The Multidirectional Influence of Feed-Borne Deoxynivalenol and Zearalenone on Animal Health.

Mycotoxins are secondary fungal metabolites which pose a significant threat for global food and feed security [...].

Influence of deoxynivalenol and zearalenone on the immunohistochemical expression of oestrogen receptors and liver enzyme genes in vivo in prepubertal gilts.

Deoxynivalenol (DON) and zearalenone (ZEN) are often detected in plant materials used to produce feed for pre-pubertal gilts. Daily exposure to small amounts of these mycotoxins causes subclinical conditions in pigs and affects various biological processes (e.g. mycotoxin biotransformation). The aim of this preclinical study was to evaluate the effect of low monotonic doses of DON and ZEN (12 µg/kg body weight-BW-and 40 µg/kg BW, respectively), administered alone or in combination to 36 prepubertal gilts for 42 days, on the degree of immunohistochemical expression of oestrogen receptors (ERs) in the liver and the mRNA expression of genes encoding selected liver enzymes during biotransformation processes. The level of expression of the analysed genes proves that the tested mycotoxins exhibit variable biological activity at different stages of biotransformation. The biological activity of low doses of mycotoxins determines their metabolic activity. Therefore, taking into account the impact of low doses of mycotoxins on energy-intensive processes and their endogenous metabolism, it seems that the observed situation may lead to the activation of adaptation mechanisms.

A Cohort Study Investigating Zearalenone Concentrations and Selected Steroid Levels in Patients with Sigmoid Colorectal Cancer or Colorectal Cancer.

THE AIM: In this study was to determine if sigmoid colorectal cancer (SCC) and colorectal cancer (CRC) in women (W) and men (M) is accompanied by zearalenone (ZEN) mycotoxicosis and changes in selected steroid levels. MATERIALS AND METHODS: This cohort study was conducted on female and male patients selected from a population based on the presence of SCC or CRC, which was accompanied by the presence or absence (control group) of ZEN in their blood. The control group consisted of 17 patients with symptoms of SCC and CRC, where ZEN and its metabolites were not detected in the peripheral blood. The experimental groups comprised a total of 16 patients with SCC and CRC, where ZEN, but not its metabolites, was detected in their peripheral blood samples. RESULTS: In groups SCC and CRC, the ZEN levels were very high, in the range from 214 to 289 ng/mL of blood. Considerable variations were observed in the concentrations of steroid hormones. Estradiol (E2) levels ranged from 166.25 (group C) to 325 pg/mL (group CRC) in women and from 98 (group C) to 95.5 pg/mL (group CRC) in men. Progesterone (P4) levels ranged from 12.09 (group C) to 13.64 ng/mL (group SCC) in women and from 6.98 (group CRC) to 12.01 ng/mL (group C) in men. CONCLUSIONS: These results indicate that post-menopausal women and similarly aged elderly men have a high and individualized demand for estrogen that is relatively effectively met by ZEN, which triggers qualitative changes in estrogen receptors. The shortage of ZEN metabolites (values under the sensitivity of the method) confirmed the high estrogen demand in the studied subjects. The presence or absence of ZEN could have influenced the therapeutic outcomes in the analyzed patients.

Carry-Over of Zearalenone and Its Metabolites to Intestinal Tissues and the Expression of CYP1A1 and GSTπ1 in the Colon of Gilts before Puberty.

The objective of this study was to evaluate whether low doses of zearalenone (ZEN) affect the carry-over of ZEN and its metabolites to intestinal tissues and the expression of CYP1A1 and GSTπ1 in the large intestine. Prepubertal gilts (with a BW of up to 14.5 kg) were exposed in group ZEN to daily ZEN5 doses of 5 µg/kg BW (n = 15); in group ZEN10, 10 µg/kg BW (n = 15); in group ZEN15, 15 µg/kg BW (n = 15); or were administered a placebo (group C, n = 15) throughout the experiment. After euthanasia, tissues were sampled on exposure days 7, 21, and 42 (D1, D2, and D3, respectively). The results confirmed that the administered ZEN doses (LOAEL, NOAEL, and MABEL) were appropriate to reliably assess the carry-over of ZEN. Based on the observations made during 42 days of exposure to pure ZEN, it can be hypothesized that all mycotoxins (ZEN, α-zearalenol, and ß-zearalenol) contribute to a balance between intestinal cells and the expression of selected genes encoding enzymes that participate in biotransformation processes in the large intestine; modulate feminization processes in prepubertal gilts; and elicit flexible, adaptive responses of the macroorganism to mycotoxin exposure at the analyzed doses.

The Effect of 42-Day Exposure to a Low Deoxynivalenol Dose on the Immunohistochemical Expression of Intestinal ERs and the Activation of CYP1A1 and GSTP1 Genes in the Large Intestine of Pre-pubertal Gilts.

Deoxynivalenol (DON) is a mycotoxin that contaminates various plant materials. Exposure to DON can disrupt hormonal homeostasis, decrease body weight gains and modulate the immune system in pigs. It can also cause diarrhea, vomiting, leukocytosis, hemorrhaging or even death. Prolonged exposure to low doses of DON can have serious health implications in mammals. This is the first in vivo study to show that per os administration of low DON doses probably contributes to specific dysfunctions in steroidogenesis processes by inducing the immunohistochemical expression of estrogen receptors alpha (ERα) in the entire gastrointestinal tract in strongly stained cells (3 points) and estrogen receptors beta (ERß), but only in both investigated segments of the duodenum in pre-pubertal gilts. Therefore, the aim of this study was to determine whether a NOAEL dose of DON (12 µg DON/kg BW) administered per os over a period of 42 days induces changes in the immunohistochemical expression of ER in different intestinal segments and the transcriptional activation of CYP1A1 and GSTP1 genes in the large intestine of pre-pubertal gilts. This is the first report to demonstrate the expression of ER, in particular ERß, with the associated consequences. The expression of ER was accompanied by considerable variations in the activation of CYP1A1 and GSTP1 genes, but it supported the maintenance of a stable consensus between the degree of mycotoxin exposure and the detoxifying effect in pre-pubertal gilts.

Occurrence of Zearalenone and Its Metabolites in the Blood of High-Yielding Dairy Cows at Selected Collection Sites in Various Disease States.

Zearalenone (ZEN) and its metabolites, alpha-zearalenol (α-ZEL) and beta-zearalenol (ß-ZEL), are ubiquitous in plant materials used as feed components in dairy cattle diets. The aim of this study was to confirm the occurrence of ZEN and its selected metabolites in blood samples collected from different sites in the hepatic portal system (posthepatic-external jugular vein EJV; prehepatic-abdominal subcutaneous vein ASV and median caudal vein MCV) of dairy cows diagnosed with mastitis, ovarian cysts and pyometra. The presence of mycotoxins in the blood plasma was determined with the use of combined separation methods involving immunoaffinity columns, a liquid chromatography system and a mass spectrometry system. The parent compound was detected in all samples collected from diseased cows, whereas α-ZEL and ß-ZEL were not identified in any samples, or their concentrations were below the limit of detection (LOD). Zearalenone levels were highest in cows with pyometra, where the percentage share of average ZEN concentrations reached 44%. Blood sampling sites were arranged in the following ascending order based on ZEN concentrations: EJV (10.53 pg/mL, 44.07% of the samples collected from this site), ASV (14.20 pg/mL, 49.59% of the samples) and MCV (26.67 pg/mL, 67.35% of the samples). The results of the study indicate that blood samples for toxicological analyses should be collected from the MCV (prehepatic vessel) of clinically healthy cows and/or cows with subclinical ZEN mycotoxicosis. This sampling site increases the probability of correct diagnosis of subclinical ZEN mycotoxicosis.

Concentration of Zearalenone, Alpha-Zearalenol and Beta-Zearalenol in the Myocardium and the Results of Isometric Analyses of the Coronary Artery in Prepubertal Gilts.

The carry-over of zearalenone (ZEN) to the myocardium and its effects on coronary vascular reactivity in vivo have not been addressed in the literature to date. Therefore, the objective of this study was to verify the hypothesis that low ZEN doses (MABEL, NOAEL and LOAEL) administered per os to prepubertal gilts for 21 days affect the accumulation of ZEN, α-ZEL and ß-ZEL in the myocardium and the reactivity of the porcine coronary arteries to vasoconstrictors: acetylcholine, potassium chloride and vasodilator sodium nitroprusside. The contractile response to acetylcholine in the presence of a cyclooxygenase (COX) inhibitor, indomethacin and / or an endothelial nitric oxide synthase (e-NOS) inhibitor, L-NAME was also studied. The results of this study indicate that the carry-over of ZEN and its metabolites to the myocardium is a highly individualized process that occurs even at very low mycotoxin concentrations. The concentrations of the accumulated ZEN metabolites are inversely proportional to each other due to biotransformation processes. The levels of vasoconstrictors, acetylcholine and potassium chloride, were examined in the left anterior descending branch of the porcine coronary artery after oral administration of ZEN. The LOAEL dose clearly decreased vasoconstriction in response to both potassium chloride and acetylcholine (P < 0.05 for all values) and increased vasodilation in the presence of sodium nitroprusside (P = 0.021). The NOAEL dose significantly increased vasoconstriction caused by acetylcholine (P < 0.04), whereas the MABEL dose did not cause significant changes in the vascular response. Unlike higher doses of ZEN, 5 µg/kg had no negative influence on the vascular system.

Correlations between Low Doses of Zearalenone, Its Carryover Factor and Estrogen Receptor Expression in Different Segments of the Intestines in Pre-Pubertal Gilts-A Study Protocol.

Plant materials can be contaminated with Fusarium mycotoxins and their derivatives, whose toxic effects on humans and animals may remain subclinical. Zearalenone (ZEN), a low-molecular-weight compound, is produced by molds in crop plants as a secondary metabolite. The objective of this study will be to analyze the in vivo correlations between very low monotonic doses of ZEN (5, 10, and 15 µg ZEN/kg body weight-BW for 42 days) and the carryover of this mycotoxin and its selected metabolites from the intestinal contents to the intestinal walls, the mRNA expression of estrogen receptor alfa (ERα) and estrogen receptor beta (ERß) genes, and the mRNA expression of genes modulating selected colon enzymes (CYP1A1 and GSTP1) in the intestinal mucosa of pre-pubertal gilts. An in vivo experiment will be performed on 60 clinically healthy animals with initial BW of 14.5 ± 2 kg. The gilts will be randomly divided into a control group (group C, n = 15) and three experimental groups (group ZEN5, group ZEN10, and group ZEN15; n = 15). Group ZEN5 will be administered per os 5 µg ZEN/kg BW (MABEL), group ZEN10-10 µg ZEN/kg BW (NOAEL), and group ZEN15-15 µg ZEN/kg BW (low LOAEL). In each group, five animals will be euthanized on analytical dates 1 (exposure day 7), 2 (exposure day 21), and 3 (exposure day 42). Samples for in vitro analyses will be collected from an intestinal segment resected from the following regions: the third (horizontal) part of the duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, and descending colon. The experimental material will be collected under special conditions, and it will be transported to specialist laboratories where samples will be obtained for further analyses.

The Presence of Mycotoxins in Feed and Their Influence on Animal Health.

Mycotoxins are secondary metabolites of fungi [...].

The Effect of Different Doses of Zearalenone in Feed on the Bioavailability of Zearalenone and Alpha-Zearalenol, and the Concentrations of Estradiol and Testosterone in the Peripheral Blood of Pre-Pubertal Gilts.

The objective of this study was to determine the effect of long-term (48 days), per os administration of specific zearalenone (ZEN) doses (20 and 40 µg ZEN/kg BW in experimental groups EI and EII, which were equivalent to 200% and 400% of the upper range limit of the no-observed-adverse-effect-level (NOAEL), respectively) on the bioavailability of ZEN and the rate of changes in estradiol and testosterone concentrations in the peripheral blood of pre-pubertal gilts. ZEN and α-ZEL levels were similar until day 28. After day 28, α-ZEL concentrations increased significantly in group EI, whereas a significant rise in ZEN levels was noted in group EII. The presence of estradiol in peripheral blood plasma was not observed until day 20 of the experiment. Spontaneous secretion of estradiol was minimal, and it was determined at very low levels of up to 10 pg/mL in EI and EII groups. Testosterone concentrations ranged from 4 to 9 ng/mL in all groups. A decrease in the concentrations of both analyzed hormones was reported in the last stage of the experiment. The results of the experiment indicate that: (i) The bioavailability of ZEN in peripheral blood has low diagnostic value, (ii) exposure to low doses of ZEN induces minor changes in the concentrations of the analyzed hormones, which could lead to situational supraphysiological hormone levels and changes in endogenous hormonal balance.

Correlations between exposure to deoxynivalenol and zearalenone and the immunohistochemical expression of estrogen receptors in the intestinal epithelium and the mRNA expression of selected colonic enzymes in pre-pubertal gilts.

Plant-based materials used in the production of pig feed are very often contaminated with deoxynivalenol and zearalenone. Daily intake of small amounts of these mycotoxins with feed induces various subclinical states in gilts and influences different biological processes. The aim of this preclinical study was to determine the correlation between monotonic doses of zearalenone and deoxynivalenol (40 µg/kg body weight and 12 µg/kg body weight, respectively, administered over a period of 42 days) and the immunohistochemical expression of estrogen receptors in the intestinal tract and the mRNA expression of selected colonic enzymes. The immunohistochemical expression of estrogen receptor alpha was observed in the colon, but its intensity varied in different weeks of exposure. A minor increase in estrogen receptor beta expression was noted only in the colon, whereas the expression of cytochrome P450 1A1 enzyme mRNA and mRNA isoform of the glutathione S-transferase π gene decreased. The observed correlations suggest that the risk of loss of control over the biotransformation and biological activity of the parent compounds in distal intestinal mucosa is delayed.

Imbalance in the Blood Concentrations of Selected Steroids in Pre-pubertal Gilts Depending on the Time of Exposure to Low Doses of Zearalenone.

Zearalenone (ZEN) is a mycotoxin that not only binds to estrogen receptors, but also interacts with steroidogenic enzymes and acts as an endocrine disruptor. The aim of this study was to verify the hypothesis that low doses, minimal anticipated biological effect level (MABEL), no-observed-adverse-effect level (NOAEL) and lowest-adverse-effect level (LOAEL), of ZEN administered orally for 42 days can induce changes in the peripheral blood concentrations of selected steroid hormones (estradiol, progesterone and testosterone) in pre-pubertal gilts. The experiment was performed on 60 clinically healthy gilts with average BW of 14.5 ± 2 kg, divided into three experimental groups and a control group. Group ZEN5 animals were orally administered ZEN at 5 µg ZEN/kg BW, group ZEN10 - at 10 µg ZEN/kg BW, group ZEN15 - at 15 µg ZEN/kg BW, whereas group C received a placebo. Five gilts from every group were euthanized on analytical dates 1, 2 and 3 (days 7, 14 and 42 of the experiment). Qualitative and quantitative changes in the biotransformation of low ZEN doses were observed. These processes were least pronounced in group ZEN5 (MABEL dose) where ZEN metabolites were not detected on the first analytical date, and where ß-ZEL was the predominant metabolite on successive dates. The above was accompanied by an increase in the concentration of estradiol (E2) which, together with "free ZEN", probably suppressed progesterone (P4) and testosterone (T) levels.

Time-Dependent Changes in the Intestinal Microbiome of Gilts Exposed to Low Zearalenone Doses.

Zearalenone is a frequent contaminant of cereals and their by-products in regions with a temperate climate. This toxic molecule is produced naturally by Fusarium fungi in crops. The aim of this study was to determine the influence of low zearalenone doses (LOAEL, NOAEL and MABEL) on the intestinal microbiome of gilts on different days of exposure (days 7, 21 and 42). Intestinal contents were sampled from the duodenal cap, the third part of the duodenum, jejunum, caecum and the descending colon. The experiment was performed on 60 clinically healthy gilts with average BW of 14.5 ± 2 kg, divided into three experimental groups and a control group. Group ZEN5 animals were orally administered ZEN at 5 µg /kg BW, group ZEN10-10 µg ZEN/kg BW and group ZEN15-15 µg ZEN/kg BW. Five gilts from every group were euthanized on analytical dates 1, 2 and 3. Differences in the log values of microbial counts, mainly Escherichia coli and Enterococcus faecalis, were observed between the proximal and distal segments of the intestinal tract on different analytical dates as well as in the entire intestinal tract. Zearalenone affected the colony counts of intestinal microbiota rather than microbiome diversity, and its effect was greatest in groups ZEN10 and ZEN15. Microbial colony counts were similar in groups ZEN5 and C. In the analysed mycobiome, ZEN exerted a stimulatory effect on the log values of yeast and mould counts in all intestinal segments, in particular in the colon, and the greatest increase was noted on the first analytical date.

The Genotoxicity of Caecal Water in Gilts Exposed to Low Doses of Zearalenone.

Zearalenone is a toxic low-molecular-weight molecule that is naturally produced by moulds on crops as a secondary metabolite. The aim of this study was to determine the genotoxicity of caecal water collected successively from the caecal contents of gilts exposed to low doses (LOAEL, NOAEL, and MABEL) of zearalenone. The experiment was performed on 60 clinically healthy gilts with average BW of 14.5 ± 2 kg, divided into three experimental groups and a control group. Group ZEN5 were orally administered ZEN at 5 µg/kg BW, group ZEN10-10 µg ZEN/kg BW and group ZEN15-15 µg ZEN/kg BW. Five gilts from every group were euthanized on analytical dates 1, 2, and 3. Caecal water samples for in vitro analysis were collected from the ileocaecal region. The genotoxicity of caecal water was noted, particularly after date 1 in groups ZEN10 and ZEN15 with a decreasing trend. Electrophoresis revealed the presence of numerous comets without tails in groups C and ZEN5 and fewer comets with clearly expressed tails in groups ZEN10 and ZEN15. The distribution of LLC-PK1 cells ranged from 15% to 20% in groups C and ZEN5, and from 30% to 60% in groups ZEN10 and ZEN15. The analysis of caecal water genotoxicity during exposure to very low doses of ZEN revealed the presence of a counter response and a compensatory effect in gilts.

Mycotoxin levels in the digestive tissues of immature gilts exposed to zearalenone and deoxynivalenol.

Most plant materials are contaminated with small doses of Fusarium mycotoxins and its modified forms that exert subclinical toxic effects on humans and animals. The aim of this study was to evaluate the carry-over of zearalenone and deoxynivalenol (pure parent compounds) to intestinal and liver tissues during 6 weeks of exposure to mycotoxins administered per os to gilts. The experiment was performed on 36 gilts with average body weight of 25 ±â€¯2 kg, divided into 2 groups: an experimental group (group E, administered zearalenone at 40 µg/kg BW and deoxynivalenol at 12 µg/kg BW daily with feed) and a control group administered placebo. Tissue saturation with mycotoxins was analysed by liquid chromatography in samples collected at weekly intervals. Six gilts were euthanized in each week of the study. The conducted analyses revealed: (i) a non-uniform increase in zearalenone levels in the duodenum, jejunum, ascending colon and the liver; and (ii) an increase in deoxynivalenol levels, mainly in the ileum, caecum, ascending colon and the transverse colon, and a minor increase in the liver. The degree of tissue saturation was determined by the type of mycotoxin, but not by the time of exposure.

Growth performance, selected blood biochemical parameters and body weights of pre-pubertal gilts fed diets supplemented with different doses of zearalenone (ZEN).

The aim of this study was to determine whether exposure to low doses of zearalenone (ZEN) induces changes in the serum biochemical profile and body weights (BW). Pre-pubertal gilts (with BW of up to 14.5 kg) were administered ZEN in daily doses of 5 µg/kg BW (group 1, n = 15), 10 µg/kg BW (group 2, n = 15), 15 µg/kg BW (group 3, n = 15) or placebo (control group C, n = 15) throughout the experiment. Blood was sampled for analysis on 10 dates (at five-day intervals). Minor but statistically significant differences in the analysed serum biochemical parameters (ALT, AST, ALP, total cholesterol, total bilirubin, glucose, total protein, iron, BUN and urea) were observed in the studied groups. The biochemical parameters of the analysed gilts indicate that the maintenance of homeostasis and biotransformation of ZEN require considerable energy expenditure. Beginning on the fourth analytical date, BW gains were consistently higher in the experimental groups than in group C. The observed decrease in glucose and total protein levels can probably be attributed to higher BW gains and the ongoing ZEN biotransformation processes in the enterocytes and the liver.