Melinda Kovács

Absorption, distribution and elimination of fumonisin B1 metabolites in weaned piglets

The absorption, distribution and elimination of fumonisin B1 (and B2) after oral administration of Fusarium verticillioides (MRC 826) fungal culture, mixed into the experimental feed for 10 days, was studied in weaned barrows. In order to determine the absorption of FB1 from the feed marked by chromium oxide, a special T-cannula was implanted into the distal part of pigs’ ileum. During the feeding of toxin-containing diet (45 mg FB1 kg−1) and until the tenth day after the end of treatment, the total quantity of urine and faeces was collected and their toxin content analysed. At the end of the trial, samples of lung, liver, kidney, brain, muscle, and fat were also collected and their fumonisin content analysed by LC-MS. The fumonisins appeared to decrease the reduced glutathione content in blood plasma and red blood cell haemolysate, possibly associated with in vivo lipid peroxidation. From a data set of 80 individual data and the concentration and rate of C r and fumonisins (FB1, partially hydrolysed FB1 and aminopentol) in the chymus, it could be established that the accumulative absorption of fumonisin B1 was 3.9% ± 0.7%. In the chymus, the FB1 conversions into aminopentol and partially hydrolysed FB1 were 1.0 and 3.9%, respectively. The degree of metabolism in faeces was variable, although the main product was the partially hydrolysed form, with very small amounts of the aminopentol moiety being recovered. In the investigated tissues the FB1 conversion to aminopentol and partially hydrolysed FB1 was 30 and 20%, respectively.

Effect of dietary supplementation of Spirulina (Arthrospira platensis) and Thyme (Thymus vulgaris) on rabbit meat appearance, oxidative stability and fatty acid profile during retail display

The objective of this study was to evaluate the effect of Spirulina and Thyme supplementation on rabbit meat during retail display. At weaning 294 rabbits were allocated to 7 different treatments (42 rabbits/treatment). Rabbits of the control group (C) received a diet without any supplementation throughout the experiment (5-11 weeks of age). The other groups were fed diets containing 5% Spirulina (S), 3% Thyme (T) or both supplements (ST) for the whole trial (5-11 weeks; treatments S, T and ST), or for a part of the growing period (8-11 weeks; treatments C-S, C-T and C-ST). Colour parameters, pH, water holding capacity and drip loss were determined on fresh and stored Longissimus dorsi muscle of 5 rabbits/treatment. Spirulina- and Thyme-supplemented diets had a significant effect on redness and yellowness of Longissimus dorsi. Drip loss was significantly reduced in C-T and T groups that also showed the highest content of α-tocopherol and n-3 fatty acids content and the lower lipid oxidation.

Residue formation of fumonisin B1 in porcine tissues

The residues derived from the uptake of fumonisin B1, a toxic metabolite of Fusarium verticillioides frequently occurring in corn and corn products, were determined in growing pigs. After oral administration of 100 mg FB1/animal/day for 5–11 days, serum, bile, lung, liver, kidney, brain, spleen, pancreas, heart, muscle, eye, and fat samples were collected immediately and analysed by LC-MS. The highest values were measured in kidney (833±1329 µgkg−1, mean±SD), liver (231±163 µgkg−1), lung (170±311 µgkg−1) and spleen (854±2212 µgkg−1). Muscle contained 26±41 µgkg−1, while in fat only 2±3 µgkg−1 were traceable. Despite the potential accumulation over extended feeding periods as well as the large variations in the residue formation of FB1, a carry-over in edible tissues from swine was considered not to be of toxicological relevance.

The cytotoxic effect of fumonisin B1 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.

Distribution and elimination of fumonisin analogues in weaned piglets after oral administration of Fusarium verticillioides fungal culture

The distribution and elimination of fumonisins after oral administration of 50 mg FB1, 20 mg FB2 and 5 mg FB3 per animal day–1 for 22 days was studied in weaned barrows. At the end of the trial, the lung, heart, liver, kidney, spleen, brain, serum, bile, muscle, fat, urine and faeces samples were collected and their content of fumonisins (FB1, FB2) determined by LC-MS. The highest FB1 concentrations were found in the liver (99.4 ± 37.5 ng g–1) and kidneys (30.6 ± 10.1 ng g–1), whilst the highest average amount of FB2 was in the liver (1.4 ± 2.3 ng g–1) and fat (2.6 ng g–1 ± 4.8) samples. Comparing the FB1/FB2 ratio in different organs (19/1), it was found that the ratio in the abdominal and subcutaneous fat samples (4/1) was markedly different from those in all other tissues, namely the relative proportion of FB2 was higher in latter cases. Of the total quantity of FB1, the 13% taken up during 5 days was excreted unchanged with the faeces and urine. On average, in the urine and faeces, FB1 was detected in nine- and 14-fold quantities, as compared with FB2.

In vitro microbial metabolism of fumonisin B1

There is a lack of information on the effect of swine caecal microbiota on fumonisin metabolism. In this in vitro study, the biotransformation of fumonisin B1 (FB1) by the gut microbiota of adult, healthy pigs was examined. Suspensions of caecal contents and McDougall buffer solution were incubated anaerobically with pure FB1 for 0, 12, 24, 48 and 72 h. After 48 h, the conversion of FB1 to partially hydrolysed FB1 (46%) was nearly equal to the percentage ratio of FB1, while by 72 h it was 49%. In vitro, the conversion of fumonisin B1 to aminopentol was less than 1%. The results show that the caecal microbiota are capable of transforming fumonisin B1 to the above metabolites. Further studies on FB1 metabolism in the small intestine are clearly justified.

Dietary Spirulina (Arthrospira platensis) and Thyme (Thymus vulgaris) supplementation to growing rabbits: Effects on raw and cooked meat quality, nutrient true retention and oxidative stability

The study evaluated the effect of Spirulina and Thyme dietary supplementation on rabbit meat quality, nutrient true retention and protection against oxidative stress. Rabbits in the control group (C-C) received a non-supplemented pellet throughout the experiment (5-11weeks of age). In the other groups, the pellet contained 5% Spirulina (S), 3% Thyme (T), or both (ST) for either the entire (groups S-S, T-T, ST-ST) or only the final part of the growing period (8-11weeks: groups C-S, C-T, C-ST). Spirulina supplementation increased the γ-linolenic acid content of rabbit meat, whereas Thyme improved the oxidative stability of raw and freeze-dried meat.

Individual and combined haematotoxic effects of fumonisin B1 and T-2 mycotoxins in rabbits

Weaned rabbits were fed diets contaminated with 2 mg/kg diet T-2 toxin alone, or 10 mg/kg diet fumonisin B1 (FB1) alone, and both toxins in combination (2+10 mg/kg, resp.), as compared to a toxin free control. Samplings were performed after 2 and 4 weeks. Bodyweight of the T-2 fed group was lower after 4 weeks; the liver weight increased dramatically. Red blood cell (RBC) Na(+)/K(+) ATPase activity decreased after 4 weeks in the T-2 group, it increased in the FB1 group and antagonism was found by the combined treatment. The RBC membrane fatty acid profile was modified by both toxins similarly during the entire feeding. After 4 weeks T-2 alone and in combination (with FB1) was found to increase mean cell volume (MCV). The time-dependent alterations in the T-2 group were significant for MCV (increase) and the mean cell haemoglobin (increase). The active monovalent cation transport was altered by both mycotoxins. Most probably FB1 exerts its sodium pump activity modification via an altered ceramide metabolism (behenic acid decrease in the RBC membrane), while for T-2 toxin a moderate membrane disruption and enzyme (protein) synthesis inhibition was supposed (ca. 75% decrease of the sodium pump activity).

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

Pannon White (n=12) male rabbits (weight: 4050 to 4500 g, age: 9 months) received 2 ml 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 (n=12) 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 (n=10) 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 gonadotropin-releasing 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).