consequently, grain by-products have come under scrutiny as possible sources of feed-borne Fusarium mycotoxins. Evaluation of the effects of Fusarium mycotoxins on productivity of fish used in aquaculture has been the subject of recent research.     

Determination of aflatoxins,deoxynivalenol, ochratoxin A and zearalenone in wheat and oat based bran supplements sold in the Spanish market

The aflatoxins (AFs), deoxynivalenol (DON), ochratoxin A (OTA) and zearalenone (ZEA) are mycotoxins produced by fungal species which can contaminate, alone or simultaneously, cereal-based raw materials. Usually, the higher mycotoxins concentrations in cereals are found in the external layers of the grain (bran). Nowadays bran is increasingly consumed for its high fibre concentration. The objectives of this study were determining the concentration of these mycotoxins in bran samples intended for direct human consumption and to study the influence of some characteristics of the samples that may affect the mycotoxins content, there are not studies about fibre for direct human consumption. 67 bran samples from shops and supermarkets from two different Spanish cities were analyzed, being 37 samples of wheat bran and the remaining of oat bran. The results showed a major presence of DON in the analyzed samples, with levels above the EU legislation in some samples. Presence of DON was more frequent in wheat samples, compared to oats ones (p < 0.05). Extruded or toasted samples, subjected to a heat treatment during processing, presented a significantly lower concentration of OTA, and differences between the organically and conventionally produced samples were also detected in OTA, which showed higher levels in the organic samples. Co-occurrence was frequently found between the Fusarium mycotoxins (ZEA and DON).Due to the high levels of DON in the analyzed samples, a calculation of DON intake has been made and it has been demonstrated that bran can account for an important percentage of DON exposure in the total diet.     

Examination of Chinese and U.S.S.R. cereals for the Fusarium mycotoxins, nivalenol, deoxynivalenol and zearalenone

Cereals, foods and feeds sampled in Taiwan, China and the U.S.S.R. were contaminated with nivalenol, deoxynivalenol and zearalenone. The frequencies and levels of contamination are similar to those observed in the cereals of Japan and Korea. This is the first report on the natural occurrence of nivalenol, deoxynivalenol and zearalenone in Chinese and U.S.S.R. cereals, foods and feeds.     

Kinetics and metabolism of the Fusarium toxin deoxynivalenol in farm animals: Consequences for diagnosis of exposure and intoxication and carry over

The knowledge of factors influencing the kinetics, metabolism and bioavailability of the Fusarium toxin deoxynivalenol (DON) is a basic prerequisite for evaluation of the transfer (carry over) of the toxin and its metabolites into edible tissues, and for a physiological specimen-based diagnosis of intoxication. These aspects are addressed in the present review, and potentials and pitfalls of the suitability of analysis of physiological samples for evaluation of the DON exposure as a veterinary tool are discussed. For example, the farm animal species was shown to be a determining factor influencing the metabolic profile and the bioavailability of DON. Although linear relationships were derived between DON exposure of ad libitum and restrictively fed animals and DON or de-epoxy-DON concentration in the blood of pigs, dairy cows and sheep, it has to be considered that individual values might markedly deviate from these relationships, which makes interpretation of measured concentrations of DON and its metabolites difficult. The situation is further complicated by the lack of established relationships between DON residues in physiological matrices and the adverse effects of DON on the health and performance of farm animals.     

Comparison of the trichothecenes deoxynivalenol and T-2 toxin for their effects on brain biogenic monoamines in the rat

Male Sprague-Dawley rats (180 g) were orally dosed with deoxynivalenol (DON) and T-2 toxin at 2.5 mg kg⁻¹ body weight. Brains were collected 24 h postdosing, dissected into five brain regions and analyzed for biogenic monoamines by high performance liquid chromatography with electrochemical detection. DON and T-2 toxin treatment resulted in significantly elevated concentrations of the indoleamines, serotonin (HT) and 5-hydroxy-3-indoleacetic acid (HIAA) in all brain regions examined, whereas norepinephrine (NE) and dopamine (DA) regional concentrations were not significantly altered. These results indicate that DON and T-2 toxin influence brain biogenic monoamine metabolism, and suggest that the central nervous system (CNS) actions of these trichothecenes are similar.     

The effect of feeding a diet naturally contaminated with deoxynivalenol (DON) and zearalenone (ZON) on the spleen and liver of sow and fetus from day 35 to 70 of gestation

Pregnant sows were fed a control diet (CON, 0.15 mg deoxynivalenol (DON) and 0.0035 mg zearalenone (ZON) per kg diet) or diet containing 15% of Fusarium toxin contaminated triticale (MYCO, 4.42 mg DON and 0.048 mg ZON per kg diet) during days 35-70 of gestation. All sows were fed in a restricted feeding regimen with the same amount of feed (2000 g/d) over the whole study. At the end of the experiment, fetuses were delivered by Caesarian section and samples of spleen and liver of euthanized sows and fetuses were analyzed. At terminal necropsy, no macroscopic lesion was observed in any organ of either sows or fetuses. The histopathological data indicated significant alteration only in elevated iron staining in the red pulp of spleens in sows of MYCO group after 35 days of feeding. The presence of hemosiderin particles in the spleen sections was confirmed by transmission electron microscopical investigation and by an enhanced Fe2+ concentration in spleen. A glycogen increase (p<0.05) was found in liver cells of fetuses in the experimental group. Together, the results provide evidence of spleen dysfunction (hemosiderosis) in sows fed a Fusarium toxin-contaminated wheat, however, with absence of clinical signs. Enhanced glycogen and an impairment of mitochondria in liver of fetuses was present when their mothers consumed the MYCO diet.     

Impact of deoxynivalenol (DON) contaminated feed on intestinal integrity and immune response in swine

This study was performed to characterize the influence of consuming DON naturally contaminated feeds on pig's intestinal immune defenses, antibody response and cellular immunity. Sixteen 4-week-old piglets were randomly allocated to two dietary treatments: control diet or diet contaminated with 3.5 mg DON/kg. At days 7 and 21, animals were immunized with ovalbumin (OVA). On day 42, intestinal samples were collected for measurement of gene expression involved in immune response, oxidative status and barrier function. Primary IgG antibody response to OVA was increased in pigs fed DON diet compared to control animals. In the ileum of pigs fed DON diet, claudin, occludin, and vimentin genes involved in integrity and barrier function were down-regulated compared to controls. Results also revealed that expression of two chemokines (IL-8, CXCL10), interferon-γ, and major antioxidant glutathione peroxidase 2 (GPX-2) were up-regulated whereas expression of genes encoding enzymatic antioxidants including GPX-3, GPX-4 and superoxide dismutase 3 (SOD-3) were down-regulated in pigs fed DON-contaminated diet. These results strongly suggest that ingestion of DON naturally contaminated feed impaired intestinal barrier and immunological functions by modulating expression of genes coding for proteins involved in tight junctions, tissue remodelling, inflammatory reaction, oxidative stress reaction and immune response.     

Effects of the Fusarium toxin deoxynivalenol from naturally contaminated wheat given subchronically or as one single dose on the in vivo protein synthesis of peripheral blood lymphocytes and plasma proteins in the pig

Trichothecenes, such as deoxynivalenol (DON), are known to inhibit the protein synthesis in vitro by binding at the 60S subunit of eukaryotic ribosomes. Therefore, cells and tissues with high protein turnover, such as lymphocytes and the liver (albumin and fibrinogen synthesis), were suggested to react most sensitively to DON. However, to the author's knowledge this observation was not proven in vivo in pigs, which were regarded as the farm animals most susceptible to DON. A total of 31 castrated male, crossbred German Landrace x Pietrain pigs weighing approx. 40 kg were fed a DON contaminated diet (5.7 mg/kg) either acutely (one single dose) or subchronically (4 weeks) or a control diet (0.1 mg/kg). In addition, one group received an intravenous injection of 53 microg DON/kg LW. One hour after feeding, a "flooding dose" of the stable isotope l-[(2)H(5)]-phenylalanine (125 mg/kg LW) was given and frequent blood samples (permanent catheter) were collected over a 60 min period. The molar percent excess (MPE) of plasma free and protein-bound phenylalanine were measured by GC/MS. No differences could be observed in the plasma concentrations of total protein, albumin, fibrinogen and serum enzymes between the groups. On the other hand, fractional synthesis rates (FSR, %/d) of albumin were significantly decreased by 43%, 45% and 26% and FSR of lymphocytes declined by 27%, 19% and 24%, whereas fibrinogen was not significantly affected after subchronic or one single oral and intravenous DON exposure, respectively. Additionally, the absolute synthesis rate (ASR, g/d) of albumin and the proportion of albumin to total body protein synthesis were reduced in the same manner, whereas the albumin secretion time ranged between 6.8 and 34.4 min and was not affected by treatment. In conclusion, the flooding dose technique appeared to be suitable for distinguishing DON-related effects on the protein synthesis, while determination of plasma protein concentrations seemed not to be an appropriate parameter.     

Toxicological interactions between the mycotoxins beauvericin, deoxynivalenol and T-2 toxin in CHO-K1 cells in vitro

Beauvericin (BEA), deoxynivalenol (DON) and T-2 toxin (T-2) are important food-borne mycotoxins that have been implicated in human health. In this study, the acute toxicity of individual and combined mycotoxins (BEA, DON and T-2) were tested in immortalized hamster ovarian cells (CHO-K1) at 24, 48 and 72 h of exposure, by the tetrazolium salt (MTT) and neutral red (NR) assays. The IC₅₀ values obtained for all mycotoxins by the MTT and NR assays ranged from 0.017 to 12.08 μM and from 0.042 to 17.22 μM, respectively. Both, individual and combined mycotoxins demonstrated a significant cytotoxic effect in CHO-K1 cells in a dose-dependent manner. When mycotoxins were assayed individually, T-2 showed the strongest IC₅₀ values (from 0.017 to 0.052 μM), by both endpoints tested, followed by DON (0.53-2.30 μM) and BEA, showing this last one, the weakest IC₅₀ values (from 2.77 to 17.22 μM). On the other hand, cytotoxicity interactions were evaluated by the isobologram method. In acute binary tests, DON + BEA (CI = 1.60-25.07) and DON + T-2 (CI = 1.74-7.71) showed antagonism at 24, 48 and 72 h of exposure. By contrast, the binary BEA + T-2 combination (CI = 0.35-0.78) showed synergism at all time of exposure tested. The tertiary BEA + DON + T-2 combination demonstrated synergism effect (CI = 0.47-0.86) after 24 and 48 h of exposure; however moderate antagonistic effect (CI = 1.14-1.60) was presented after 72 h of exposure at the lower doses. These results provide quantitative evidence regarding potentially important interactions between BEA, DON and T-2 depending of the time of exposure. The combination index-isobologram equation method can serve as a useful tool in food risk assessment. Due to the potent toxic effects of BEA, DON and T-2, its combined exposure might be an important trigger for development of several diseases in humans, from the mycotoxicological point of view, especially after long period of exposure time.