Toxins — Open Access Journal

The study applied ¹H NMR-based plasma metabolomics to identify candidate biomarkers of aflatoxin B1 (AFB₁) ingestion in dairy cows fed no sequestering agents and evaluate the effect of supplementing clay and/or a Saccharomyces cerevisiae fermentation product (SCFP) on such biomarkers. Eight lactating cows were randomly assigned to 1 of 4 treatments in a balanced 4 × 4 Latin square design with 2 squares. Treatments were: control, toxin (T; 1725 µg AFB₁/head/day), T with clay (CL; 200 g/head/day), and CL with SCFP (CL + SCFP; 35 g of SCFP/head/day). Cows in T, CL, and CL + SCFP were dosed with AFB₁ from d 26 to 30. The sequestering agents were top-dressed from d 1 to 33. On d 30 of each period, 15 mL of blood was taken from the coccygeal vessels and plasma samples were prepared by centrifugation. Compared to the control, T decreased plasma concentrations of alanine, acetic acid, leucine, arginine and valine. In contrast, T increased plasma ethanol concentration 3.56-fold compared to control. Treatment with CL tended to reduce sarcosine concentration, whereas treatment with CL + SCFP increased concentrations of mannose and 12 amino acids. Based on size of the area under the curve (AUC) of receiver operating characteristic and fold change (FC) analyses, ethanol was the most significantly altered metabolite in T (AUC = 0.88; FC = 3.56); hence, it was chosen as the candidate biomarker of aflatoxin ingestion in dairy cows fed no sequestering agent. Full article

Venomous animals are found through a wide taxonomic range including cartilaginous fish such as the freshwater stingray Potamotrygon motoro occurring in South America, which can injure people and cause venom-related symptoms. Ensuring the efficacy of drug development to treat stingray injuries can be assisted by the knowledge of the venom composition. Here we performed a detailed transcriptomic characterization of the venom gland of the South American freshwater stingray Potamotrygon motoro. The transcripts retrieved showed 418 hits to venom components (comparably to 426 and 396 hits in other two Potamotrygon species), with high expression levels of hyaluronidase, cystatin and calglandulin along with hits uniquely found in P. motoro such as DELTA-alicitoxin-Pse1b, Augerpeptide hhe53 and PI-actitoxin-Aeq3a. We also identified undescribed molecules with extremely high expression values with sequence similarity to the SE-cephalotoxin and Rapunzel genes. Comparative analyses showed that despite being closely related, there may be significant variation among the venoms of freshwater stingrays, highlighting the importance of considering elicit care in handling different envenomation cases. Since hyaluronidase represents a major component of fish venom, we have performed phylogenetic and selective pressure analyses of this gene/protein across all fish with the available information. Results indicated an independent recruitment of the hyaluronidase into the stingray venom relative to that of venomous bony fish. The hyaluronidase residues were found to be mostly under negative selection, but 18 sites showed evidence of diversifying positive selection (P < 0.05). Our data provides new insight into stingray venom variation, composition, and selective pressure in hyaluronidase. Full article

Aflatoxins are fungal metabolites that contaminate foods and feeds, causing adverse health effects in humans and animals. This study determined the occurrence of aflatoxins in fish feeds and their potential effects on fish. Eighty-one fish feeds were sampled from 70 farms and 8 feed manufacturing plants in Nyeri, Kenya for aflatoxin analysis using competitive enzyme-linked immunosorbent assay. Fish were sampled from 12 farms for gross and microscopic pathological examination. Eighty-four percent of feeds sampled tested positive for aflatoxins, ranging from 1.8 to 39.7 µg/kg with a mean of 7.0 ± 8.3 µg/kg and the median of 3.6 µg/kg. Fifteen feeds (18.5%) had aflatoxins above the maximum allowable level in Kenya of 10 µg/kg. Homemade and tilapia feeds had significantly higher aflatoxin levels than commercial and trout feeds. Feeds containing maize bran and fish meal had significantly higher aflatoxin levels than those without these ingredients. Five trout farms (41.7%) had fish with swollen abdomens, and enlarged livers with white or yellow nodules, which microscopically had large dark basophilic hepatic cells with hyperchromatic nuclei in irregular cords. In conclusion, aflatoxin contamination of fish feeds is prevalent in Nyeri, and may be the cause of adverse health effects in fish in this region. Full article

For many of the most important human bacterial infections, invasive disease severity is fueled by the cell damaging and pro-inflammatory effects of secreted pore-forming toxins (PFTs). Isogenic PFT-knockout mutants, e.g., Staphylococcus aureus lacking α-toxin or Streptococcus pneumoniae deficient in pneumolysin, show attenuation in animal infection models. This knowledge has inspired multi-model investigations of strategies to neutralize PFTs or counteract their toxicity as a novel pharmacological approach to ameliorate disease pathogenesis in clinical disease. Promising examples of small molecule, antibody or nanotherapeutic drug candidates that directly bind and neutralize PFTs, block their oligomerization or membrane receptor interactions, plug establishment membrane pores, or boost host cell resiliency to withstand PFT action have emerged. The present review highlights these new concepts, with a special focus on β-PFTs produced by leading invasive human Gram-positive bacterial pathogens. Such anti-virulence therapies could be applied as an adjunctive therapy to antibiotic-sensitive and -resistant strains alike, and further could be free of deleterious effects that deplete the normal microflora. Full article

Host defense peptides (HDPs) are efficient defense components of the innate immune system, playing critical roles in intestinal homeostasis and protection against pathogens. This study aims to investigate the interference effects of DON on the intestinal porcine HDPs expression in piglets and intestinal porcine epithelial cell line (IPEC-J2) cells, and elucidate the underlying mechanisms through which it functions. In an animal experiment, intestinal HDPs were determined in weaned piglets fed control and 1.28 mg/kg or 2.89 mg/kg DON-contaminated diets. Dietary exposure to DON significantly decreased piglet average daily gain, increased intestinal permeability and depressed the expression of porcine β-defensin1 (pBD1), pBD2, pBD3, epididymis protein 2 splicing variant C (pEP2C), PMAP23, and proline/arginine-rich peptide of 39 amino acids (PR39) in the intestine (p < 0.05). In IPEC-J2 cells, DON decreased cell viability and inhibited the expression of pBD1, pBD3, pEP2C, PG1-5, and PR39 (p < 0.05). NOD2, key regulator that is responsible for HDPs production, was markedly downregulated, whereas caspase-12 was activated in the presence of DON. In conclusion, DON induced caspase-12 activation and inhibited the NOD2-mediated HDPs production, which led to an impaired intestinal barrier integrity of weaned piglets. Our study provides a promising target for future therapeutic strategies to prevent the adverse effects of DON. Full article

Staphylococcal enterotoxins (SEs) are a food safety concern. Existing methods for biologically active SE detection rely on the emetic response in live kittens or monkeys. This method suffers from low sensitivity, poor reproducibility, and causes ethical concerns regarding the use of experimental animals. The Lautenberg Chemical Safety Act encourages the development and adoption of alternatives to testing on animals for chemical toxicity methodologies. In this study, we utilized the superantigenic effect of SE type A (SEA) and used an ex vivo bioassay as an alternative to live animal testing. We found that interleukin-2 (IL-2) secreted by splenocyte can be utilized for quantifiable detection of SEA in food products. To avoid food matrix interference and attenuation of signal, we separated SEA from spiked food products by employing immunomagnetic beads that were coated with an anti-SEA antibody. This ex vivo method has achieved the detection of 1 ng mL⁻¹ of SEA, which is 10⁷ times more sensitive than the existing live animal testing methods. However, this ex vivo bioassay requires sacrificing of mice. To overcome this limitation, we established a cell based in vitro assay using CCRF-CEM, a human CD4⁺ T-cell line, for the quantitative detection of SEA. Incubation of SEA with CCRF-CEM human T-cells and Raji cells led to quantifiable and dose dependent secretion of IL-2. This novel cell-based assay is highly specific to biologically active SEA, compared with the related SE toxin subtypes B, D, and E or heat inactivated SEA, which produce no secretion of IL-2. This is the first demonstration of an alternative assay that completely eliminates the use of animals for quantitative detection of active SEA. Full article

Snakebite envenomation is an affliction currently estimated to be killing upwards of 100,000 people annually. Snakebite is associated with a diverse pathophysiology due to the magnitude of variation in venom composition that is observed worldwide. The haemolytic (i.e., lysis of red blood cells) actions of snake venoms are well documented, although the direct impact of venoms on haemoglobin is not fully understood. Here we report on the varied ability of a multitude of snake venoms to oxidise haemoglobin into methaemoglobin. Moreover, our results demonstrate that the venom of an elapid, the black necked spitting cobra, Naja nigricollis, oxidises oxyhaemoglobin (Fe²⁺) into methaemoglobin (Fe³⁺) in a time- and concentration-dependent manner that is unparalleled within the 47 viper and elapid venoms evaluated. The treatment of venom with a reducing agent, dithiothreitol (DTT) is observed to potentiate this effect at higher concentrations, and the use of denatured venom demonstrates that this effect is dependent upon the heat-sensitive proteinaceous elements of the venom. Together, our results suggest that Naja nigricollis venom appears to promote methaemoglobin production to a degree that is rare within the Elapidae family, and this activity appears to be independent of proteolytic activities of venom components on haemoglobin. Full article

The sensitive detection of the mycotoxin citrinin (CIT) utilizing its fluorescence requires approaches to enhance the emission. In this respect, we studied the complexation of CIT and ochratoxin A (OTA) with Al³⁺ in methanol using absorption and fluorescence spectroscopy. In this context, an isocratic high performance liquid chromatography (HPLC) method using a polymer column and a fluorescence detector was also developed that enables the separation of the metal ion complexes from the free ligands and non-complexed Al³⁺. CIT and OTA showed distinct changes in their absorption and fluorescence properties upon Al³⁺-coordination, and the fluorescence of CIT was considerably enhanced. Analysis of the photometrically assessed titration of CIT and OTA with Al³⁺ using the Job plot method revealed 1:2 and 1:1 stoichiometries for the Al³⁺ complexes of CIT (Al:CIT) and OTA (Al:OTA), respectively. In the case of CIT, only one β-diketone moiety participates in Al³⁺ coordination. These findings can be elegantly exploited for signal amplification and provide the base to reduce the limit of detection for CIT quantification by about an order of magnitude, as revealed by HPLC measurements using a fluorescence detector. Full article

Palytoxin (PlTX) and analogues are produced by certain dinoflagellates, sea anemones, corals and cyanobacteria. PlTX can accumulate in the food chain and when consumed it may cause intoxication with symptoms like myalgia, weakness, fever, nausea, and vomiting. The analysis of PlTXs is challenging, and because of the large molecular structure, it is difficult to develop a sensitive and selective liquid chromatography-mass spectrometry (LC-MS/MS) method. In this work, an LC-MS/MS method was developed to analyse PlTXs with use of lithium iodine and formic acid as additives in the mobile phase. For method development, initially, LC-hrMS was used to accurately determine the elemental composition of the precursor and product ions. The main adduct formed was [M + H + 2Li]³⁺. Fragments were identified with LC-hrMS and these were incorporated in the LC-MS/MS method. A method of 10 min was developed and a solid phase extraction clean-up procedure was optimised for shellfish matrix. The determined limits of detection were respectively 8 and 22 µg PlTX kg⁻¹ for mussel and oyster matrix. Oysters gave a low recovery of approximately 50% for PlTX during extraction. The method was successfully in-house validated, repeatability had a relative standard deviation less than 20% (n = 5) at 30 µg PlTX kg⁻¹ in mussel, cockle, and ensis, and at 60 µg PlTX kg⁻¹ in oyster. Full article

Microcystin-LR (MC-LR) is the most widely distributed microcystin (MC) that is hazardous to environmental safety and public health, due to high toxicity. Microbial degradation is regarded as an effective and environment-friendly method to remove it, however, the performance of MC-degrading bacteria in environmentally relevant pollution concentrations of MC-LR and the degradation pathways remain unclear. In this study, one autochthonous bacterium, Sphingopyxis sp. m6 which exhibited high MC-LR degradation ability, was isolated from Lake Taihu, and the degrading characteristics in environmentally relevant pollution concentrations were demonstrated. In addition, degradation products were identified by utilizing the full scan mode of UPLC-MS/MS. The data illustrated that strain m6 could decompose MC-LR (1–50 μg/L) completely within 4 h. The degradation rates were significantly affected by temperatures, pH and MC-LR concentrations. Moreover, except for the typical degradation products of MC-LR (linearized MC-LR, tetrapeptide, and Adda), there were 8 different products identified, namely, three tripeptides (Adda-Glu-Mdha, Glu-Mdha-Ala, and Leu-MeAsp-Arg), three dipeptides (Glu-Mdha, Mdha-Ala, and MeAsp-Arg) and two amino acids (Leu, and Arg). To our knowledge, this is the first report of Mdha-Ala, MeAsp-Arg, and Leu as MC-LR metabolites. This study expanded microbial degradation pathways of MC-LR, which lays a foundation for exploring degradation mechanisms and eliminating the pollution of microcystins (MCs). Full article

Botulinum neurotoxin type-A (BoNT-A) blocks the release of acetylcholine from peripheral cholinergic nerve terminals and is an important option for the treatment of disorders characterised by excessive cholinergic neuronal activity. Several BoNT-A products are currently marketed, each with unique manufacturing processes, excipients, formulation, and non-interchangeable potency units. Nevertheless, the effects of all the products are mediated by the 150 kDa BoNT-A neurotoxin. We assessed the quantity and light chain (LC) activity of BoNT-A in three commercial BoNT-A products (Dysport^®; Botox^®; Xeomin^®). We quantified 150 kDa BoNT-A by sandwich ELISA and assessed LC activity by EndoPep assay. In both assays, we assessed the results for the commercial products against recombinant 150 kDa BoNT-A. The mean 150 kDa BoNT-A content per vial measured by ELISA was 2.69 ng/500 U vial Dysport^®, 0.90 ng/100 U vial Botox^®, and 0.40 ng/100 U vial Xeomin^®. To present clinically relevant results, we calculated the 150 kDa BoNT-A/US Food and Drug Administration (FDA)-approved dose in adult upper limb spasticity: 5.38 ng Dysport^® (1000 U; 2 × 500 U vials), 3.60 ng Botox^® (400 U; 4 × 100 U vials), and 1.61 ng Xeomin^® (400 U; 4 × 100 U vials). EndoPep assay showed similar LC activity among BoNT-A products. Thus, greater amounts of active neurotoxin are injected with Dysport^®, at FDA-approved doses, than with other products. This fact might explain the long duration of action reported across multiple indications, which benefits patients, caregivers, clinicians, and healthcare systems. Full article

Snakebite envenoming is a neglected tropical disease that each year claims the lives of 80,000–140,000 victims worldwide. The only effective treatment against envenoming involves intravenous administration of antivenoms that comprise antibodies that have been isolated from the plasma of immunized animals, typically horses. The drawbacks of such conventional horse-derived antivenoms include their propensity for causing allergenic adverse reactions due to their heterologous and foreign nature, an inability to effectively neutralize toxins in distal tissue, a low content of toxin-neutralizing antibodies, and a complex manufacturing process that is dependent on husbandry and procurement of snake venoms. In recent years, an opportunity to develop a fundamentally novel type of antivenom has presented itself. By using modern antibody discovery strategies, such as phage display selection, and repurposing small molecule enzyme inhibitors, next-generation antivenoms that obviate the drawbacks of existing plasma-derived antivenoms could be developed. This article describes the conceptualization of a novel therapeutic development strategy for biosynthetic oligoclonal antivenom (BOA) for snakebites based on recombinantly expressed oligoclonal mixtures of human monoclonal antibodies, possibly combined with repurposed small molecule enzyme inhibitors. Full article

This study aimed to assess post-harvest contamination with mycotoxins in the context of the geographic and agroclimatic conditions in Romania in 2012–2015, a period that was characterized by extreme meteorological events and the effects of climate change. The samples were randomly sampled from five agricultural regions of Romania and analyzed for mycotoxins by enzyme-linked immunosorbent assay. An SPSS analysis was done to explore correlations between mycotoxins (deoxynivalenol—DON, aflatoxins—AF, ochratoxin A—OTA, zearalenone—ZEA), product types (raw cereal, processed cereal, cereal-based food), geographic coordinates (latitude, longitude, agricultural region), and agroclimatic factors (air temperature, precipitation, soil moisture reserve, aridity index, soil type). In the southeast part of the Southern Plain and Dobrogea (Baragan Plain, located at 44–45° N, 26–27° E), contamination with AF and OTA was higher in raw and processed cereals (maize in silo, silo corn germs) in the dry years (2012 and 2013), and contamination with DON was high in processed cereals (wheat flour type 450) in the rainy year (2014). DON and OTA contamination were significantly correlated with cumulative precipitation in all years, while AF and ZEA contamination were non-significantly correlated with climatic factors and aridity indices. The distribution of mycotoxins by product type and the non-robust correlations between post-harvest mycotoxins and agrometeorological factors could be explained by the use of quality management systems that control cereal at warehouse receptions, performant processing technologies, and the quality of storage spaces of agri-food companies. The Baragan Plain is Romania’s most sensitive area to the predicted climate change in southeast Europe, which may be associated with its increased cereal contamination with AF and OTA. Full article

Foot dystonia (FD) is a disabling condition causing pain, spasm and difficulty in walking. We treated fourteen (14) adult patients experiencing FD with onabotulinum toxin A injection into the dystonic foot muscles. We analyzed the spatiotemporal gait utilizing the GaitRite system pre- and 3 weeks post-botulinum toxin injection along with measuring dystonia by the Fahn–Marsden Dystonia Scale (FMDS), pain by the Visual Analog Scale (VAS) and other lower limb functional outcomes such as gait velocity, the Berg Balance Scale (BBS), the Unified Parkinson’s Disease Rating Scale–Lower Limb Score (UPDRS–LL), the Timed Up and Go (TUG) test and the Goal Attainment Scale (GAS). We found that stride length increased significantly in both the affected (p = 0.02) and unaffected leg (p = 0.01) after treatment, and the improvement in stride length was roughly the same in each leg. Similar results were found for step length (p = 0.02) with improvement in the step length differential (p = 0.01). The improvements in the lower limb functional outcomes were also significant—FMDS, VAS, TUG, and UPDRS–LL decreased significantly after treatment (all p < 0.001), and BBS (p = 0.001), GAS (p < 0.001) except cadence (p = 0.37). BT injection improved walking in foot dystonia as evidenced through gait analysis, pain and lower limb functional outcomes. Main study limitations were small sample size and lack of control. Full article

Phα1β, a purified peptide from the venom of the spider Phoneutria nigriventer, and its recombinant form CTK 01512-2 are voltage-dependent calcium channel (Ca_V) blockers of types N, R, P/Q, and L with a preference for type N. These peptides show analgesic action in different pain models in rats. The aim of this study was to evaluate the acute intrathecal toxicity of the native and recombinant Phα1β toxin in Wistar rats. Clinical signs, serum biochemistry, organ weight, and histopathological alterations were evaluated in male and/or female rats. Dyspnea was observed in males, hyporesponsiveness in females, and Straub tail and tremors in both genders. There were no significant differences in male organ weight, although significant differences in the female relative weight of the adrenal glands and spleen have been observed; these values are within the normal range. Serum biochemical data revealed a significant reduction within the physiological limits of species related to urea, ALT, AST, and FA. Hepatic and renal congestion were observed for toxin groups. In renal tissue, glomerular infiltrates were observed with increased glomerular space. These histological alterations were presented in focal areas and in mild degree. Therefore, Phα1β and CTK 01512-2 presented a good safety profile with transient toxicity clinical signals in doses higher than used to obtain the analgesic effect. Full article

Chestnut drying is used to prevent postharvest losses and microorganism contamination during storage. Several studies reported the contamination by aflatoxins (AFs) produced by Aspergillus spp. in chestnuts. The effect of drying temperatures (from 30 to 50 °C) was evaluated on the growth of A. flavus and the production of aflatoxins in chestnuts. The influence of the treatment on the proximate composition, the total phenol content and antioxidant activity of chestnuts was considered. Fungal colonization was observed on the nuts dried at 30, 35, and 40 °C; the incidence was lower at 40 °C. The highest concentrations of AFB₁ and AFB₂ were produced at 40 °C. No aflatoxins were detected at 45 or 50 °C. At 40 °C A. flavus was under suboptimal conditions for growth (a_w 0.78), but the fungus was able to synthesize aflatoxins. As the temperatures applied increased, the total phenol content increased, while the antioxidant activity decreased. A drying treatment at 45 °C for seven days (a_w 0.64) could be a promising method to effectively control both the growth of aflatoxigenic fungi and the production of aflatoxins. This study provides preliminary data useful to improve the current drying conditions used in chestnut mills, to reduce both fungal growth and aflatoxin production. Full article

Tetrodotoxin (TTX) is a marine biotoxin that has high scientific value. However, the lack of efficient TTX extraction and preparation methods has led to a scarcity of TTX samples for clinical application. In this study, TTX from the liver of Takifugu pseudommus was ultrasound-assisted extracted with acidified organic solvents. The extraction process was analyzed and optimized by single factor method and response surface methodology (RSM). The optimal extraction conditions predicted by a response surface model were as follows: liquid:material ratio, 2.8:1; extraction temperature, 60 °C; extraction time, 23.3 min. Under these conditions, the extraction of TTX had a yield of 89.65%, and the results were further verified by experimental extraction, and analyzed by ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). It was found that the extracts of T. pseudommus liver contained TTX and its four analogues at certain proportions (TTX: 10.4%; 5,6,11-trideoxyTTX: 83.3%; 5,11-dideoxyTTX:2.4%; 4,9-anhydro TTX:2.6%; 5-deoxyTTX:1.3%). This study demonstrates a stable and efficient extraction process of TTX from pufferfish liver, which can be helpful for further research and analysis, as well as the utilization of TTX from pufferfish. Full article

Aspergillus flavus is the most important mycotoxin-producing fungus involved in the global episodes of aflatoxin B₁ contamination of crops at both the pre-harvest and post-harvest stages. However, in order to effectively control aflatoxin contamination in crops using antiaflatoxigenic and/or antifungal compounds, some of which are photosensitive, a proper understanding of the photo-sensitive physiology of potential experimental strains need to be documented. The purpose of the study is therefore to evaluate the effect of visible (VIS) light illumination on growth and conidiation, aflatoxin production ability and modulation of A. flavus oxidative status during in vitro experiment. Aflatoxigenic A. flavus strain was inoculated in aflatoxin-inducing YES media and incubated under three different VIS illumination regimes during a 168 h growth period at 29 °C. VIS illumination reduced A. flavus mycelia biomass yield, both during growth on plates and in liquid media, promoted conidiation and increased the aflatoxin production. Furthermore, aflatoxin production increased with increased reactive oxidative species (ROS) levels at 96 h of growth, confirming illumination-driven oxidative stress modulation activity on A. flavus cells. Full article

The non-proteinogenic amino acid beta-methyl-amino-l-alanine (BMAA) is a neurotoxin produced by cyanobacteria. BMAA accumulation in the brain of animals via biomagnification along the food web can contribute to the development of neurodegenerative diseases such as Amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC), the latter being associated with a loss of dopaminergic neurons. Daphnia magna is an important microcrustacean zooplankton species that plays a key role in aquatic food webs, and BMAA-producing cyanobacteria often form part of their diet. Here, we tested the effects of BMAA on putative neurodegeneration of newly identified specific dopaminergic neurons in the optic ganglia/brain complex of D. magna using quantitative tyrosine-hydroxylase immunohistochemistry and fluorescence cytometry. The dopaminergic system was analysed in fed and starved isogenic D. magna adults incubated under different BMAA concentrations over 4 days. Increased BMAA concentration showed significant decrease in the stainability of dopaminergic neurons of D. magna, with fed animals showing a more extreme loss. Furthermore, higher BMAA concentrations tended to increase offspring mortality during incubation. These results are indicative of ingested BMAA causing neurodegeneration of dopaminergic neurons in D. magna and adversely affecting reproduction. This may imply similar effects of BMAA on known human neurodegenerative diseases involving dopaminergic neurons. Full article