Preliminary investigations on the single and combined cytotoxic effect of T-2 and HT-2 measured by Methyl Thiazol Tetrazolium (MTT) cytotoxicity test using pig lymphocytes

Fusariotoxins are frequently existing contaminants in cereal and other plant products. High relationship was found between the levels of the trichothecene T-2 and HT-2 toxin detected in contaminated commodities. Many in vitro studies investigated the cytotoxicity of T-2 and HT-2, but the interaction between them has not been thoroughly studied yet. The aim of the study was to examine the dose dependent cytotoxic effect of T-2 and HT-2 toxins, single and in combination, using pig lymphocytes in the methyl thiazol tetrazolium (MTT ) assay. The mycotoxins were added at various concentrations, i.e. 0.5, 0.1, 0.05, 0.01 and 0.001 μM of T-2; 1.0, 0.5, 0.2., 0.1 and 0.05 μM of HT-2; and both mycotoxins combined. Two exposure times (6 and 24 h) were tested. Both T-2 and HT-2 toxins exerted a dose dependent effect. After 6 h incubation, the increase in concentration of T-2 from 0.001 to 0.5 μM and HT-2 for 0.05 to 1.0 μM resulted in lower cell viability by 22 and 17%, respectively. After 24 h cell viability was significantly lower compared to values obtained at 6 h, except 0.5 μM T-2 and 0.05, 1.0 μM HT-2, respectively. Measured cell viability for combinations of T-2 and HT-2 was higher compared to the calculated expected values. The two toxins caused lower cell survival when applied together than in single administration after 6 h incubation. After 24 h incubation this tendency was not consistent

Subsequent effect of subacute T-2 toxicosis on spermatozoa, seminal plasma and testosterone production in rabbits

Pannon White (n512) male rabbits (weight: 4050 to 4500 g, age: 9 months) received 2ml of a suspension containing purified T-2 toxin by gavage for 3 days. The daily toxin intake was 4 mg/animal (0.78 to 0.99 mg/kg body weight (BW)). Control animals (n512) received toxin-free suspension for 3 days. Since a feed-refusal effect was observed on the second day after T-2 administration, a group of bucks (n510) were kept as controls (no toxin treatment) but on a restricted feeding schedule, that is, the same amount of feed was provided to them as was consumed by the exposed animals. On day 51 of the experiment (i.e. 48 days after the 3-day toxin treatment), semen was collected, and pH, concentration, motility and morphology of the spermatozoa, as well as concentration of citric acid, zinc and fructose in the seminal plasma, were measured. After gonadotropinreleasing hormone (GnRH) analogue treatment, the testosterone level was examined. One day of T-2 toxin treatment dramatically decreased voluntary feed intake (by 27% compared to control, P,0.05) and remained lower ( P,0.05) during the first 2 weeks after the withdrawal of the toxin. BW of the contaminated rabbits decreased by 88% on days 17 and 29 compared to controls ( P,0.05). No effect of toxin treatment was detected on pH and quantity of the semen or concentration of spermatozoa. The ratio of spermatozoa showing progressive forward motility decreased from 65% to 53% in the semen samples of toxin-treated animals compared to controls ( P.0.05). The ratio of spermatozoa with abnormal morphology increased ( P,0.05) in the ejaculates collected from the toxin-treated animals. T-2 toxin applied in high doses decreased the concentration of citric acid in seminal plasma ( P,0.05). No effect of T-2 toxin on the concentrations of the other seminal plasma parameters (fructose and zinc) was observed. T-2 toxin decreased the basic testosterone level by 45% compared to control ( P,0.01) and resulted in lower ( P,0.05) GnRH-induced testosterone concentration. Feed restriction, that is, less nutrient intake, resulted in more morphologically abnormal spermatozoa in the semen, but it did not cause significant loss in BW, motility of the spermatozoa, composition of the seminal plasma or testosterone concentration – its effect needs further examination

Effect of chronic T-2 toxin exposure in rabbit bucks, determination of the No Observed Adverse Effect Level (NOAEL)

T-2 toxin (T-2) was administered to adult Pannon White (n = 10/group) male rabbits for 65 days, first in a suspension by gavage (0.05, 0.1 or 0.2 mg/animal/day), and secondly mixed into the feed (0.33 and 0.66 mg/kg feed). In the first experiment 0.1 mg T-2 exposure resulted in temporary decrease in feed intake, slower increase in the gonadotropin-releasing hormone (GnRH) induced testosterone synthesis, slight centrolobular infiltration in the liver and a slight hyperplasia of the Leydig cells. In addition to the temporary feed refusal effect, 0.2 mg T-2 caused a temporary decrease in plasma albumin and urea concentrations, lesser glutathione peroxidase (GPx) activity in the seminal plasma, a greater (by 320%) ratio of spermatozoa with cytoplasmic droplets, slower increase in the GnRH-induced testosterone synthesis, centrolobular infiltration in the liver, slightly hyperaemic testes and increased proliferative activity of the Leydig cells. The two smaller doses applied in feed (0.33 and 0.66 mg/kg) did not cause any significant adverse effect, and no feed refusal was observed. According to these results the No Observed Adverse Effect Level (NOAEL) of T-2 for adult rabbit males was found to be <0.1 mg/animal/day (<0.02 mg/kg b.w./day)

Preliminary Results on the Effect of Chronic T-2 Toxin Exposure in Rabbit Bucks

The aim of the present study was to examine the chronic effect of T-2 toxin on feed consumption and sperm quality. Pannon White (n=10/group) male rabbits (weight: 4050-4500 g, age: 9 month) trained to ejaculate into artificial vagina were exposed to 0 (control), 0.05, 0.1 or 0.2 mg/animal/day of T-2 toxin by gavage for 63 days. On the 63rd day of the experiment semen was collected with an artificial vagina, and the following traits were evaluated: pH, concentration, morphology, motility with CASA, concentration of seminal plasma components such as citric acid, zinc and fructose. At the end of the experiment animals were necropsied and the testes were subjected to histopathological examination. T-2 toxin in 0.1 mg and 0.2 daily dose significantly decreased feed intake in the first two weeks but no significant difference between groups were observed from the 4th week. Among the sperm quality traits examined only the ratio of spermatozoa with cytoplasmic droplets increased by 320% in animals treated with the highest dose of T-2. The 0.1 mg/animal/day toxin exposure resulted in a slight hyperplasia of the Leydig cells, while the highest dose (0.2 mg/animal/ day) caused hyperaemia, increased proliferative activity and hyperplasia of the Leydig cells. According to the preliminary results it seems, that adult male rabbits may tolerate the concentration of 0.05 mg/animal/day T-2 toxin

Kinetics of fumonisin B ₁ in pigs and persistence in tissues after ingestion of a diet containing a high fumonisin concentration

The absorption, distribution and elimination of fumonisin B 1 (FB 1 ) and its metabolites was investigated in pigs. For the determination of the absorption and biotransformation of FB 1 , T-cannula were implanted into the distal part of experimental pigs’ ileum and the total urine and faeces moiety was collected during the toxin feeding (45 mg FB 1 /kg diet, duration: 10 days) and in the subsequent elimination period (10 days). At the end of trial several organs, muscle and fat samples were also collected. The accumulative absorption of fumonisin B 1 was 4%. In the chymus, the FB 1 conversion to aminopentol (totally hydrolysed FB 1 ; AP 1 ) and partially hydrolysed FB 1 (PHFB 1 ) was 1% and 3.9%, respectively. Derivatives of FB 1 were mostly accumulated in the liver and kidney, while in negligible concentration could be detected in the muscle and fat samples. In the organs the efficacy of the FB 1 to AP 1 and PHFB 1 conversion was 30% and 20%, respectively. In the faecal content the main hydrolised product was PHFB 1 (47%), with 12% of AP 1 . 1.5% of the FB 1 quantity taken up was excreted with the urine, about 35% in hydrolyzed form. Detectable amounts of FB 1 and its metabolites were measured in most of the organs, in faeces and urine even 10 days after the feeding of the noncontaminated diet. As a general conclusion, the intestinal microbiota of pigs is able to transform the intact FB 1 to a similarly toxic substance (partially hydrolyzed FB 1 ) or to a more toxic metabolite (aminopentol)