Dietary Risk Assessment and Consumer Awareness of Mycotoxins among Household Consumers of Cereals, Nuts and Legumes in North-Central Nigeria.

This study characterized the health risks due to the consumption of mycotoxin-contaminated foods and assessed the consumer awareness level of mycotoxins in households in two north-central Nigerian states during the harvest and storage seasons of 2018. Twenty-six mycotoxins and 121 other microbial and plant metabolites were quantified by LC-MS/MS in 250 samples of cereals, nuts and legumes. Aflatoxins were detected in all food types (cowpea, maize, peanut and sorghum) except in millet. Aflatoxin B1 was the most prevalent mycotoxin in peanut (64%) and rice (57%), while fumonisin B1 occurred most in maize (93%) and beauvericin in sorghum (71%). The total aflatoxin concentration was highest in peanut (max: 8422 µg/kg; mean: 1281 µg/kg) and rice (max: 955 µg/kg; mean: 94 µg/kg), whereas the totals of the B-type fumonisins and citrinin were highest in maize (max: 68,204 µg/kg; mean: 2988 µg/kg) and sorghum (max: 1335 µg/kg; mean: 186 µg/kg), respectively. Citrinin levels also reached 51,195 µg/kg (mean: 2343 µg/kg) in maize. Aflatoxin and citrinin concentrations in maize were significantly (p < 0.05) higher during storage than at harvest. The estimated chronic exposures to aflatoxins, citrinin and fumonisins were high, resulting in as much as 247 new liver cancer cases/year/100,000 population and risks of nephrotoxicity and esophageal cancer, respectively. Children who consumed the foods were the most vulnerable. Mycotoxin co-occurrence was evident, which could increase the health risk of the outcomes. Awareness of mycotoxin issues was generally low among the households.

A 28-day repeated oral dose toxicity study of enniatin complex in mice.

Enniatins are so-called "emerging mycotoxins" that commonly occur in milligrams per kilogram levels in grains and their derived products, as well as in fish, dried fruits, nuts, spices, cocoa, and coffee. The present study investigated the 28-day repeated oral dose toxicity of enniatin complex in CD1(ICR) mice. Enniatin B, enniatin B1, and enniatin A1 at a ratio of 4:4:1 were administered to male and female mice at doses of 0 (vehicle controls), 0.8, 4, and 20 mg/kg body weight/day. In life parameters did not change during the study period, with the exception of slight reductions in food consumption in male mice administered 4 and 20 mg/kg and in female mice administered 20 mg/kg. Body and organ weights did not change, and no alterations in hematology, blood biochemistry, or histopathology parameters were observed at the end of the administration period. Thus, we determined that the no-observed-adverse-effect level of enniatin complex was 20 mg/kg/day for both sexes under the present experimental conditions.

Oral subchronic exposure to the mycotoxin ochratoxin A induces key pathological features of Parkinson's disease in mice six months after the end of the treatment.

Some epidemiological studies with different levels of evidence have pointed to a higher risk of Parkinson's disease (PD) after exposure to environmental toxicants. A practically unexplored potential etiological factor is a group of naturally-occurring fungal secondary metabolites called mycotoxins. The mycotoxin ochratoxin A (OTA) has been reported to be neurotoxic in mice. To further identify if OTA exposure could have a role in PD pathology, Balb/c mice were orally treated with OTA (0.21, 0.5 mg/kg bw) four weeks and left for six months under normal diet. Effects of OTA on the onset, progression of alpha-synuclein pathology and development of motor deficits were evaluated. Immunohistochemical and biochemical analyses showed that oral subchronic OTA treatment induced loss of striatal dopaminergic innervation and dopaminergic cell dysfunction responsible for motor impairments. Phosphorylated alpha-synuclein levels were increased in gut and brain. LAMP-2A protein was decreased in tissues showing alpha-synuclein pathology. Cell cultures exposed to OTA exhibited decreased LAMP-2A protein, impairment of chaperone-mediated autophagy and decreased alpha-synuclein turnover which was linked to miRNAs deregulation, all reminiscent of PD. These results support the hypothesis that oral exposure to low OTA doses in mice can lead to biochemical and pathological changes reported in PD.

The effects of feed naturally contaminated with Fusarium mycotoxins on the thymus in suckling piglets.

In this study, feed naturally containing Fusarium mycotoxins was fed to gilts during the perinatal period, and the effects on the thymus were investigated in one-week-old piglets. Twenty gilts were divided into equal control (0.26 mg deoxynivalenol, DON) and experimental (5.08 mg DON, 0.09 mg zearalenone and 21.61 mg fusaric acid per kg of feed) groups. One suckling piglet from each litter (n = 20) was sacrificed at one week of age to obtain thymus samples for further analysis. The cortex to medulla ratio of the thymus was morphometrically analysed using NIS Elements BR (Nikon) software. Paraffin-embedded thymus sections were stained to quantify apoptosis (with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling - TUNEL method), cellular proliferation (Ki-67) and macrophages (MAC 387). The results showed that the thymus cortex (P = 0.023) to medulla (P = 0.023) ratio was significantly lower in the experimental group. The number of apoptotic cells (cortex, P = 0.010, medulla, P = 0.001) and the number of proliferating cells in the thymus cortex (P = 0.001) and medulla (P < 0.001) were significantly higher in the experimental group. Our results indicate that feeding Fusarium mycotoxins to a parent animal during the perinatal period induces significant alterations in the thymus of one-week-old piglets, which indicates an immunosuppressive effect in piglets.

Maternal Supplementation of Food Ingredient (Prebiotic) or Food Contaminant (Mycotoxin) Influences Mucosal Immune System in Piglets.

The early life period is crucial for the maturation of the intestinal barrier, its immune system, and a life-long beneficial host-microbiota interaction. The study aims to assess the impact of a beneficial dietary (short-chain fructooligosaccharides, scFOS) supplementation vs. a detrimental dietary environment (such as mycotoxin deoxynivalenol, DON) on offspring intestinal immune system developmental profiles. Sows were given scFOS-supplemented or DON-contaminated diets during the last 4 weeks of gestation, whereas force-feeding piglets with DON was performed during the first week of offspring life. Intestinal antigen-presenting cell (APC) subset frequency was analyzed by flow cytometry in the Peyer's patches and in lamina propria and the responsiveness of intestinal explants to toll-like receptor (TLR) ligands was performed using ELISA and qRT-PCR from post-natal day (PND) 10 until PND90. Perinatal exposure with scFOS did not affect the ontogenesis of APC. While it early induced inflammatory responses in piglets, scFOS further promoted the T regulatory response after TLR activation. Sow and piglet DON contamination decreased CD16+ MHCII+ APC at PND10 in lamina propria associated with IFNγ inflammation and impairment of Treg response. Our study demonstrated that maternal prebiotic supplementation and mycotoxin contamination can modulate the mucosal immune system responsiveness of offspring through different pathways.

Oral exposure of pigs to the mycotoxin deoxynivalenol does not modulate the hepatic albumin synthesis during a LPS-induced acute-phase reaction.

The sensitivity of pigs to deoxynivalenol (DON) might be influenced by systemic inflammation (SI) which impacts liver. Besides following acute-phase proteins, our aim was to investigate both the hepatic fractional albumin (ALB) synthesis rate (FSR) and the ALB concentration as indicators of ALB metabolism in presence and absence of SI induced by LPS via pre- or post-hepatic venous route. Each infusion group was pre-conditioned either with a control diet (CON, 0.12 mg DON/kg diet) or with a DON-contaminated diet (DON, 4.59 mg DON/kg diet) for 4 wk. A depression of ALB FSR was observed 195 min after LPS challenge, independent of feeding group or LPS application route, which was not paralleled by a down-regulated ALB mRNA expression but by a reduced availability of free cysteine. The drop in ALB FSR only partly explained the plasma ALB concentrations which were more depressed in the DON-pre-exposed groups, suggesting that ALB levels are influenced by further mechanisms. The abundances of haptoglobin, C-reactive protein, serum amyloid A, pig major acute-phase protein, fibrinogen and LPS-binding protein mRNA were up-regulated upon LPS stimulation but not accompanied by increases in the plasma concentrations of these proteins, pointing at an imbalance between synthesis and consumption.

Efficacy of Mycotoxin Detoxifiers on Health and Growth of Newly-Weaned Pigs under Chronic Dietary Challenge of Deoxynivalenol.

The efficacy of yeast-based mycotoxin detoxifiers on health and growth performance of newly-weaned pigs (27-d-old) fed diets naturally contaminated with deoxynivalenol was investigated. Sixty pigs were individually assigned to five treatments for 34 d: NC (negative control, 1.2 mg/kg of deoxynivalenol); PC (positive control, 3.2 mg/kg of deoxynivalenol); CYC (PC + clay/yeast culture-based product, 0.2%); CYE (PC + clay/yeast cell wall/plant extracts/antioxidants-based product, 0.2%); and CYB (PC + clay/inactivated yeast/botanicals/antioxidants-based product, 0.2%). Blood and jejunal mucosa were sampled, and data were analyzed using Proc Mixed of SAS with pre-planned contrasts. Deoxynivalenol reduced the average daily gain (ADG) in phase 3. Pigs fed CYC had greater overall ADG, average daily feed intake during phase 3, and gain to feed ratio during phase 2 than PC. At d 14, deoxynivalenol reduced blood urea nitrogen/creatinine and tended to reduce blood urea nitrogen. Pigs fed CYB tended to have greater aspartate aminotransferase than PC. At d 34, pigs fed CYC and CYB tended to have lower serum creatine phosphokinase than PC. Pigs fed CYE had lower blood urea nitrogen/creatinine than PC. In jejunal mucosa, deoxynivalenol tended to increase malondialdehydes and decrease glutathione. Pigs fed CYE and CYB had lower malondialdehydes, pigs fed CYB had greater glutathione and tended to have lower immunoglobulin A than PC. Pigs fed CYC and CYE tended to have lower interleukin 8 than PC. In summary, deoxynivalenol challenge (1.2 vs. 3.2 mg/kg) mildly compromised growth performance and increased the oxidative stress of pigs. Mycotoxin detoxifiers could partially overcome deoxynivalenol toxicity enhancing liver health, whereas CYE and CYB reduced oxidative stress, and CYC and CYB reduced immune activation. In conclusion, yeast-based detoxifiers with functional components as clay/inactivated yeast/botanicals/antioxidants had increased detoxifying properties in newly-weaned pigs challenged with deoxynivalenol, potentially by enhancing adsorbability, immune function, gut health, and reducing oxidative stress.

In Vitro Toxicokinetics and Phase I Biotransformation of the Mycotoxin Penitrem A in Dogs.

The tremorgenic mycotoxin penitrem A is produced by Penicillium species as a secondary metabolite on moldy food and feed. Dogs are sometimes exposed to penitrem A by consumption of spoiled food waste or fallen fruit. The lipophilic toxin crosses the blood-brain barrier and targets neuroreceptors and neurotransmitter release mechanisms in the central and peripheral nervous systems. Typical symptoms of penitrem A intoxication are periodical or continuous tremors, which can be passing, persistent or lethal, depending on the absorbed dose. There is presently no information on the biotransformation and toxicokinetics of penitrem A in dogs. The aim of the present study was therefore to identify potential metabolites of the toxin by performing in vitro biotransformation assays in dog liver microsomes. Analyses by liquid chromatography coupled to high-resolution mass spectrometry led to the provisional identification of eleven penitrem A phase I metabolites, which were tentatively characterized as various oxidation products. Furthermore, elimination parameters determined in in vitro assays run under linear kinetics were used for in vitro-to-in vivo extrapolation of the toxicokinetic data, predicting a maximal bioavailability of more than 50%. The metabolite profile detected in the in vitro assays was similar to that observed in the plasma of an intoxicated dog, confirming the predictive capability of the in vitro approach.

Dietary exposure to mycotoxins in the French infant total diet study.

The present study evaluated the exposure of children aged from one to 36 months to seven groups of mycotoxins, in the context of the infant French Total Diet Study (iTDS). Exposure was then compared to the health-based guidance values (HBGVs) for each mycotoxin. The value of the 90th percentile of exposure to nivalenol, patulin, fumonisins and zearalenone was less than 40% of the HBGV considered relevant for children. On the other hand, a risk could not be excluded for ochratoxin A and aflatoxins as exposure was close to the HBGV for ochratoxin A and the margin of exposure was much lower than the critical margin of 10,000 for aflatoxins. The HBGVs for toxins T2 and HT2, and for deoxynivalenol (DON) and its acetylated compounds were exceeded. Five percent to 10% of the children aged 5-12 months exceeded the HBGV considering the lower bound hypothesis for toxins T2 and HT2 and 7.5%-27% of the children aged 5 months and above exceeded the HBGV for DON. Consequently, the exposure of young children raises safety concerns for T2/HT2 and DON. Efforts should therefore be pursued to decrease their exposure to these molecules.

Evaluation of the cytotoxic and genotoxic effects of mycotoxin fusaric acid.

Fusaric acid (FA) is produced by several Fusarium species and is commonly found in grains. This investigation was performed to evaluate the cytotoxic and genotoxic effects of FA either in human cervix carcinoma (HeLa) cell line using 3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT) assay and in human lymphocytes using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (MN) as well as comet assay in vitro. The cells were treated with 0.78, 1.56, 3.125, 6.25, 12.50, 25, 50, 100, 200, and 400 µg/mL concentrations of FA. It has potent cytotoxic effect on HeLa cell line measured by MTT assay especially at higher concentrations (200, 400 µg/mL). The half of inhibitory concentration (IC50) evidenced by FA in the HeLa cells was 200 µg/mL at 24 h and between 200 and 400 µg/mL at 48 h. It was also observed that FA produced a significant decrease in mitotic index (MI) at 12.50 µg/mL compared to solvent control. Furthermore, it indicated a cytotoxic effect at the concentrations ranging from 25 to 400 µg/mL in human lymphocytes. The results of this research point out that being exposed to FA at high concentrations show cytotoxicity. Besides FA induced comet tail intensity at 3.125, 6.25, and 12.50 µg/mL concentrations in isolated human lymphocytes. On the other hand, no genotoxic effects were seen in human lymphocytes in vitro using CA, SCE and MN assays.

Mycotoxin management in a developing country context: A critical review of strategies aimed at decreasing dietary exposure to mycotoxins in Zimbabwe.

Mycotoxins are unavoidable environmental contaminants, which are found throughout the food chain, particularly in cereals. Mycotoxin management is not effective in developing countries, such as Zimbabwe, due to resource constraints, yet human health risk is evident. Various practical mitigation strategies that can be employed to decrease human dietary exposure to mycotoxins as a means of preliminary steps towards risk management are discussed. These strategies were stratified into two categories. First, crop/commodity-centred strategies, mainly the pre-harvest actions of cultivar selection, bio-control, as well as good agricultural practices (GAP), and the post-harvest actions including timeous harvesting, appropriate drying and storage technologies, are elaborated making use of hazard analysis critical control points (HACCP) principles. The role of legislation is also explored as a crop/commodity centred mitigation strategy. Second, human-centred strategies anchored on dietary diversity and the use of socio-cultural approaches as a direct means of reducing mycotoxin exposure are discussed. Finally, an integrated science-based mycotoxin management strategy, encompassing targeted legislation on mycotoxins, consumer education and information sharing, human and institutional capacity building, training and financing, is suggested in addition to GAP, as a means of reducing human health risk associated with mycotoxin exposure in Zimbabwe.HighlightsFarm-to-fork HACCP-based mycotoxin managementHuman-centred mycotoxin management approaches are keyAgronomy, technology and legislation critical in reducing mycotoxin exposure.

Metabolic Profile, Bioavailability and Toxicokinetics of Zearalenone-14-Glucoside in Rats after Oral and Intravenous Administration by Liquid Chromatography High-Resolution Mass Spectrometry and Tandem Mass Spectrometry.

Zearalenone-14-glucoside (ZEN-14G), a key modified mycotoxin, has attracted a great deal of attention due to the possible conversion to its free form of zearalenone (ZEN) exerting toxicity. In this study, the toxicokinetics of ZEN-14G were investigated in rats after oral and intravenous administration. The plasma concentrations of ZEN-14G and its major five metabolites were quantified using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The data were analyzed via non-compartmental analysis using software WinNonlin 6.3. The results indicated that ZEN-14G was rapidly hydrolyzed into ZEN in vivo. In addition, the major parameters of ZEN-14G following intravenous administration were: area under the plasma concentration-time curve (AUC), 1.80 h·ng/mL; the apparent volume of distribution (VZ), 7.25 L/kg; and total body clearance (CL), 5.02 mL/h/kg, respectively. After oral administration, the typical parameters were: AUC, 0.16 h·ng/mL; VZ, 6.24 mL/kg; and CL, 4.50 mL/h/kg, respectively. The absolute oral bioavailability of ZEN-14G in rats was about 9%, since low levels of ZEN-14G were detected in plasma, which might be attributed to its extensive metabolism. Therefore, liquid chromatography high-resolution mass spectrometry (LC-HRMS) was adopted to clarify the metabolic profile of ZEN-14G in rats' plasma. As a result, eight metabolites were identified in which ZEN-14-glucuronic acid (ZEN-14GlcA) had a large yield from the first time-point and continued accumulating after oral administration, indicating that ZEN-14-glucuronic acid could serve a potential biomarker of ZEN-14G. The obtained outcomes would prompt the accurate safety evaluation of ZEN-14G.

Naturally occurring mixtures of Alternaria toxins: anti-estrogenic and genotoxic effects in vitro.

Alternaria molds can produce a variety of different mycotoxins, often resulting in food contamination with chemical mixtures, posing a challenge for risk assessment. Some of these metabolites possess estrogenic properties, an effect whose toxicological relevance is questioned in the light of the strong genotoxic and cytotoxic properties of co-occurring toxins. Thus, we tested a complex extract from A. alternata for estrogenic properties in Ishikawa cells. By assessing alkaline phosphatase activity, we did not observe estrogen receptor (ER) activation at non-cytotoxic concentrations (≤ 10 µg/ml). Furthermore, an extract stripped of highly genotoxic perylene quinones also did not mediate estrogenic effects, despite diminished genotoxic properties in the comet assay (≥ 10 µg/ml). Interestingly, both extracts impaired the estrogenicity of 17ß-estradiol (E2) at non-cytotoxic concentrations (5-10 µg/ml), indicating anti-estrogenic effects which could not be explained by the presence of known mycoestrogens. A mechanism for this unexpected result might be the activation of the aryl hydrocarbon receptor (AhR) by Alternaria metabolites, as indicated by the induction of CYP1A1 transcription. While a direct influence on the metabolism of E2 could not be confirmed by LC-MS/MS, literature describing a direct interplay of the AhR with estrogenic pathways points to a corresponding mode of action. Taken together, the present study indicates AhR-mediated anti-estrogenic effects as a novel mechanism of naturally co-occurring Alternaria toxin mixtures. Furthermore, our results confirm their genotoxic activity and raise questions about the contribution of still undiscovered metabolites to toxicological properties.

Lack of Toxic Interaction Between Fusariotoxins in Broiler Chickens Fed throughout Their Life at the Highest Level Tolerated in the European Union.

Fusarium mycotoxins (FUS) occur frequently in poultry diets, and regulatory limits are laid down in several countries. However, the limits were established for exposure to a single mycotoxin, whereas multiple contamination is more realistic, and different studies have demonstrated that it is not possible to predict interactions between mycotoxins. The purpose of this study was thus to compare the toxic effect of deoxynivalenol (DON), fumonisins (FB) and zearalenone (ZON), alone and in combination on broiler chickens, at the maximum tolerated level established by the EU for poultry feed. Experimental corn-soybean diets incorporated ground cultured toxigenic Fusarium strains. One feed was formulated for chickens 0 to 10 days old and another for chickens 11 to 35 days old. The control diets were mycotoxin free, the DON diets contained 5 mg DON/kg, the FB diet contained 20 mg FB1 + FB2/kg, and the ZON diet contained 0.5mg ZON/kg. The DONFBZON diet contained 5, 20, and 0.5 mg/kg of DON, FB1 + FB2, and ZON, respectively. Diets were distributed ad libitum to 70 broilers (male Ross PM3) separated into five groups of 14 chickens each reared in individual cages from one to 35 days of age. On day 35, after a starvation period of 8 h, a blood sample was collected, and all the animals were killed and autopsied. No difference between groups that could be attributed to FUS was observed in performances, the relative weight of organs, biochemistry, histopathology, intestinal morphometry, variables of oxidative damage, and markers of testicle toxicity. A significant increase in sphinganine and in the sphinganine to sphingosine ratio was observed in broilers fed FB. Taken together, these results suggest that the regulatory guidelines established for single contamination of broiler chickens fed with DON, FB, and ZON can also be used in the case of multiple contamination with these toxins.

Treatment with potassium bromide mitigates ataxia and reduces tremor in lambs with perennial ryegrass toxicosis.

Aims: To assess the use of potassium bromide (KBr) as a therapeutic intervention for perennial ryegrass toxicosis (PRGT) in lambs fed ryegrass seed containing lolitrem B. Methods: Male lambs aged 10-12 months (n = 43) were assigned to receive ryegrass seed containing lolitrem B, at a dose of 0.16 mg/kg/day (Groups 2, 3 and 4), or lucerne chaff and molasses (Groups 1 and 5). Lambs in Groups 2 and 3 were observed for clinical signs and gait changes until defined signs of PGRT were observed, when they were transferred, with lambs in Group 1, to the Testing phase of the trial. Lambs in Group 3 were then treated with a single oral dose of 300 mg/kg bromide. Lambs in Groups 4 and 5 received KBr daily from the start of the trial (540 mg/kg bromide over 3 days then 20 mg/kg daily) and were transferred to the Testing phase after 18 days. Clinical examination and gait assessment, and surface electromyography of the triceps muscle, measuring root-mean-square (RMS) voltages, were carried out on Days 0, 1 and 2 of the Testing phase followed by necropsy, histopathology, measurement of concentrations of bromide in serum and CSF and faecal cortisol metabolites (FCM). Results: In Group 3 lambs, mean composite gait scores decreased between Testing phase Day 0 and Days 1 and 2 (p < 0.001), but increased in lambs in Group 2 between Day 0 and Day 2 (p = 0.015). Scores for lambs in Group 3 on Day 2 were lower than for lambs in Group 2 (p < 0.001). Mean RMS voltages on Day 2 were higher in lambs in Group 2 than Group 3 (p = 0.045). Mean concentrations of bromide in serum were >800 µg/mL in lambs in Groups 3 and 4 on Day 2. Concentrations of FCM were higher in lambs from Group 2 than in Groups 1 or 5, but were similar in Groups 2, 3 and 4. Histopathological findings in the cerebellum of lambs from Groups 2, 3 and 4 were similar, showing pyknosis of neurons within the granular layer of the cerebellum and Purkinje neuron proximal axonal spheroid formation. Conclusions and clinical relevance: A single oral dose of 300 mg/kg bromide in lambs with neurological signs of PRGT resulted in reduced composite gait scores and reduced RMS voltages, indicating a significant improvement in clinical signs of ataxia, movement disorder and muscle tremor associated with the neurotoxic effects of lolitrem B.

Functional impairment triggered by altertoxin II (ATXII) in intestinal cells in vitro: cross-talk between cytotoxicity and mechanotransduction.

Intestinal cells are able to continuously integrate response to multiple stimuli/stressors; these include the concomitant activation of "chemically driven" pathways, of paramount importance in the response to toxicants, as well as physical stimulation derived from motility. Altertoxin II (ATXII, 0.1, 1 and 10 µM), a mycotoxin produced by the food contaminant fungus Alternaria alternata was studied in HT-29 intestinal adenocarcinoma cells and in non-transformed intestinal epithelial cells, HCEC. One-hour incubation with ATXII was sufficient to trigger irreversible cytotoxicity in both cell types, as well as to modify cellular responses to concomitant pro-oxidant challenge (H2O2, 100-500 µM, DCF-DA assay) suggesting that even relatively short-time exposure of the intestinal cells could be sufficient to alter their functionality. Combination of ATXII (1 µM) with physical stimulation typical of the intestinal compartment (shear stress) revealed differential response of tumor-derived epithelial cells HT-29 in comparison to HCEC, in particular in the localization of the transcription factor Nrf2 (NF-E2-related factor 2). Moreover, ATXII reduced the migratory potential of HCEC as well as their membrane fluidity, but had no respective impact on HT-29 cells. Taken together, ATXII appeared to alter predominantly membrane functionality in HCEC thus hampering crucial functions for cellular motility/turnover, as well as barrier function of healthy intestinal cells and had very limited activity on the tumor counterparts.

Toxicological effects of fumonisin B1 in combination with other Fusarium toxins.

Fusarium is a fungal genus spread worldwide commonly associated to the production of several mycotoxins, where fumonisins (FBs) are of major importance due to its prevalence. Since mycotoxins have been reported to cause deleterious effects on mammalians, including carcinogenic, neurotoxic, estrogenic, and immune-suppressive, many countries had established regulations on the tolerated concentrations of such substances in foods and animal feed. Even though many mycotoxins - especially fusariotoxins - are concomitantly found in a single matrix, there is no regulation on co-occurrence levels. This is possibly a result of the lack of data in the literature on the toxicological interactions between different mycotoxins. Considering this, it is of utmost importance to gather what is currently known about the combination of FBs, considered to be the most ubiquitous mycotoxins, with other frequently reported fusariotoxins, such as zearalenone (ZEA), deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T-2), and other emerging mycotoxins. This paper gives an overview about the toxic effects of fusariotoxins individually and combined to FB1, also gathering the mechanisms and probable interactions between them. This important information may help to develop regulations covering multi-mycotoxins contamination, a growing concern of current days.

In silico and in vitro prediction of the toxicological effects of individual and combined mycotoxins.

3-Acetyldeoxynivalenol (3-AcDON) and 15-acetyldeoxynivalenol (15-AcDON) are converted to deoxynivalenol (DON) in vivo and their simultaneous presence may increase DON intake. Mixtures of DON and its derivatives are a public health concern. In this study DON, 3-AcDON and 15-AcDON were evaluated in vitro and in silico. The in vitro cytotoxicity of DON and its derivatives individually and combined was determined by the Neutral Red (NR) assay in human hepatocarcinoma (HepG2) cells. The concentrations tested were from 1.25 to 15 µM (DON) and from 0.937 to 7.5 µM (DON derivatives). The IC50 values were from >15 to 2.55 µM (DON), from 1.77 to 1.02 µM (3-AcDON), and from 4.05 to 1.68 µM (15-AcDON). 3-AcDON was the most cytotoxic molecule in HepG2 cells. The concentrations tested in combinations ranged from 0.5625 to 4.5 µM (DON), and from 0.094 to 0.75 µM (DON derivatives), with ratios of 1:6 (DON+3-AcDON and DON+15-AcDON), 1:1 (3-AcDON+15-AcDON) and 1:6:6 (DON+3-AcDON+15-AcDON). The DON+15-AcDON mixture exhibited additive effects, while the rest showed synergistic effects. In silico methods assess individual mycotoxins. Absorption, Distribution, Metabolism, Excretion and Toxicity of mycotoxins were predicted using in silico SwissADME tools. Absorption, Distribution, Metabolism and Excretion profile prediction shows high gastrointestinal absorption and CYP3A4 mediated metabolism.

Cytotoxic effects induced by patulin, deoxynivalenol and toxin T2 individually and in combination in hepatic cells (HepG2).

Patulin (PAT), deoxynivalenol (DON) and toxin T-2 (T-2) are mycotoxins distributed worldwide in food and feed. Cytotoxicity of the three mycotoxins individually or in combination in human hepatocellular carcinoma (HepG2) cells was evaluated by MTT assay over 24, 48 and 72 h of exposure. The concentration ranges used were 0.625-15 µM for DON, 1.25-50 nM for T-2 and 0.45-7.5 µM for PAT. The IC50 values obtained ranged from 9.30 to 2.53 µM, from 33.69 to 44.37 nM and from 2.66 to 1.17 µM for DON, T-2 and PAT, respectively. The most cytotoxic mycotoxin to HepG2 cells was T-2 followed by PAT and DON. The combination ratios used for the mixtures were 1:3 (DON: T-2), 1:5 (DON: PAT), 1:1.7 (T-2: PAT) and 1:3:5 (DON: T-2: PAT). The mixture with the highest cytotoxic effect was T-2+PAT, followed by DON + T-2+PAT, DON + T-2 and DON + PAT respect to the cytotoxic effect of their individuals. In the combinations, at low fa an antagonistic effect was detected, and this effect changes the shape of the combination to additive effect at high fa in the mixtures.

Development of a model for investigation of perennial ryegrass toxicosis in sheep.

AIMS To develop a clinical model of perennial ryegrass toxicosis (PRGT) based on feeding a known dose of lolitrem B and ergotamine, and to produce a consistent clinical presentation for assessment of disease pathophysiology, neurological changes and neurohistopathology. METHODS Male lambs, aged between 10-12 months, were randomly assigned to either Treatment (n=9) or Control (n=9) groups. Lambs in the Treatment group received feed containing a novel endophyte-infested perennial ryegrass seed, commencing on Day 0 of the Feeding phase with a low induction dose, then increasing after 3 days to provide 0.16 mg/kg live bodywight (LBW)/day of lolitrem B and 0.054 mg/kg LBW/day ergotamine. Lambs were examined daily and when defined signs of PRGT were observed they were transferred to the Testing phase. Neurological examinations, assessment of gait, surface electromyography (EMG) and mechanosensory nociceptive threshold testing were carried out and blood samples collected during both phases of the trial, with a full necropsy, histopathological examination and measurement of faecal cortisol metabolites (FCM) performed on Day 2 of the Testing phase. RESULTS Typical clinical signs of PRGT, including ataxia of vestibulocerebellar origin leading to stumbling, were observed in all Treatment lambs. The median interval from the start of the Feeding phase to entry into the Testing phase was 21 (min 18, max 34) days. Histopathological characterisation of neurological lesions included the presence of Purkinje cell vacuolation, pyknotic granular layer neurons and proximal axonal Purkinje cell spheroids. Lesions were most apparent within the vestibulocerebellum. Mean root-mean-square voltages from triceps EMG increased in Treatment lambs between Feeding phase Day 0 and Testing phase Day 2 (p<0.001). Daily water intake during the Testing phase for the Treatment group was less than in Control group lambs (p=0.002), and concentrations of FCM at necropsy were higher in Treatment compared to Control lambs (p=0.02). CONCLUSIONS AND CLINICAL RELEVANCE Lolitrem B and ergotamine dosing in feed on a live weight basis combined with neurological/gait assessment provides an effective model for investigation of PRGT and potential therapeutics. Assessment of gait changes using defined criteria and RMS voltages from EMG appear to be useful tools for the assessment of the severity of neurological changes.