Metabolism of the masked mycotoxin deoxynivalenol-3-β-D-glucoside (D3G) in rats

Das Fusarium Mykotoxin Deoxynivalenol (DON) löst in Tieren zahlreiche Krankheitssymptome aus und verursacht beträchtliche wirtschaftliche Schäden. Pflanzen besitzen einen wirksamen Verteidigungsmechanismus gegenüber diesem Toxin, indem sie Glukose an DON konjugieren. Das resultierende maskierte Mykotoxin Deoxynivalenol-3-β-D-Glukosid (D3G) wurde sowohl in Nahrungs- als auch in Futtermitteln nachgewiesen. Eine mögliche Hydrolyse von D3G im Verdauungstrakt von Säugetieren könnte zu einer Erhöhung der Gesamtbelastung an DON führen und somit gesundheitsschädigende Wirkung aufweisen. Aufgrund fehlender in vivo Daten wurde dieses maskierte Mykotoxin bislang nicht in die EU-Höchstmengenregelungen für DON inkludiert. Ziel der vorliegenden Arbeit war es daher abzuklären, ob oral verabreichtes D3G in Ratten hydrolysiert wird und ob es in der Folge zu einer Absorption von freigesetztem DON kommt. In einem Messwiederholungsdesign wurde sechs Sprague-Dawley Ratten Wasser, DON (2,0 mg/kg KG) und die äquimolare Menge an D3G (3,1 mg/kg KG) an den Tagen 1, 8 und 15 oral verabreicht. Nach jeder Applikation wurden die Tiere für 48 h einzeln in Stoffwechselkäfigen gehalten, um Kot und Urin zu sammeln. Die darin enthaltenen Mengen an D3G, DON, Deoxynivalenol-Glukuronid (DON-GlcA) und Deepoxy-deoxynivalonol (DOM-1) wurden anhand einer validierten LC-MS/MS Analysenmethode bestimmt. Nach Verabreichung von D3G konnten sowohl das maskierte Mykotoxin selbst, als auch DON, DON-GlcA und DOM-1 im Urin der Ratten detektiert werden. D3G repräsentierte hierbei lediglich 0,3 ± 0,1% der verabreichten Dosis, was eine äußerst geringe Bioverfügbarkeit indiziert. Insgesamt konnten im Urin nach Applikation von D3G und DON 3,7 ± 0,7% und 14,9 ± 5,0% der verabreichten Toxinmengen wiedergefunden werden. Der Hauptteil an verabreichtem D3G wurde in Form von DON und DOM-1 im Kot der Tiere wiedergefunden. Die Studie konnte belegen, dass D3G im Verdauungstrakt von Ratten hydrolysiert und DON freigesetzt wird. Dieses wird zum Teil zu DON-GlcA und DOM-1 metabolisiert, jedoch nur in geringen Mengen resorbiert. Unsere Daten weisen daher darauf hin, dass D3G in Ratten eine geringere toxikologische Relevanz als DON besitzt.The Fusarium mycotoxin Deoxynivalenol (DON) leads to numerous adverse health effects in animals and causes serious economic losses. Plants can defend themselves against this toxin by conjugating glucose to DON. The resulting masked mycotoxin deoxynivalenol-3-β-D-glucoside (D3G) is frequently occurring in food and feed. There are major concerns that D3G is hydrolyzed in the digestive tract of mammals, thus increasing the total DON load of an individual. Due to a lack of in vivo data D3G has not been included in the regulatory limits established by the European Commission for DON. Therefore, the aim of our study was to clarify whether orally administered D3G is hydrolyzed in rats and liberated DON is subsequently absorbed. Using a repeated measures design, six Sprague-Dawley rats received water, DON (2.0 mg/kg body weight; bw) and the equimolar amount of D3G (3.1 mg/kg bw) by gavage on days 1, 8 and 15, respectively. After each application, the animals were housed individually for 48 h in metabolic cages to collect urine and feces. The concentrations of D3G, DON, deoxynivalenol-glucuronide (DON-GlcA) and de-epoxydeoxynivalenol (DOM-1) in the excreta were determined by a validated LC-MS/MS based method. After administration of D3G, the masked mycotoxin itself as well as DON, DON-GlcA and DOM-1 were detected in the urine of rats. In total, 3.7 ± 0.7% and 14.9 ± 5.0% of the administered dose were recovered in urine after application of D3G and DON, respectively. Urinary eliminated D3G represented only 0.3 ± 0.1% of the given dose, thus indicating a very low bioavailability of this masked mycotoxin in rats. The majority of administered D3G was recovered as DON and DOM-1 in feces. This study clearly demonstrated that D3G is hydrolyzed in the digestive tract of rats. The liberated DON is metabolized to DOM-1 and DON-GlcA, but only poorly absorbed. Our data indicate that D3G is of considerably lower toxicological relevance than DON in rats

Mycotoxin occurrence in maize silage : a neglected risk for bovine gut health?

Forages are important components of dairy cattle rations but might harbor a plethora of mycotoxins. Ruminants are considered to be less susceptible to the adverse health effects of mycotoxins, mainly because the ruminal microflora degrades certain mycotoxins. Yet, impairment of the ruminal degradation capacity or high ruminal stability of toxins can entail that the intestinal epithelium is exposed to significant mycotoxin amounts. The aims of our study were to assess (i) the mycotoxin occurrence in maize silage and (ii) the cytotoxicity of relevant mycotoxins on bovine intestinal cells. In total, 158 maize silage samples were collected from European dairy cattle farms. LC-MS/MS-based analysis of 61 mycotoxins revealed the presence of emerging mycotoxins (e.g., emodin, culmorin, enniatin B1, enniatin B, and beauvericin) in more than 70% of samples. Among the regulated mycotoxins, deoxynivalenol and zearalenone were most frequently detected (67.7%). Overall, 87% of maize silages contained more than five mycotoxins. Using an in vitro model with calf small intestinal epithelial cells B, the cytotoxicity of deoxynivalenol, nivalenol, fumonisin B1 and enniatin B was evaluated (0-200 mu M). Absolute IC50 values varied in dependence of employed assay and were 1.2-3.6 mu M, 0.8-1.0 mu M, 8.6-18.3 mu M, and 4.0-6.7 mu M for deoxynivalenol, nivalenol, fumonisin B1, and enniatin B, respectively. Results highlight the potential relevance of mycotoxins for bovine gut health, a previously neglected target in ruminants

Effects of orally administered fumonisin B1 (FB1), partially hydrolysed FB1, hydrolysed FB1 and N-(1-deoxy-D-fructos-1-yl) FB1 on the sphingolipid metabolism in rats

Fumonisin B1 (FB1) is a Fusarium mycotoxin frequently occurring in maize-based food and feed. Alkaline processing like nixtamalisation of maize generates partially and fully hydrolysed FB1 (pHFB1 and HFB1) and thermal treatment in the presence of reducing sugars leads to formation of N-(1-deoxy-D-fructos- 1-yl) fumonisin B1 (NDF). The toxicity of these metabolites, in particular their effect on the sphingolipid metabolism, is either unknown or discussed controversially.We produced high purity FB1, pHFB1a+b, HFB1 and NDF and fed them to male Sprague Dawley rats for three weeks. Once a week, urine and faeces samples were collected over 24 h and analysed for fumonisin metabolites as well as for the sphinganine (Sa) to sphingosine (So) ratio by validated LC–MS/MS based methods. While the latter was significantly increased in the FB1 positive control group, the Sa/So ratios of the partially and fully hydrolysed fumonisins were indifferent from the negative control group. Although NDF was partly cleaved during digestion, the liberated amounts of FB1 did not raise the Sa/So ratio. These results show that the investigated alkaline and thermal processing products of FB1 were, at the tested concentrations, non-toxic for rats, and suggest that according food processing can reduce fumonisin toxicity for humans

Influence of Canopy Disturbances on Runoff and Landslide Disposition after Heavy Rainfall Events

As protective forests have a major control function on runoff and erosion, they directly affect the risk from hydrogeomorphic processes such as sediment transport processes or debris flows. In this context, future scenarios of climate-related canopy disturbances and their influence on the protective effect remain, however, an unsolved problem. With the individual-based forest landscape and disturbance model iLand, an ensemble of forest landscape simulations was carried out and the effects of future changes in natural disturbance regimes were evaluated. To determine peak runoff, hydrological simulations have been conducted, using the conceptual hydrological model ZEMOKOST as well as the deterministic model GEOtop. Effects of forest disturbances on hillslope stability were investigated, based on a modified Coulomb landslide model. Our results suggest no influence of the disturbance regime on the runoff. The climate-related increase in the frequency of disturbances is not reflected in increased runoff during the period under consideration. Contrary, slope stability analyses indicate that the availability of shallow landslides in steep forested torrent catchments might be decreased by the occurrence of disturbances – especially for a warm and dry climate projection. Canopy disturbances seem to accelerate the adaptation of tree species to future climate conditions, which is likely to be accompanied by a change in root systems away from flat roots that currently predominate in torrential catchments. In terms of managing the protective effect of forests against shallow landslides, such natural disturbances can thus be considered as positive interventions in the existing forest ecosystem by promoting natural succession

Exposure to Zearalenone Leads to Metabolic Disruption and Changes in Circulating Adipokines Concentrations in Pigs

Zearalenone (ZEN) is a mycotoxin classified as an endocrine disruptor. Many endocrine disruptors are also metabolic disruptors able to modulate energy balance and inflammatory processes in a process often involving a family of protein hormones known as adipokines. The aim of our study was to elucidate the role of ZEN as metabolic disruptor in pigs by investigating the changes in energy balance and adipokines levels in response to different treatment diets. To this end, weaned piglets (n = 10/group) were exposed to either basal feed or feed contaminated with 680 and 1620 µg/kg ZEN for 28 days. Serum samples collected at days 7 and 21 were subjected to biochemistry analysis, followed by determination of adipokine levels using a combined approach of protein array and ELISA. Results indicate that ZEN has an impact on lipid and glucose metabolism that was different depending on the dose and time of exposure. In agreement with these changes, ZEN altered circulating adipokines concentrations, inducing significant changes in adiponectin, resistin, and fetuin B. Our results suggest that ZEN may function as a natural metabolism-disrupting chemical

Metabolism of the masked mycotoxin deoxynivalenol-3-β-D-glucoside (D3G) in rats

Das Fusarium Mykotoxin Deoxynivalenol (DON) löst in Tieren zahlreiche Krankheitssymptome aus und verursacht beträchtliche wirtschaftliche Schäden. Pflanzen besitzen einen wirksamen Verteidigungsmechanismus gegenüber diesem Toxin, indem sie Glukose an DON konjugieren. Das resultierende maskierte Mykotoxin Deoxynivalenol-3-β-D-Glukosid (D3G) wurde sowohl in Nahrungs- als auch in Futtermitteln nachgewiesen. Eine mögliche Hydrolyse von D3G im Verdauungstrakt von Säugetieren könnte zu einer Erhöhung der Gesamtbelastung an DON führen und somit gesundheitsschädigende Wirkung aufweisen. Aufgrund fehlender in vivo Daten wurde dieses maskierte Mykotoxin bislang nicht in die EU-Höchstmengenregelungen für DON inkludiert. Ziel der vorliegenden Arbeit war es daher abzuklären, ob oral verabreichtes D3G in Ratten hydrolysiert wird und ob es in der Folge zu einer Absorption von freigesetztem DON kommt. In einem Messwiederholungsdesign wurde sechs Sprague-Dawley Ratten Wasser, DON (2,0 mg/kg KG) und die äquimolare Menge an D3G (3,1 mg/kg KG) an den Tagen 1, 8 und 15 oral verabreicht. Nach jeder Applikation wurden die Tiere für 48 h einzeln in Stoffwechselkäfigen gehalten, um Kot und Urin zu sammeln. Die darin enthaltenen Mengen an D3G, DON, Deoxynivalenol-Glukuronid (DON-GlcA) und Deepoxy-deoxynivalonol (DOM-1) wurden anhand einer validierten LC-MS/MS Analysenmethode bestimmt. Nach Verabreichung von D3G konnten sowohl das maskierte Mykotoxin selbst, als auch DON, DON-GlcA und DOM-1 im Urin der Ratten detektiert werden. D3G repräsentierte hierbei lediglich 0,3 ± 0,1% der verabreichten Dosis, was eine äußerst geringe Bioverfügbarkeit indiziert. Insgesamt konnten im Urin nach Applikation von D3G und DON 3,7 ± 0,7% und 14,9 ± 5,0% der verabreichten Toxinmengen wiedergefunden werden. Der Hauptteil an verabreichtem D3G wurde in Form von DON und DOM-1 im Kot der Tiere wiedergefunden. Die Studie konnte belegen, dass D3G im Verdauungstrakt von Ratten hydrolysiert und DON freigesetzt wird. Dieses wird zum Teil zu DON-GlcA und DOM-1 metabolisiert, jedoch nur in geringen Mengen resorbiert. Unsere Daten weisen daher darauf hin, dass D3G in Ratten eine geringere toxikologische Relevanz als DON besitzt.The Fusarium mycotoxin Deoxynivalenol (DON) leads to numerous adverse health effects in animals and causes serious economic losses. Plants can defend themselves against this toxin by conjugating glucose to DON. The resulting masked mycotoxin deoxynivalenol-3-β-D-glucoside (D3G) is frequently occurring in food and feed. There are major concerns that D3G is hydrolyzed in the digestive tract of mammals, thus increasing the total DON load of an individual. Due to a lack of in vivo data D3G has not been included in the regulatory limits established by the European Commission for DON. Therefore, the aim of our study was to clarify whether orally administered D3G is hydrolyzed in rats and liberated DON is subsequently absorbed. Using a repeated measures design, six Sprague-Dawley rats received water, DON (2.0 mg/kg body weight; bw) and the equimolar amount of D3G (3.1 mg/kg bw) by gavage on days 1, 8 and 15, respectively. After each application, the animals were housed individually for 48 h in metabolic cages to collect urine and feces. The concentrations of D3G, DON, deoxynivalenol-glucuronide (DON-GlcA) and de-epoxydeoxynivalenol (DOM-1) in the excreta were determined by a validated LC-MS/MS based method. After administration of D3G, the masked mycotoxin itself as well as DON, DON-GlcA and DOM-1 were detected in the urine of rats. In total, 3.7 ± 0.7% and 14.9 ± 5.0% of the administered dose were recovered in urine after application of D3G and DON, respectively. Urinary eliminated D3G represented only 0.3 ± 0.1% of the given dose, thus indicating a very low bioavailability of this masked mycotoxin in rats. The majority of administered D3G was recovered as DON and DOM-1 in feces. This study clearly demonstrated that D3G is hydrolyzed in the digestive tract of rats. The liberated DON is metabolized to DOM-1 and DON-GlcA, but only poorly absorbed. Our data indicate that D3G is of considerably lower toxicological relevance than DON in rats

Milk Thistle Extract and Silymarin Inhibit Lipopolysaccharide Induced Lamellar Separation of Hoof Explants in Vitro

The pathogenesis of laminitis is not completely identified and the role of endotoxins (lipopolysaccharides, LPS) in this process remains unclear. Phytogenic substances, like milk thistle (MT) and silymarin, are known for their anti-inflammatory and antioxidant properties and might therefore have the potential to counteract endotoxin induced effects on the hoof lamellar tissue. The aim of our study was to investigate the influence of endotoxins on lamellar tissue integrity and to test if MT and silymarin are capable of inhibiting LPS-induced effects in an in vitro/ex vivo model. In preliminary tests, LPS neutralization efficiency of these phytogenics was determined in an in vitro neutralization assay. Furthermore, tissue explants gained from hooves of slaughter horses were tested for lamellar separation after incubation with different concentrations of LPS. By combined incubation of explants with LPS and either Polymyxin B (PMB; positive control), MT or silymarin, the influence of these substances on LPS-induced effects was assessed. In the in vitro neutralization assay, MT and silymarin reduced LPS concentrations by 64% and 75%, respectively, in comparison PMB reduced 98% of the LPS concentration. In hoof explants, LPS led to a concentration dependent separation. Accordantly, separation force was significantly decreased by 10 µg/mL LPS. PMB, MT and silymarin could significantly improve tissue integrity of explants incubated with 10 µg/mL LPS. This study showed that LPS had a negative influence on the structure of hoof explants in vitro. MT and silymarin reduced endotoxin activity and inhibited LPS-induced effects on the lamellar tissue. Hence, MT and silymarin might be used to support the prevention of laminitis and should be further evaluated for this application

Concentration Dependent Influence of Lipopolysaccharides on Separation of Hoof Explants and Supernatant Lactic Acid Concentration in an Ex Vivo/In Vitro Laminitis Model.

Laminitis is one of the most common diseases in horses. It is not only painful for the animal, but also has a significant financial impact on the equine industry. This multifactorial disease affects the connective tissue of the hoof. However, the pathogenesis of laminitis is still not fully understood. Endotoxins, also known as lipopolysaccharides (LPS), and bacterial exotoxins seem to play an important role during the development of laminitis. The aim of our study was to investigate the effect of increasing LPS concentrations (0, 2.5, 5, 10, and 100 μg/mL) on cell viability of isolated epidermal and dermal hoof cells as well as on the tissue integrity of hoof explants. Furthermore, glucose, acetic acid, lactic acid, and propionic acid concentrations in explant supernatants were measured to evaluate the energy metabolism in the hoof tissue. LPS did not exhibit cytotoxic effects on epidermal or dermal cells. Force required to separate LPS treated hoof explants decreased in a concentration dependent manner. Specifically, explants incubated with 10 and 100 μg/mL needed significantly less force to separate compared to control explants. Lactic acid concentrations were significantly decreased in explants incubated with 5, 10, or 100 μg/mL LPS, while glucose, acetic acid and propionic acid concentrations were unaffected by LPS treatment. Our study indicates that LPS has no cytotoxic effect on epidermal and dermal cells isolated from hoof tissue, but impairs integrity of hoof explants. In addition, LPS led to an alteration of the lactic acid production in the lamellar tissue. Since our data highlight that LPS can affect the integrity of the equine hoof tissue in vitro, endotoxins should be further explored for their contribution to facilitate the development of laminitis

Fumonisin B1 (FB1) Induces Lamellar Separation and Alters Sphingolipid Metabolism of In Vitro Cultured Hoof Explants

One of the most important hoof diseases is laminitis. Yet, the pathology of laminitis is not fully understood. Different bacterial toxins, e.g. endotoxins or exotoxins, seem to play an important role. Additionally, ingestion of mycotoxins, toxic secondary metabolites of fungi, might contribute to the onset of laminitis. In this respect, fumonsins are of special interest since horses are regarded as species most susceptible to this group of mycotoxins. The aim of our study was to investigate the influence of fumonisin B1 (FB1) on primary isolated epidermal and dermal hoof cells, as well as on the lamellar tissue integrity and sphingolipid metabolism of hoof explants in vitro. There was no effect of FB1 at any concentration on dermal or epidermal cells. However, FB1 significantly reduced the separation force of explants after 24 h of incubation. The Sa/So ratio was significantly increased in supernatants of explants incubated with FB1 (2.5–10 µg/mL) after 24 h. Observed effects on Sa/So ratio were linked to significantly increased sphinganine concentrations. Our study showed that FB1 impairs the sphingolipid metabolism of explants and reduces lamellar integrity at non-cytotoxic concentrations. FB1 might, therefore, affect hoof health. Further in vitro and in vivo studies are necessary to elucidate the effects of FB1 on the equine hoof in more detail