Effect of deoxynivalenol on the levels of toll-like receptors 2 and 9 and their mRNA expression in enterocytes in the porcine large intestine: a preliminary study.

Deoxynivalenol (DON), one of the most prevalent mycotoxins in the world, and is capable of inducing immune disorders in humans and animals. The aim of this study was to determine the effect of feed contaminated with DON on the number of TLR2- and TLR9-positive cells and their mRNA expression in the porcine large intestine. The experiment was conducted on two equal groups of pigs (n=4). The experimental group (E) was administered feed contaminated with DON (1008 µg/kg of feed) for 6 weeks, and the control group (C) was administered non-contaminated feed over the same period of time. A decrease in the expression of TLR2 mRNA was noted in the cecum. The percentage of TLR9-positive enterocytes increased in the ascending colon and decreased in the cecum. The results of this study indicate that DON can modify the local immune response by changing the expression of TLRs.

Immunohistochemical evaluation of apoptosis and proliferation in the mucous membrane of selected uterine regions in pre-pubertal bitches exposed to low doses of zearalenone.

Mycotoxins are toxic secondary metabolites produced by fungi. Those biologically active compounds occur naturally and they include zearalenone (ZEN), a mycotoxin that contaminates plant material, including the ingredients used in the production of commercial dog food. The influence of monotonic, low-dose and long-term exposure to ZEN on pre-pubertal bitches has not been fully explored to date. This paper describes a 42-day experiment performed on clinically healthy female dogs aged approximately 70 days, with estimated body weight of 8 kg. The animals were randomly divided into two experimental groups (EI and EII) and a control group (C) of 10 animals each. Group EI received 50 µg ZEN/kg (of body weight) per os, group EII received 75 µg ZEN/kg BW per os, and the control group was administered placebo. The bitches were ovariohysterectomized at the end of the experiment (at around 112 days of age), and selected sections of the uterine wall were subjected to immunohistochemical analyses (TUNEL and PCNA). A shift towards higher apoptotic (AI) and proliferative index (PI) was observed, in particular in group EI. Higher AI and PI values were noted in the epithelium of all uterine regions analysed and in the uterine glands in the uterine horn proximal to the body of the uterus.

The effect of selected environmental Fusarium mycotoxins on the ovaries in the female wild boar (Sus scrofa).

The contamination of agricultural crops with Fusarium mycotoxins poses one of the greatest problems in food production. Wild boars live in specific habitats and are physiologically sensitive to Fusarium mycotoxins, therefore, they are an interesting model for studies investigating the effects of the discussed toxin, in particular under low-dose exposure. The objective of this study was to determine potential effects of Fusarium mycotoxins ingested with naturally contaminated food on reproductive function based on the proliferation and apoptotic indices of ovarian follicles in female wild boars. The experiment was conducted on 40 wild boars inhabiting north-eastern Poland. The effect of seasonal variations in the quantity and quality of ingested food on the concentrations of Fusarium mycotoxins and their metabolites in the blood of wild boars was analyzed. The observed differences in toxin levels were accompanied by changes in proliferation and apoptotic indices. Proliferation processes were most intense in autumn-winter and were least advanced in winter-spring. The intensity of apoptotic processes was inversely correlated with proliferation.

Evaluation of selected serum biochemical and haematological parameters in gilts exposed per os to 100 ppb of zearalenone.

Zearalenone (ZEN) widely contaminates animal feed of plant origin. The recommended safe concentrations of ZEN in feeds for various animal species are set mainly based on the mycotoxin's hormonal properties (NOEL). Our growing knowledge about biologically active concentrations of ZEN, molecular mechanisms and cells/tissues targeted by ZEN indicates that the harmful effects exerted by this mycotoxin on animals may be far greater than previously believed. This experiment was performed on pre-pubertal gilts divided into a control group (n=9) and an experimental group (ZEN, n=9). The control group received placebo, whereas the experimental group was administered ZEN at a dose of 0.1 mg/kg feed (equivalent to 5 µg/kg BW/day) for 42 days. On days 14, 28 and 42 blood samples were collected from the animals to determine the concentrations of selected zearalenols, serum biochemical and haematological parameters. Conjugated ZEN was found in the blood serum of the experimental gilts. Changes in the analysed biochemical parameters included a transient increase in albumin and cholesterol levels. A statistically significant increase in the concentrations of neutrophilic and acidophilic granulocytes was observed in the white blood cell system. The results indicate that long-term per os exposure of pre-pubertal gilts to low doses of ZEN (below NOEL) has a modulatory effect on liver function and white blood cells.

Cecal enzyme activity in gilts following experimentally induced Fusarium mycotoxicosis.

The objective of the presented study was to examine the influence of Fusarium mycotoxins (zearalenone--ZEN and deoxynivalenol--DON), administered separately and in combination, on the activity of cecal enzymes (ß-glucosidase and ß-glucuronidase) in gilts which were fed fodder con- taminated with these mycotoxins. The activity of ß-glucosidase and ß-glucuronidase varied in the range of 0.170-1.236 µmol · h(-1) · mg(-1) and 8.701-96.704 µmol · h(-1) · mg(-1), respectively. In the first two weeks, the toxins had no significant effect on the activity of ß-glucosidase and ß-glucuronidase in the ascending and descending colon. After week 3 and later on, ZEN and DON administered as a mix- ture led to the highest increase in the activity of both enzymes. Administered separately, DON affected the activity of enzymes more than ZEN. From the third week of the experiment, an increase in the activity of CW ß-glucosidase and ß-glucuronidase was observed.

Polymorphisms of DNA repair genes and risk of squamous cell carcinoma of the head and neck in young adults.

Squamous cell carcinoma of the head and neck (HNSCC) most frequently arise in the epithelial tissues of the upper aerodigestive tract. Patients with HNSCC, aged <45 years are categorized as young adults (YA). They are characterized by more severe form of this disease and often lack of classical, causative risk factors (tobacco smoking, alcohol abusing) in comparison to older (typical) patients (OP). The study purpose was to establish an anticipated protective role of DNA repair genes polymorphisms against cancer-causing agents. It was assumed that the polymorphisms in these genes may have a significant role in the etiology of HNSCC in YA. Studies were carried out on three groups: YA group with HNSCC (n = 90), young healthy group without cancer (YH, n = 160) and OP with HNSCC (n = 205). Three polymorphisms in DNA repair genes were analyzed: XPD ex23: A35931C, XRCC1 ex10: G28152A, and XRCC3 ex7: C18067T. The choice of these genes was connected with their involvement in three different DNA repair pathways. Genotyping was carried out by polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) technique. Statistical analysis included: calculation of odds ratio (ORs), 95 % confidence intervals (CIs) and p value. There was no significant difference in the distribution of XPD genotypes in YA compared to OP or YH. The XRCC1 AA genotype variant was observed less frequently in HNSCC YA (4.7 %) than in YH and in OP group (17.1 and 10.8 %, respectively). XRCC3 CT genotype variant was observed more frequently in HNSCC YA (61.8 %) than in YH (36.3 %) and this result is statistically significant. This variant was associated with the borderline increased risk of HNSCC development in an early age, however, a similar tendency was not observed in case of double mutated TT variant. The established differences of genotypes distribution do not seem to differentiate substantially YA and OP in head and neck cancer risk.

The effects of experimental administration of low doses of zearalenone on the histology of ovaries in pre-pubertal bitches.

The experiment involved 30 clinically healthy Beagle bitches aged approximately 70 days with an initial body weight of approximately 8 kg. The animals were randomly divided into two experimental groups (EI and EII) and a control group of 10 animals each. Group EI was administered 50 microg of body weight zearalenone/kg per os for 42 days, group EII received 75 microg of body weight zearalenone/kg per os for 42 days, and the control group was administered placeboper os for 42 days. The bitches were ovariectomized at the end of the treatment period for anatomopathological examination. At the same time, peripheral blood samples were collected for endocrinological analyses (17beta-estradiol and progesterone). Administartion of zearalenone particularly higher doses, resulted in the hyperestrogenism degeneration and atrophy of ovarian cells and tissues with accompanying edema and blood extravasation, leading to increased 17beta-estradiol concentrations and an insignificant decrease in progesterone levels.

The effect of experimental low zearalenone intoxication on ovarian follicles in pre-pubertal bitches.

Companion animals, including bitches, may be exposed to zearalenone (ZEN) toxins that are often present in feed, and ZEN intoxication may lead to ovarian dysfunction. This study involved evaluation of the degree of ZEN-induced hypo stimulation of ovary by determination of proliferative and apoptotic indices and description of the ultra-structural organization of ovarian follicles in pre-pubertal bitches subjected to experimental, long-term exposure to low-dose ZEN mycotoxicosis. The experiment involved 30 clinically healthy, immature Beagle bitches aged approximately 70 days with initial average body weight of 8 kg, randomly divided into three groups of 10 animals each: two experimental groups (EI and EII) and a control group (C). Over a period of 42 days, ZEN was administered per os to EI animals at a dose of 50 microg/kg BW, and to EII bitches at a dose of 75 microg/kg BW. Control group animals were fed placebo containing no ZEN for 42 days. Analytical samples of the mycotoxin were administered daily in gelatin capsules before morning feeding. All the bitches were subjected to ovario-hysterectomy at the end of the experiment. Proliferation index (PCNA method) and apoptotic index (TUNEL test) values were determined by immune-histological analyses. The median for apoptotic index was determined at 13.45 for group EI, 17.84 for group EII, and 8.59 for group C. The median for proliferation index was determined at 35.25 for group EII, 42.44 for group EI, and 70.60 for group C. The results of ultra-structural examinations of the ovaries revealed that experimental, ZEN-induced hyperestrogenism enhanced apoptosis and lowered the proliferative ability of follicular cells what contributed to organelle destruction in pre-pubertal bitches. The changes observed were particularly advanced in EII animals, which were administered a higher dose of ZEN.

Immunocytochemical expression of 3beta- and 17beta-hydroxysteroid dehydrogenase in bitch ovaries exposed to low doses of zearalenone.

Companion animals, including bitches, are exposed to intoxication with zearalenone (ZEN) present in feed, and the above may lead to reproductive disorders which are pften noted in veterinary practice. The aim of this study was to determine the effect of long-term and low-dose ZEN intoxication on the immunoexpression (optical density) of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in the ovaries of pre-pubertal bitches. The experiment involved 30 clinically healthy, immature Beagle bitches aged approximately 70 days with initial average body weight of 8 kg. The animals were randomly divided into two experimental groups (EI and EII) and a control group (C; n = 10 in all groups). Group EI was administered 50 microg ZEN/kg BW per os over a period of 42 days, group EII received 75 microg ZEN/kg BW per os for 42 days, and group C was administered placebo per os over a period of 42 days. Weighed amounts of the analyzed mycotoxin were administered every morning in gel capsules before feeding. The animals were subjected to ovariohysterectomy at the end of the experiment. Immunocytochemical analyses were performed to localize 3beta-HSD and 17beta-HSD enzymes and determine their optical density. The results revealed that changes in the immunoexpression of the above enzymes were inversely proportional to the applied dose of ZEN substrate.

The effect of T-2 toxin on percentages of CD4+, CD8+, CD4+ CD8+ and CD21+ lymphocytes, and mRNA expression levels of selected cytokines in porcine ileal Peyer's patches.

The immune system is one of the main toxicity targets of the T-2 toxin. In view of scant research data demonstrating the effect of T-2 on cellular and humoral responses in gut-associated lymphoid tissue (GALT), this study set out to investigate the effects of chronic exposure to low doses of the T-2 toxin (200 microg T-2 toxin kg(-1) feed) on percentages of CD4+ and CD8+ T lymphocytes, CD4+/CD8+ double-positive T lymphocytes, CD21+ B cells, and IL-2, IFN-gamma, IL-4 and IL-10 mRNA expression levels in porcine ileal Peyer's patches. The investigated material comprised ileum sections sampled from piglets (aged 8-10 weeks, body weight of 15-18 kg) on days 14, 28 and 42 of the experiment. After 42 days of exposure to T-2, a significant drop in the quantity of the IL-10 product was observed (R = 0.94; S.E. 0.49-0.79; p < 0.001). A gradual decrease in the amount of IL-4 and IFN-gamma cytokine transcripts was found throughout the experiment, but the reported trend was not significant. On experimental days 14 and 42, a significant increase in the percentage of CD8+ T lymphocytes was observed in comparison with the control (p = 0.04 and p = 0.05, respectively), whereas on day 28, a significant decrease in the percentage of the above subpopulation was noted (p = 0.00). The percentage of CD21+ B cells in the experimental group decreased steadily in comparison with the control, and the observed drop was significant on days 28 and 42 (p = 0.06 and p = 0.00, respectively). On days 14 and 28, the percentages of CD4+ and CD8+ T lymphocytes were lower in the experimental animals than in the control group, and the drop reported on day 28 was statistically significant (p = 0.03).

The effect of low-dose experimental zearalenone intoxication on the immunoexpression of estrogen receptors in the ovaries of pre-pubertal bitches.

Zearalenone is an estrogenic mycotoxin that often contaminates plant material used in the production of feeds for companion animals. Small daily doses of ingested zearalenone--a competitive substrate modulating the activity of enzymes participating in estrogen biosynthesis at the pre-receptor level--can induce subclinical symptoms of hyperestrogenism in bitches. The objective of this study was to determine the effects of low zearalenone doses on the presence of estrogen receptors in the ovaries of pre-pubertal Beagle bitches. The bitches were divided into three groups of 10 animals each: experimental group I--50 microg zearalenone/kg body weight administered once daily per os; experimental group II--75 microg zearalenone/kg body weight administered once daily per os; control group--placebo containing no ZEN administered per os. The animals were ovariorectomized at the end of the experiment, at 112 days of age. Estrogen receptors were detected in ovarian specimens by immunohistochemical methods. The results revealed an absence of estrogen receptors alpha in all groups. In both experimental groups a decrease in the positive response of estrogen receptors beta in specified structures of ovaries was observed. Very low alpha-zearalenol levels probably attested to the slowing down (hypostimulation) of the biotransformation process. Overall, zearalenone intoxication led to hyperestrogenism during a specific developmental stage of pre-pubertal bitches. As regards hormesis, the threshold dose of zearalenone (adaptive capability) was exceeded in the ovaries of experimental group II animals. The results obtained in both experimental groups suggest that long-term exposure to low-dose zearalenone intoxication decreased the degree of estrogen receptors beta staining in particular structures of ovaries in the experimental bitches, which initiated epigenetic modification mechanisms that inhibited ovarian development.

The low doses effect of experimental zearalenone (ZEN) intoxication on the presence of Ca2+ in selected ovarian cells from pre-pubertal bitches.

The objective of this study was to determine the effect of 42-day ZEN intoxication on the presence of Ca2+ in selected ovarian cells from beagle bitches, using the potassium pyroantimonate (PPA) method. Samples were collected from 30 clinically healthy, pre-pubertal, genetically homogeneous animals. The bitches were divided into three groups of 10 animals each: experimental group I--50 microg ZEN/kg BW (100% NOAEL) administered once daily per os; experimental group II--75 microg ZEN/kg BW (150% NOAEL) administered once dailyper os; control group--placebo containing no ZEN administered per os. An electron microscopic analysis revealed that cells died due to apoptosis, depending on the ZEN dose and the type of cells exposed to intoxication. Lower ZEN doses led to apoptosis-like changes in the cells. Cell death was a consequence of excess Ca2+ accumulation in the mitochondria, followed by cell dysfunction and a decrease in or the absence of mitochondrial metabolic activity in oocytes, follicle cells and interstitial cells in experimental bitches.

Zearalenone and deoxynivalenol mycotoxicosis in dairy cattle herds.

Mycotoxin contaminations pose a growing problem in animal production from the economic and toxicological point of view. Clinical symptoms of mycotoxicosis are relatively unspecific, making the disease difficult to diagnose. This study presents a clinical case of dairy cattle infected with natural mycotoxins produced by fungi of the genus Fusarium (zearalenone [ZEA] and deoxynivalenol [DON]) in eastern Poland. In dead and infected cows, the presence of ZEA and DON was determined in the blood serum, significant changes were observed in blood morphological and biochemical profiles, extravasations and bowel inflammations were also observed. The results reported testify to an acute autoimmune process in the intestines as well as immunosuppression.

The effect of exogenous proteases on selected hematological and serum biochemical parameters in boars.

The increasing interest in enzymatic growth promoters prompted this investigation of the effect of an enzymatic stimulating complex on selected hematological and serum biochemical parameters in boars. The enzymatic complex [five proteases (proteinases-endopeptidases) and two peptidases (exopeptidases) obtained by fermentation from Streptomyces fradiae] was added to diets for three months, at initial doses of 90 (group E1) and 120 (group E2) g/ton feed for the first seven days followed by 40 and 60 g/ton, respectively, in the subsequent weeks. The evaluation was based on selected hematological (RBC, WBC, HCT (Ht), HGB (Hb), MCV, MCH, and MCHC) and biochemical (AST, ALT, AP, Ca, Mg, urea, cholesterol, creatinine, inorganic phosphorus, and oxyglucose) parameters determined before and after the experimental period. The reported values were within physiological norms. Statistical differences were found between experimental groups with respect to WBC, HCT (Ht), MCV, MCH, AP, Ca, Mg, creatinine, and inorganic phosphorus. The study results indicate that the complex of proteolytic enzymes administered to boars caused no negative changes in their metabolic profiles. The statistically different hematological and serum biochemical parameters, while within normal limits, point to an early stage of microcytic anemia and heightened agitation that could be the result of intensified nitrogen metabolism.

The biotransformation of chosen mycotoxins.

Despite the unfavourable influence of mycotoxins on human and animal health and few toxicological aspects that have been documented, about these biologically active substances has not been explored. Aiming at more knowledge and a better understanding of the effects and mechanism of mycotoxin action in mammals would provide the basics for developing strategies to restrain different mycotoxicoses. One of the processes not fully understood is biotransformation, to which mycotoxins are subjected the animal organism. Biotransformation is the conversion of mycotoxins to non-toxic metabolites and occurs mostly in the intestinal mucosal membrane and liver, although other tissues and systems also take part in this process. Mycotoxin biotransformation reactions can be considered bioinactivation or detoxication, but mycotoxin biotransformation processes could also result in products more toxic than the mycotoxin. It can be concluded from research studies that our knowledge of mycotoxin biotransformation is scarce.

Influence of low doses of deoxynivalenol on histopathology of selected organs of pigs.

Deoxynivalenol is one of mycotoxins that are most frequently determined in animal feed manufactured in Poland. The examination of histopathological lesions concomitant with deoxynivalenol intoxication is difficult because of the common, often synergistic, reaction of this mycotoxin with other toxins, such as zearalenone or ochratoxin A, which has a strong nephrotoxic activity. The possibility of estimating histopathological lesions in the course of intoxication with pure toxin at various doses is therefore of interest. Dosages used in this experiment relate to clinical cases observed in feeding the animals with whole ration feed obtained by processing feedingstuffs contaminated with Fusarium moulds. However, concerning the fact of one-shot administration of clinically pure toxin, the main question was if it was a sufficient dose to cause changes in the histopathological picture of gastrointestinal tract organs. The experiment was carried out on 12 nursery pigs of mixed breed (Polish White Large x Polish White Ear-pendent) with an average body weigh of 35 kg. The experimental nursery pigs were divided into 3 groups: group I (n=4)--control; group II (n=4)--DON administered at a dose of 0.2 mg/kg b.w.; group III (n=4)--DON administered at a dose of 0.4 mg/kg b.w. After slaughter of the animals, macroscopic examination was performed and segments of duodenum, jejunum, ileum, liver and mesenteric lymph nodes were sampled and assigned for histopathological examination. The results obtained equate to the clinically observed signs in swine production involving some nutrient metabolism disturbances in the gastrointestinal tract in the course of deoxynivalenol mycotoxicosis. Histopathological examination of segments of the duodenum, the jejunum, the ileum, the liver and the lymph nodes indicate that the regressive lesions are more expressed in the experimental group treated with the highest concentration of deoxynivalenol.

Evaluation of the degree of pancreas activity in piglets from sows fed enzymatic stimulating complex.

One of the possibilities for estimating pancreas activity is the estimation of zymogene granule content in pancreatic follicular cells. In the present study, the degree of pancreatic activity was measured in piglets from sows receiving enzymatic stimulating complex throughout pregnancy and during the lactation period. The pancreas was collected for ultrastructural examination from 1-day-old and 21-day-old piglets. The enzyme preparation influenced the ultrastructural structure of the piglet pancreas, but the secretory cells in these animals did not confirm a more intensive course of creation and maturation processes. The accumulation of granules in extra-secretory pancreatic cells was observed, with a large volume of these granules and granular crinophagy observed in older piglets. The findings indicate a slow process of granule release, which may be the result of overproduction, lower requirements for enzymes contained in the granules, or both.

The evaluation of the efficacy of sodium carbonate as zearalenone destructor in feeding stuffs.

Contamination of feed with zearalenone (ZEA) is still a serious problem in farm animals feeding, especially in gilts, sensitive to this compound. The relative failure of current methods of decontamination and quality control lead us to look for new techniques. The commonly accepted method for breaking down ZEA was performed in controlled temperature and time conditions. Various sodium carbonate doses (0.5 - 4%) were added to feed naturally contaminated with ZEA (ZEA biosynthesis by F. graminearum isolates). These doses were found to be effective in in vitro studies. The addition of 2% sodium carbonate gave the best results in reducing the phytoestrogen in the feed.

Factors determining sensitivity of prepubertal gilts to hormonal influence of zearalenone.

Among large husbandry animals, swine are the most predisposed to zearalenone (ZEA) intoxication, mainly because cereal is an important component of their diet. Studies performed so far (in vivo, in vitro) suggest that ZEA and its metabolites, which may appear due to ZEA biotransformation (especially alpha-zearalenole; alpha-ZOL), can modify signaling cascades of endogenous sex steroids, through either receptor or non-receptor mechanisms. Of all age groups of swine, immature gilts are particularly predisposed to zearalenone intoxication, as manifested by the occurrence of genital tract tissue dysfunction on exposure to ZEA. The intensity of the adverse effects observed at either systemic or local level in gilts, when compared to sexually mature swine females, suggest that specific age-dependent physiological conditions may exist, which determine the high sensitivity of gilts to exogenous estrogen-like compounds, including ZEA.

Histopathological examination of ovaries in bitches after experimental zearalenone mycotoxicosis.

The present study deals with the influence of experimental ZEA mycotoxicosis on histopathological lesions in ovaries of bitches, which were administered zearalenone per os during anestrus phase for one hundred days. The experiment was performed on 9 sexually mature, clinically healthy bitches. The animals assigned into two experimental groups received zearalenone per os at two doses, 25 microg/kg b.w. and 50 microg/kg b.w., respectively: the bitches from control group received placebo per os. On the last day of zearalenone intoxication, the bitches were ovariohystorectomized. Histopathology and immunohistochemistry were performed. The study revealed that zearalenone and its metabolites caused profound regressive lesions: granular cells degeneration and atrophy. Numerous edemas and blood extravasations were also found. The intensity of these changes was significantly dose dependent. Furthermore, in ovarian cells and tissues of both experimental groups, no reaction for PCNA antigen was observed. In conclusion, zearalenone and its metabolites exerts unfavorable effects on the morphology of ovaries in bitches.