Toxins

959 KiB

Open AccessArticle

In Vitro Glucuronidation of Ochratoxin A by Rat Liver Microsomes

by Zheng Han, Emmanuel K. Tangni, José Diana Di Mavungu, Lynn Vanhaecke, Sarah De Saeger, Aibo Wu and Alfons Callebaut

Toxins 2013, 5(12), 2671-2685; https://doi.org/10.3390/toxins5122671 - 18 Dec 2013

Cited by 21 | Viewed by 8863

Abstract

Ochratoxin A (OTA), one of the most toxic mycotoxins, can contaminate a wide range of food and feedstuff. To date, the data on its conjugates via glucuronidation request clarification and consolidation. In the present study, the combined approaches of ultra high performance liquid [...] Read more.

Ochratoxin A (OTA), one of the most toxic mycotoxins, can contaminate a wide range of food and feedstuff. To date, the data on its conjugates via glucuronidation request clarification and consolidation. In the present study, the combined approaches of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), UHPLC-Orbitrap-high resolution mass spectrometry (HRMS) and liquid chromatography-multiple stage mass spectrometry (LC-MSⁿ) were utilized to investigate the metabolic profile of OTA in rat liver microsomes. Three conjugated products of OTA corresponding to amino-, phenol- and acyl-glucuronides were identified, and the related structures were confirmed by hydrolysis with ?-glucuronidase. Moreover, OTA methyl ester, OT? and OT?-glucuronide were also found in the reaction solution. Based on these results, an in vitro metabolic pathway of OTA has been proposed for the first time. Full article

(This article belongs to the Special Issue Recent Advances in Ochratoxins Research)

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Graphical abstract

1479 KiB

Open AccessArticle

Occurrence of Deoxynivalenol and Deoxynivalenol-3-glucoside in Hard Red Spring Wheat Grown in the USA

by Senay Simsek, Maribel Ovando-Martínez, Bahri Ozsisli, Kristin Whitney and Jae-Bom Ohm

Toxins 2013, 5(12), 2656-2670; https://doi.org/10.3390/toxins5122656 - 18 Dec 2013

Cited by 18 | Viewed by 6274

Abstract

Deoxynivalenol (DON) is a mycotoxin found in wheat that is infected with Fusarium fungus. DON may also be converted to a type of “masked mycotoxin”, named deoxynivalenol-3-glucoside (D3G), as a result of detoxification of the plant. In this study, DON and D3G were [...] Read more.

Deoxynivalenol (DON) is a mycotoxin found in wheat that is infected with Fusarium fungus. DON may also be converted to a type of “masked mycotoxin”, named deoxynivalenol-3-glucoside (D3G), as a result of detoxification of the plant. In this study, DON and D3G were measured using gas chromatographic (GC) and liquid chromatography-mass spectrometry (LC-MS) in wheat samples collected during 2011 and 2012 in the USA. Results indicate that the growing region had a significant effect on the DON and D3G (p < 0.0001). There was a positive correlation between both methods (GC and LC-MS) used for determination of DON content. DON showed a significant and positive correlation with D3G during 2011. Overall, DON production had an effect on D3G content and kernel damage, and was dependent on environmental conditions during Fusarium infection. Full article

(This article belongs to the Special Issue Recent Advances and Perspectives in Deoxynivalenol Research)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Distribution of hard red spring (HRS) wheat samples from the 2011 and 2012 Crop Surveys from Montana, North Dakota, Minnesota and South Dakota. A, B, C, D, E and F: regions in which the samples were collected from each state. The numbers inside the parenthesis represent the number of samples taken, from left to right: 2011 Crop Survey and 2012 Crop Survey. Full article ">Figure 2
Correlation GC-DON and LC-MS DON content values. (a) 2011 survey samples; (b) 2012 survey samples and (c) 2011 and 2012 survey samples combined. *** Significantly different from 1 at p < 0.001. Full article ">Figure 3
Correlation between DON and D3G levels in survey samples between 2011 and 2012; where ***, and * indicate that regression coefficients are significant at p < 0.001 and p < 0.05, respectively. Full article ">Figure 4
Correlation between GC-DON and damage levels in survey samples from (a) 2011 and (b) 2012. Full article ">Figure 5
Representative chromatograms of a wheat sample containing approximately 2.0 mg/kg DON and 1.0 mg/kg D3G. (a) gas chromatography–electron capture detection (GC-ECD) chromatogram of DON * and Mirex ** (internal standard); (b) LC-MS extracted ion (m/z of (M + H) + ion 297.1333) chromatogram of DON; and (c) LC-MS extracted ion (m/z of the (M + Na) + ion 481.1680) chromatogram of D3G. Full article ">

9748 KiB

Open AccessArticle

Venom Down Under: Dynamic Evolution of Australian Elapid Snake Toxins

by Timothy N. W. Jackson, Kartik Sunagar, Eivind A. B. Undheim, Ivan Koludarov, Angelo H. C. Chan, Kate Sanders, Syed A. Ali, Iwan Hendrikx, Nathan Dunstan and Bryan G. Fry

Toxins 2013, 5(12), 2621-2655; https://doi.org/10.3390/toxins5122621 - 18 Dec 2013

Cited by 54 | Viewed by 16296

Abstract

Despite the unparalleled diversity of venomous snakes in Australia, research has concentrated on a handful of medically significant species and even of these very few toxins have been fully sequenced. In this study, venom gland transcriptomes were sequenced from eleven species of small [...] Read more.

Despite the unparalleled diversity of venomous snakes in Australia, research has concentrated on a handful of medically significant species and even of these very few toxins have been fully sequenced. In this study, venom gland transcriptomes were sequenced from eleven species of small Australian elapid snakes, from eleven genera, spanning a broad phylogenetic range. The particularly large number of sequences obtained for three-finger toxin (3FTx) peptides allowed for robust reconstructions of their dynamic molecular evolutionary histories. We demonstrated that each species preferentially favoured different types of ?-neurotoxic 3FTx, probably as a result of differing feeding ecologies. The three forms of ?-neurotoxin [Type I (also known as (aka): short-chain), Type II (aka: long-chain) and Type III] not only adopted differential rates of evolution, but have also conserved a diversity of residues, presumably to potentiate prey-specific toxicity. Despite these differences, the different ?-neurotoxin types were shown to accumulate mutations in similar regions of the protein, largely in the loops and structurally unimportant regions, highlighting the significant role of focal mutagenesis. We theorize that this phenomenon not only affects toxin potency or specificity, but also generates necessary variation for preventing/delaying prey animals from acquiring venom-resistance. This study also recovered the first full-length sequences for multimeric phospholipase A₂ (PLA₂) ‘taipoxin/paradoxin’ subunits from non-Oxyuranus species, confirming the early recruitment of this extremely potent neurotoxin complex to the venom arsenal of Australian elapid snakes. We also recovered the first natriuretic peptides from an elapid that lack the derived C-terminal tail and resemble the plesiotypic form (ancestral character state) found in viper venoms. This provides supporting evidence for a single early recruitment of natriuretic peptides into snake venoms. Novel forms of kunitz and waprin peptides were recovered, including dual domain kunitz-kunitz precursors and the first kunitz-waprin hybrid precursors from elapid snakes. The novel sequences recovered in this study reveal that the huge diversity of unstudied venomous Australian snakes are of considerable interest not only for the investigation of venom and whole organism evolution but also represent an untapped bioresource in the search for novel compounds for use in drug design and development. Full article

(This article belongs to the Collection Evolution of Venom Systems)

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462 KiB

Open AccessReview

Towards Clinical Applications of Anti-endotoxin Antibodies; A Re-appraisal of the Disconnect

by James C. Hurley

Toxins 2013, 5(12), 2589-2620; https://doi.org/10.3390/toxins5122589 - 18 Dec 2013

Cited by 26 | Viewed by 9134

Abstract

Endotoxin is a potent mediator of a broad range of patho-physiological effects in humans. It is present in all Gram negative (GN) bacteria. It would be expected that anti-endotoxin therapies, whether antibody based or not, would have an important adjuvant therapeutic role along [...] Read more.

Endotoxin is a potent mediator of a broad range of patho-physiological effects in humans. It is present in all Gram negative (GN) bacteria. It would be expected that anti-endotoxin therapies, whether antibody based or not, would have an important adjuvant therapeutic role along with antibiotics and other supportive therapies for GN infections. Indeed there is an extensive literature relating to both pre-clinical and clinical studies of anti-endotoxin antibodies. However, the extent of disconnect between the generally successful pre-clinical studies versus the failures of the numerous large clinical trials of antibody based and other anti-endotoxin therapies is under-appreciated and unexplained. Seeking a reconciliation of this disconnect is not an abstract academic question as clinical trials of interventions to reduce levels of endotoxemia levels are ongoing. The aim of this review is to examine new insights into the complex relationship between endotoxemia and sepsis in an attempt to bridge this disconnect. Several new factors to consider in this reappraisal include the frequency and types of GN bacteremia and the underlying mortality risk in the various study populations. For a range of reasons, endotoxemia can no longer be considered as a single entity. There are old clinical trials which warrant a re-appraisal in light of these recent advances in the understanding of the structure-function relationship of endotoxin. Fundamentally however, the disconnect not only remains, it has enlarged. Full article

(This article belongs to the Special Issue Toxin-Antibody Interactions)

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Figure 1
The location of the lipopolysaccharide (endotoxin) molecule in the cell wall of Gram negative bacteria. Full article ">Figure 2
The components of the lipopolysaccharide (endotoxin) molecule. Full article ">Figure 3
Simplified diagram of TLR4-MD-2 receptor complex dimerization upon ligation of hexa-acyl lipid A. See text for description. Copyright © 2013 Maeshima and Fernandez. From reference [<a href="#B13-toxins-05-02589" class="html-bibr">13</a>], an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited. Full article ">Figure 4
L’Abbé plots of study specific mortality rates from 35 studies. Each figure shows mortality rates for studies undertaken in an ICU (triangles) or non-ICU (circles) setting with symbols proportional to group size with the line of no difference (y = x; dotted line) shown for visual reference purposes. Shown are (a) Groups 1 (endotoxemia and GN bacteremia detected) versus groups 4 (neither detected); (b) Groups 2 (GN bacteremia alone) versus groups 4 (neither detected); and (c) Groups 3 (endotoxemia alone) versus groups 4 (neither detected). GN, gram negative. From [<a href="#B77-toxins-05-02589" class="html-bibr">77</a>] © 2012 Hurley et al.; licensee BioMed Central Ltd., an open access article distributed under the terms of the Creative Commons Attribution License. Full article ">

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Open AccessArticle

Expression of VEGF and Flk-1 and Flt-1 Receptors during Blood-Brain Barrier (BBB) Impairment Following Phoneutria nigriventer Spider Venom Exposure

by Monique C. P. Mendonça, Edilene S. Soares, Leila M. Stávale, Catarina Rapôso, Andressa Coope, Evanguedes Kalapothakis and Maria Alice Da Cruz-Höfling

Toxins 2013, 5(12), 2572-2588; https://doi.org/10.3390/toxins5122572 - 18 Dec 2013

Cited by 15 | Viewed by 7570

Abstract

Apart from its angiogenic and vascular permeation activity, the vascular endothelial growth factor (VEGF) has been also reported as a potent neuronal protector. Newborn rats with low VEGF levels develop neuron degeneration, while high levels induce protective mechanisms in several neuropathological conditions. Phoneutria [...] Read more.

Apart from its angiogenic and vascular permeation activity, the vascular endothelial growth factor (VEGF) has been also reported as a potent neuronal protector. Newborn rats with low VEGF levels develop neuron degeneration, while high levels induce protective mechanisms in several neuropathological conditions. Phoneutria nigriventer spider venom (PNV) disrupts the blood-brain barrier (BBB) and causes neuroinflammation in central neurons along with excitotoxic signals in rats and humans. All these changes are transient. Herein, we examined the expression of VEGF and its receptors, Flt-1 and Flk-1 in the hippocampal neurons following envenomation by PNV. Adult and neonatal rats were evaluated at time limits of 2, 5 and 24 h. Additionally, BBB integrity was assessed by measuring the expression of occludin, ?-catenin and laminin and neuron viability was evaluated by NeuN expression. VEGF, Flt-1 and Flk-1 levels increased in PNV-administered rats, concurrently with respective mRNAs. Flt-1 and Flk-1 immunolabeling was nuclear in neurons of hippocampal regions, instead of the VEGF membrane-bound typical location. These changes occurred simultaneously with the transient decreases in BBB-associated proteins and NeuN positivity. Adult rats showed more prominent expressional increases of the VEGF/Flt-1/Flk-1 system and earlier recovery of BBB-related proteins than neonates. We conclude that the reactive expressional changes seen here suggest that VEGF and receptors could have a role in the excitotoxic mechanism of PNV and that such role would be less efficient in neonate rats. Full article

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Figure 1
Immunoblots of occludin (A), β-catenin (B) and laminin (C). Phoneutria nigriventer (PNV) intra-peritoneal (i.p) injection induced significant decreases of all three proteins at 2 h for adults, whereas at 5 h for neonates. * p ≤ 0.05 and *** p ≤ 0.001 denote significant decreases relative to controls; ## p ≤ 0.01 and ### p ≤ 0.001 indicate PNV-treated neonates with higher increase in laminin expression than their adult counterpart at 2 and 5 h. Student t-test; data are shown as means ± SEM. Full article ">Figure 2
CA1 subfield of 8–10-week-old rats: composite photomicrographs of hippocampal coronal sections stained for localization of anti-VEGF, anti-Flt-1 and anti-Flk-1 and using the immunoperoxidase technique. (A,C,E) are sections from controls (saline-treated) and (B,D,F) are from PNV-treated adult rats 5 h after i.p. injection. PNV increased the immunoreactivity of VEGF, Flt-1 and Flk-1. Py = stratum pyramidale; Or = stratum oriens; Rad = stratum radiatum; * = microvessels with perivascular edema. Scale Bars = 25 µm. Full article ">Figure 3
Western blot signals were densitometrically quantified and normalized to an internal standard (β-actin). VEGF (A), Flt-1 (B) and Flk-1 (C) expressions in PNV-treated samples (1.7 mg/kg) relative to control (CTR); * p ≤ 0.05 and *** p ≤ 0.001 indicate significant difference relative to respective controls; # p ≤ 0.05 and ## p ≤ 0.01 denote significant age-related differences between control (CTR) or PNV-treated groups at corresponding time-point. Student t-test; data were shown as means ± SEM. Full article ">Figure 4
Quantitative real-time polymerase chain reaction (PCR) analysis quantified and normalized to endogen control (GAPDH). VEGF (A), Flt-1 (B) and Flk-1 (C) mRNAs expression at time-points after PNV (1.7 mg/kg) or 0.9% saline peritoneal injection. * p ≤ 0.05 and ** p ≤ 0.01 indicate significant difference relative to respective controls; # p ≤ 0.05, ## p ≤ 0.01 and ### p ≤ 0.001, denote significant age-related differences between control (CTR) or PNV-treated groups at corresponding time-point. Student t-test; data were shown as means ± SEM. Full article ">Figure 5
NeuN labeling in the CA2 subfield of 8–10-week-old rats 5 hafter i.p. injection of saline solution (A) or PNV (B). Py = stratum pyramidale; Or = stratum oriens; Rad = stratum radiatum; * = microvessels with perivascular edema. (C) Percentage of pixel density of NeuN-labeled neurons in different time points. (D) Immunoblots of NeuN in the hippocampus of i.p.-injected saline or PNV rats at 2, 5 and 24 h. * p ≤ 0.05 and ** p ≤ 0.01 denote significant decrease of the nuclear marker relative to respective controls; # p ≤ 0.05, ## p ≤ 0.01 and ### p ≤ 0.001) denote significant difference in NeuN expression between control (CTR) or PNV-treated groups at the corresponding time-point. Student t-test; data were shown as means ± SEM. Scale Bars = 25 µm. Full article ">

10055 KiB

Open AccessReview

Secreted Phospholipases A₂ of Snake Venoms: Effects on the Peripheral Neuromuscular System with Comments on the Role of Phospholipases A₂ in Disorders of the CNS and Their Uses in Industry

by John B. Harris and Tracey Scott-Davey

Toxins 2013, 5(12), 2533-2571; https://doi.org/10.3390/toxins5122533 - 17 Dec 2013

Cited by 120 | Viewed by 17280

Abstract

Neuro- and myotoxicological signs and symptoms are significant clinical features of envenoming snakebites in many parts of the world. The toxins primarily responsible for the neuro and myotoxicity fall into one of two categories—those that bind to and block the post-synaptic acetylcholine receptors [...] Read more.

Neuro- and myotoxicological signs and symptoms are significant clinical features of envenoming snakebites in many parts of the world. The toxins primarily responsible for the neuro and myotoxicity fall into one of two categories—those that bind to and block the post-synaptic acetylcholine receptors (AChR) at the neuromuscular junction and neurotoxic phospholipases A₂ (PLAs) that bind to and hydrolyse membrane phospholipids of the motor nerve terminal (and, in most cases, the plasma membrane of skeletal muscle) to cause degeneration of the nerve terminal and skeletal muscle. This review provides an introduction to the biochemical properties of secreted sPLA₂s in the venoms of many dangerous snakes and a detailed discussion of their role in the initiation of the neurologically important consequences of snakebite. The rationale behind the experimental studies on the pharmacology and toxicology of the venoms and isolated PLAs in the venoms is discussed, with particular reference to the way these studies allow one to understand the biological basis of the clinical syndrome. The review also introduces the involvement of PLAs in inflammatory and degenerative disorders of the central nervous system (CNS) and their commercial use in the food industry. It concludes with an introduction to the problems associated with the use of antivenoms in the treatment of neuro-myotoxic snakebite and the search for alternative treatments. Full article

(This article belongs to the Special Issue Neurotoxins: Health Threats and Biological Tools)

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321 KiB

Open AccessArticle

Analysis of Deoxynivalenol and Deoxynivalenol-3-glucoside in Hard Red Spring Wheat Inoculated with Fusarium Graminearum

by Maribel Ovando-Martínez, Bahri Ozsisli, James Anderson, Kristin Whitney, Jae-Bom Ohm and Senay Simsek

Toxins 2013, 5(12), 2522-2532; https://doi.org/10.3390/toxins5122522 - 17 Dec 2013

Cited by 39 | Viewed by 7282

Abstract

Deoxynivalenol (DON) is a mycotoxin affecting wheat quality. The formation of the “masked” mycotoxin deoxinyvalenol-3-glucoside (D3G) results from a defense mechanism the plant uses for detoxification. Both mycotoxins are important from a food safety point of view. The aim of this work was [...] Read more.

Deoxynivalenol (DON) is a mycotoxin affecting wheat quality. The formation of the “masked” mycotoxin deoxinyvalenol-3-glucoside (D3G) results from a defense mechanism the plant uses for detoxification. Both mycotoxins are important from a food safety point of view. The aim of this work was to analyze DON and D3G content in inoculated near-isogenic wheat lines grown at two locations in Minnesota, USA during three different years. Regression analysis showed positive correlation between DON content measured with LC and GC among wheat lines, locality and year. The relationship between DON and D3G showed a linear increase until a certain point, after which the DON content and the D3G increased. Wheat lines having higher susceptibility to Fusarium showed the opposite trend. ANOVA demonstrated that the line and location have a greater effect on variation of DON and D3G than do their interaction among years. The most important factor affecting DON and D3G was the growing location. In conclusion, the year, environmental conditions and location have an effect on the D3G/DON ratio in response to Fusarium infection. Full article

(This article belongs to the Special Issue Recent Advances and Perspectives in Deoxynivalenol Research)

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281 KiB

Open AccessArticle

Phormidium autumnale Growth and Anatoxin-a Production under Iron and Copper Stress

by Francine M. J. Harland, Susanna A. Wood, Elena Moltchanova, Wendy M. Williamson and Sally Gaw

Toxins 2013, 5(12), 2504-2521; https://doi.org/10.3390/toxins5122504 - 16 Dec 2013

Cited by 31 | Viewed by 9265

Abstract

Studies on planktonic cyanobacteria have shown variability in cyanotoxin production, in response to changes in growth phase and environmental factors. Few studies have investigated cyanotoxin regulation in benthic mat-forming species, despite increasing reports on poisoning events caused by ingestion of these organisms. In [...] Read more.

Studies on planktonic cyanobacteria have shown variability in cyanotoxin production, in response to changes in growth phase and environmental factors. Few studies have investigated cyanotoxin regulation in benthic mat-forming species, despite increasing reports on poisoning events caused by ingestion of these organisms. In this study, a method was developed to investigate changes in cyanotoxin quota in liquid cultures of benthic mat-forming cyanobacteria. Iron and copper are important in cellular processes and are well known to affect growth and selected metabolite production in cyanobacteria and algae. The effect of iron (40–4000 ?g L^?¹) and copper (2.5–250 ?g L^?¹) on growth and anatoxin-a quota in Phormidium autumnale was investigated in batch culture. These concentrations were chosen to span those found in freshwater, as well as those previously reported to be toxic to cyanobacteria. Anatoxin-a concentrations varied throughout the growth curve, with a maximum quota of between 0.49 and 0.55 pg cell^?¹ measured within the first two weeks of growth. Growth rates were significantly affected by copper and iron concentrations (P < 0.0001); however, no statistically significant difference between anatoxin-a quota maxima was observed. When the iron concentrations were 800 and 4000 ?g L^?¹, the P. autumnale cultures did not firmly attach to the substratum. At 250 ?g L^?¹ copper or either 40 or 4000 ?g L^?¹ iron, growth was suppressed. Full article

(This article belongs to the Collection Marine and Freshwater Toxins)

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Figure 1
Mean metal concentrations in MLA with 95% confidence intervals measured in Phormidium autumnale (CYN52) growth experiments: (a) Iron <img alt="Toxins 05 02504 i001" src="/toxins/toxins-05-02504/article_deploy/html/images/toxins-05-02504-i001.png"/> control, ■ treatment (MLA2×Fe); (b) Copper <img alt="Toxins 05 02504 i001" src="/toxins/toxins-05-02504/article_deploy/html/images/toxins-05-02504-i001.png"/> control, ■ treatment (MLA2×Fe). (n = 3 for cultures, n = 2+ for controls). Full article ">Figure 2
Growth profile for Phormidium autumnale (CYN52) under (a) iron (Fe) and (b) copper (Cu) regimes over 49 days. Growth is recorded as mean number of cells per 30 mL culture with 95% confidence intervals (n = 3). Full article ">Figure 3
Intracellular anatoxin-a in Phormidium autumnale (CYN52) under (a) iron (Fe) and (b) copper (Cu) regimes. Anatoxin-a quota are recorded as mean concentrations with 95% confidence intervals (n = 3). Full article ">Figure 4
Extracellular anatoxin-a detected in Phormidium autumnale (CYN52) growth experiment under standard MLA1×Fe,1×Cu. Extracellular anatoxin-a is recorded as mean concentration with 95% confidence intervals (n = 3); nd, not detected. Full article ">Figure 5
Percentage of extracellular anatoxin-a from total intra- and extracellular anatoxin-a detected for each treatment (Fe = iron, Cu = copper) during the late exponential and stationary phase. 95% confidence intervals are included; <img alt="Toxins 05 02504 i002" src="/toxins/toxins-05-02504/article_deploy/html/images/toxins-05-02504-i002.png"/> statistically significant concentrations per day; nd, not detected. Full article ">

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Open AccessArticle

A Proteomics and Transcriptomics Investigation of the Venom from the Barychelid Spider Trittame loki (Brush-Foot Trapdoor)

by Eivind A. B. Undheim, Kartik Sunagar, Volker Herzig, Laurence Kely, Dolyce H. W. Low, Timothy N. W. Jackson, Alun Jones, Nyoman Kurniawan, Glenn F. King, Syed A. Ali, Agostino Antunes, Tim Ruder and Bryan G. Fry

Toxins 2013, 5(12), 2488-2503; https://doi.org/10.3390/toxins5122488 - 13 Dec 2013

Cited by 61 | Viewed by 11324

Abstract

Although known for their potent venom and ability to prey upon both invertebrate and vertebrate species, the Barychelidae spider family has been entirely neglected by toxinologists. In striking contrast, the sister family Theraphosidae (commonly known as tarantulas), which last shared a most recent [...] Read more.

Although known for their potent venom and ability to prey upon both invertebrate and vertebrate species, the Barychelidae spider family has been entirely neglected by toxinologists. In striking contrast, the sister family Theraphosidae (commonly known as tarantulas), which last shared a most recent common ancestor with Barychelidae over 200 million years ago, has received much attention, accounting for 25% of all the described spider toxins while representing only 2% of all spider species. In this study, we evaluated for the first time the venom arsenal of a barychelid spider, Trittame loki, using transcriptomic, proteomic, and bioinformatic methods. The venom was revealed to be dominated by extremely diverse inhibitor cystine knot (ICK)/knottin peptides, accounting for 42 of the 46 full-length toxin precursors recovered in the transcriptomic sequencing. In addition to documenting differential rates of evolution adopted by different ICK/knottin toxin lineages, we discovered homologues with completely novel cysteine skeletal architecture. Moreover, acetylcholinesterase and neprilysin were revealed for the first time as part of the spider-venom arsenal and CAP (CRiSP/Allergen/PR-1) were identified for the first time in mygalomorph spider venoms. These results not only highlight the extent of venom diversification in this neglected ancient spider lineage, but also reinforce the idea that unique venomous lineages are rich pools of novel biomolecules that may have significant applied uses as therapeutics and/or insecticides. Full article

(This article belongs to the Collection Evolution of Venom Systems)

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Magnetic resonance imaging of Trittame loki venom glands. Full article ">Figure 2
Phylogenetic reconstruction of Trittame loki and related inhibitor cystine knot (ICK)/knottin peptide toxins, conserved ancestral cysteines are shown in black, newly evolved cysteines are in red. Sequences obtained in this study are in green. Signal peptides are shown in lowercase. Full article ">Figure 3
Sequence alignment of spider venom colipase venom peptides: (1) Trittame loki COLIPASE-1; (2) D2Y2E5 Haplopelma hainanum; (3) Q5D233 Hadronyche infensa; (4) Q5D231 Hadronyche sp. (strain 20); (5) Q5D232 Hadronyche sp. (strain 20); (6) B1P1J0 Chilobrachys jingzhao; and (7) B1P1J2 Chilobrachys jingzhao. Signal peptides are shown in lowercase. Full article ">Figure 4
Sequence alignment of spider venom CAP (CRiSP/Allergen/PR-1) venom peptides: (1) Trittame loki CAP-1; and (2) A9QQ26 Lycosa singoriensis. Signal peptides are shown in lowercase. Full article ">Figure 5
Sequence alignment of spider venom kunitz venom peptides: (1) Trittame loki KUNITZ-1; and (2) E7D1N7 Latrodectus hesperus. Signal peptides are shown in lowercase. Full article ">Figure 6
Sequence alignment of the Trittame loki venom acetylcholinesterase and the non-venom homologue P56161 Anopheles stephensi. Signal peptides are shown in lowercase. Full article ">Figure 7
Sequence alignment of the Trittame loki venom neprilysin and the snake venom convergent neprilysin homologue T1E4Z0 Crotalus horridus. Signal peptides are shown in lowercase. Full article ">

5767 KiB

Open AccessArticle

Evolution Stings: The Origin and Diversification of Scorpion Toxin Peptide Scaffolds

by Kartik Sunagar, Eivind A. B. Undheim, Angelo H. C. Chan, Ivan Koludarov, Sergio A. Muñoz-Gómez, Agostinho Antunes and Bryan G. Fry

Toxins 2013, 5(12), 2456-2487; https://doi.org/10.3390/toxins5122456 - 13 Dec 2013

Cited by 75 | Viewed by 14470

Abstract

The episodic nature of natural selection and the accumulation of extreme sequence divergence in venom-encoding genes over long periods of evolutionary time can obscure the signature of positive Darwinian selection. Recognition of the true biocomplexity is further hampered by the limited taxon selection, [...] Read more.

The episodic nature of natural selection and the accumulation of extreme sequence divergence in venom-encoding genes over long periods of evolutionary time can obscure the signature of positive Darwinian selection. Recognition of the true biocomplexity is further hampered by the limited taxon selection, with easy to obtain or medically important species typically being the subject of intense venom research, relative to the actual taxonomical diversity in nature. This holds true for scorpions, which are one of the most ancient terrestrial venomous animal lineages. The family Buthidae that includes all the medically significant species has been intensely investigated around the globe, while almost completely ignoring the remaining non-buthid families. Australian scorpion lineages, for instance, have been completely neglected, with only a single scorpion species (Urodacus yaschenkoi) having its venom transcriptome sequenced. Hence, the lack of venom composition and toxin sequence information from an entire continent’s worth of scorpions has impeded our understanding of the molecular evolution of scorpion venom. The molecular origin, phylogenetic relationships and evolutionary histories of most scorpion toxin scaffolds remain enigmatic. In this study, we have sequenced venom gland transcriptomes of a wide taxonomical diversity of scorpions from Australia, including buthid and non-buthid representatives. Using state-of-art molecular evolutionary analyses, we show that a majority of CS?/? toxin scaffolds have experienced episodic influence of positive selection, while most non-CS?/? linear toxins evolve under the extreme influence of negative selection. For the first time, we have unraveled the molecular origin of the major scorpion toxin scaffolds, such as scorpion venom single von Willebrand factor C-domain peptides (SV-SVC), inhibitor cystine knot (ICK), disulphide-directed beta-hairpin (DDH), bradykinin potentiating peptides (BPP), linear non-disulphide bridged peptides and antimicrobial peptides (AMP). We have thus demonstrated that even neglected lineages of scorpions are a rich pool of novel biochemical components, which have evolved over millions of years to target specific ion channels in prey animals, and as a result, possess tremendous implications in therapeutics. Full article

(This article belongs to the Collection Evolution of Venom Systems)

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938 KiB

Open AccessArticle

Appearance of Planktothrix rubescens Bloom with [D-Asp³, Mdha⁷]MC–RR in Gravel Pit Pond of a Shallow Lake-Dominated Area

by Gábor Vasas, Oszkár Farkas, Gábor Borics, Tamás Felföldi, Gábor Sramkó, Gyula Batta, István Bácsi and Sándor Gonda

Toxins 2013, 5(12), 2434-2455; https://doi.org/10.3390/toxins5122434 - 12 Dec 2013

Cited by 27 | Viewed by 9344

Abstract

Blooms of toxic cyanobacteria are well-known phenomena in many regions of the world. Microcystin (MC), the most frequent cyanobacterial toxin, is produced by entirely different cyanobacteria, including unicellular, multicellular filamentous, heterocytic, and non-heterocytic bloom-forming species. Planktothrix is one of the most important MC-producing [...] Read more.

Blooms of toxic cyanobacteria are well-known phenomena in many regions of the world. Microcystin (MC), the most frequent cyanobacterial toxin, is produced by entirely different cyanobacteria, including unicellular, multicellular filamentous, heterocytic, and non-heterocytic bloom-forming species. Planktothrix is one of the most important MC-producing genera in temperate lakes. The reddish color of cyanobacterial blooms viewed in a gravel pit pond with the appearance of a dense 3 cm thick layer (biovolume: 28.4 mm³ L^?1) was an unexpected observation in the shallow lake-dominated alluvial region of the Carpathian Basin. [d-Asp³, Mdha⁷]MC–RR was identified from the blooms sample by MALDI-TOF and NMR. Concentrations of [d-Asp³, Mdha⁷]MC–RR were measured by capillary electrophoresis to compare the microcystin content of the field samples and the isolated, laboratory-maintained P. rubescens strain. In analyzing the MC gene cluster of the isolated P. rubescens strain, a deletion in the spacer region between mcyE and mcyG and an insertion were located in the spacer region between mcyT and mcyD. The insertion elements were sequenced and partly identified. Although some invasive tropical cyanobacterial species have been given a great deal of attention in many recent studies, our results draw attention to the spread of the alpine organism P. rubescens as a MC-producing, bloom-forming species. Full article

(This article belongs to the Collection Marine and Freshwater Toxins)

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(a) Location of Kocka pond in Hungary, indicated by a filled circle; (b) The Planktothrix rubescens bloom in the gravel pit pond; and (c) a microscopic observation of Planktothrix rubescens trichomes from the pond. Full article ">Figure 2
(A) Maximum likelihood trees showing the phylogenetic position of BGSD-500 based on the 16S rRNA gene and (B) the phycocyanin operon. In the case of the 16S rRNA gene, 1329 nt positions were involved in the analysis that was performed with the HKY + G substitution model, while for the construction of the cpcBA-IGS tree, 464 nt were used and the Kimura 2-parameter model was applied. Type strains of Planktothrix species according to Suda et al. [<a href="#B29-toxins-05-02434" class="html-bibr">29</a>] are marked with superscript T. Arthrospira platensis PCC 7345 was used as an outgroup in both phylogenetic analyses. Bootstrap values lower than 70 are not shown (based on 500 replicates). Full article ">Figure 3
DEAE-52 chromatography and Blue-Green Sinapis Test of [d-Asp3, Mdha7]MC–RR from Planktothrix rubescens. Absorbance at 239 nm (-○-); hypocotyl length of three-day-old mustard seedlings (-●-), gradient between 0 and 0.2 M NaCl in 5 mM Tris-HCl buffer (---). Full article ">Figure 4
Chemical structure of the identified cyanobacterial heptapeptide [d-Asp3, Mdha7]MC–RR. Full article ">Figure 5
Effect of crude P. rubescens-dominated bloom-sample extract (-●-) and the isolated P. rubescens BGSD-500 (- <img alt="Toxins 05 02434 i001" src="/toxins/toxins-05-02434/article_deploy/html/images/toxins-05-02434-i001.png"/>-) on the growth of Sinapis alba etiolated seedlings (Blue-Green-Sinapis-Test). Full article ">Figure 6
Capillary electrophoresis of P. rubescens-dominated bloom-sample extract (A) and the isolated P. rubescens BGSD-500 (B). Peak of [d-Asp3, Mdha7]MC–RR is indicated by black arrow. (separation conditions: capillary: 64.5 cm, 50 µm i.d., buffer electrolyte: 25 mM borate and 75 mM SDS, pH 9.3, applied voltage: +25 kV, detection: UV absorption at 238 nm). Full article ">Figure 7
Localization of the detected and partly identified 1194 nt length insertion element in the spacer region between mcyT and mcyD of mcy gene cluster of Planktothrix. Full article ">

993 KiB

Open AccessArticle

Preliminary Results of the in Vivo and in Vitro Characterization of a Tentacle Venom Fraction from the Jellyfish Aurelia aurita

by Dalia Ponce, Estuardo López-Vera, Manuel B. Aguilar and Judith Sánchez-Rodríguez

Toxins 2013, 5(12), 2420-2433; https://doi.org/10.3390/toxins5122420 - 6 Dec 2013

Cited by 18 | Viewed by 8357

Abstract

The neurotoxic effects produced by a tentacle venom extract and a fraction were analyzed and correlated by in vivo and in vitro approaches. The tentacle venom extract exhibited a wide range of protein components (from 24 to >225 kDa) and produced tetanic reactions, [...] Read more.

The neurotoxic effects produced by a tentacle venom extract and a fraction were analyzed and correlated by in vivo and in vitro approaches. The tentacle venom extract exhibited a wide range of protein components (from 24 to >225 kDa) and produced tetanic reactions, flaccid paralysis, and death when injected into crabs. Two chromatography fractions also produced uncontrolled appendix movements and leg stretching. Further electrophysiological characterization demonstrated that one of these fractions potently inhibited ACh-elicited currents mediated by both vertebrate fetal and adult muscle nicotinic acetylcholine receptors (nAChR) subtypes. Receptor inhibition was concentration-dependent and completely reversible. The calculated IC50 values were 1.77 ?g/?L for fetal and 2.28 ?g/?L for adult muscle nAChRs. The bioactive fraction was composed of a major protein component at ~90 kDa and lacked phospholipase A activity. This work represents the first insight into the interaction of jellyfish venom components and muscle nicotinic receptors. Full article

(This article belongs to the Collection Marine and Freshwater Toxins)

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Figure 1
Micrograph of A. aurita nematocysts along fishing tentacles. Atrichous ishorizas (white arrow) measured 6 × 4 µm wide and presented a disperse distribution along the tentacle tissue. Heterotrichous microbasic euryteles (black arrow) were approximately 12 × 9 µm wide and were arranged in clusters. 400× magnification, bar = 50 µm. Full article ">Figure 2
Protein content and fractionation of A. aurita TVE. (A) SDS-PAGE protein profile of TVE was performed in 12% polyacrylamide gel stained with Coomassie brilliant blue R-250. M indicates molecular mass standards (Amersham Rainbow Marker high-range, GE Healthcare); Lane 1 corresponds to 20 μg total protein of TVE. (B) Chromatogram of TVE fractionation by C18 reversed-phase column using a gradient of solution B from 5% to 95% (dashed line) at a flow rate of 1 mL/min over 60 min; Baseline indicated with a thin line. (C) SDS-PAGE protein profile of chromatography fractions. Analysis was performed in 10% polyacrylamide gels stained with Coomassie brilliant blue R-250. M indicates molecular mass standards (Kaleidoscope, Bio-Rad); Lanes 1–10 correspond to the eluted peaks equally labeled from HPLC fractionation. Full article ">Figure 3
Activity of fraction 4 on fetal muscle nAChR expressed in X. laevis oocytes. Concentration-response effects can be compared through the various panels. Arrows indicate the first current elicited after the 5 min static bath of toxin equilibration. Control membrane depolarisations elicited by 1–5 μM ACh pulses are indicated as 10% of total value (nA); (A) 1.5 μg total protein of fraction 4 inhibited 25% of ACh-elicited currents; (B) 3 μg total protein of fraction 4 blocked 50% of ACh currents; (C) 4.5 μg total protein of fraction 4 caused 96% blocking effect. Full article ">Figure 4
Concentration-response curves for fraction 4 on the adult (up-triangle) and fetal (down-triangle) subtypes of mouse muscle nAChR. Curves were generated by plotting current amplitude after toxin application as a percentage of current amplitude prior to toxin application (% response). Each data point represents the average value ± S.E. of the responses from three oocytes. Full article ">

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Open AccessArticle

Molecular Cloning and Pharmacological Properties of an Acidic PLA₂ from Bothrops pauloensis Snake Venom

by Francis Barbosa Ferreira, Mário Sérgio Rocha Gomes, Dayane Lorena Naves De Souza, Sarah Natalie Cirilo Gimenes, Letícia Eulalio Castanheira, Márcia Helena Borges, Renata Santos Rodrigues, Kelly Aparecida Geraldo Yoneyama, Maria Inês Homsi Brandeburgo and Veridiana M. Rodrigues

Toxins 2013, 5(12), 2403-2419; https://doi.org/10.3390/toxins5122403 - 4 Dec 2013

Cited by 28 | Viewed by 7593

Abstract

In this work, we describe the molecular cloning and pharmacological properties of an acidic phospholipase A₂ (PLA₂) isolated from Bothrops pauloensis snake venom. This enzyme, denominated BpPLA₂-TXI, was purified by four chromatographic steps and represents 2.4% of [...] Read more.

In this work, we describe the molecular cloning and pharmacological properties of an acidic phospholipase A₂ (PLA₂) isolated from Bothrops pauloensis snake venom. This enzyme, denominated BpPLA₂-TXI, was purified by four chromatographic steps and represents 2.4% of the total snake venom protein content. BpPLA₂-TXI is a monomeric protein with a molecular mass of 13.6 kDa, as demonstrated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis and its theoretical isoelectric point was 4.98. BpPLA₂-TXI was catalytically active and showed some pharmacological effects such as inhibition of platelet aggregation induced by collagen or ADP and also induced edema and myotoxicity. BpPLA₂-TXI displayed low cytotoxicity on TG-180 (CCRF S 180 II) and Ovarian Carcinoma (OVCAR-3), whereas no cytotoxicity was found in regard to MEF (Mouse Embryonic Fibroblast) and Sarcoma 180 (TIB-66). The N-terminal sequence of forty-eight amino acid residues was determined by Edman degradation. In addition, the complete primary structure of 122 amino acids was deduced by cDNA from the total RNA of the venom gland using specific primers, and it was significantly similar to other acidic D49 PLA₂s. The phylogenetic analyses showed that BpPLA₂-TXI forms a group with other acidic D49 PLA₂s from the gender Bothrops, which are characterized by a catalytic activity associated with anti-platelet effects. Full article

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343 KiB

Open AccessArticle

Different Assay Conditions for Detecting the Production and Release of Heat-Labile and Heat-Stable Toxins in Enterotoxigenic Escherichia coli Isolates

by Letícia B. Rocha, Christiane Y. Ozaki, Denise S. P. Q. Horton, Caroline A. Menezes, Anderson Silva, Irene Fernandes, Fabio C. Magnoli, Tania M. I. Vaz, Beatriz E. C. Guth and Roxane M. F. Piazza

Toxins 2013, 5(12), 2384-2402; https://doi.org/10.3390/toxins5122384 - 2 Dec 2013

Cited by 16 | Viewed by 7954

Abstract

Enterotoxigenic Escherichia coli (ETEC) produce heat-labile (LT) and/or heat-stable enterotoxins (ST). Despite that, the mechanism of action of both toxins are well known, there is great controversy in the literature concerning the in vitro production and release of LT and, for ST, no [...] Read more.

Enterotoxigenic Escherichia coli (ETEC) produce heat-labile (LT) and/or heat-stable enterotoxins (ST). Despite that, the mechanism of action of both toxins are well known, there is great controversy in the literature concerning the in vitro production and release of LT and, for ST, no major concerns have been discussed. Furthermore, the majority of published papers describe the use of only one or a few ETEC isolates to define the production and release of these toxins, which hinders the detection of ETEC by phenotypic approaches. Thus, the present study was undertaken to obtain a better understanding of ST and LT toxin production and release under laboratory conditions. Accordingly, a collection of 90 LT-, ST-, and ST/LT-producing ETEC isolates was used to determine a protocol for toxin production and release aimed at ETEC detection. For this, we used previously raised anti-LT antibodies and the anti-ST monoclonal and polyclonal antibodies described herein. The presence of bile salts and the use of certain antibiotics improved ETEC toxin production/release. Triton X-100, as chemical treatment, proved to be an alternative method for toxin release. Consequently, a common protocol that can increase the production and release of LT and ST toxins could facilitate and enhance the sensitivity of diagnostic tests for ETEC using the raised and described antibodies in the present work. Full article

(This article belongs to the Special Issue Advances in Toxin Detection)

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Reactivity of ST MAb by immunoblotting. Bacterial lysates from strains 30 (Lane 1), 127 (Lane 2), and 3321-4 (Lane 3) (3 µg) were separated by SDS-PAGE (10% gel; tricine) and transferred to a PVDF membrane. Each strip was incubated with anti-ST MAb followed by goat anti-mouse IgG peroxidase-conjugate. Immunodetection signals were visualized by addition of DAB/H2O2. Molecular markers are indicated as kilodaltons (kDa) at the left side of the panel. The arrow indicates the pre-pro-peptide form of ST toxin. ETEC isolates 30 and 127 had spontaneously lost the estA gene. Full article ">Figure 2
LT (A) and ST (B) production after chemical treatments. The ETEC H10407 strain was cultivated in EC broth. After 16–18 h, the culture was centrifuged and cell pellets were treated with 0.1 M EDTA or 0.2 mg/mL polymyxin B sulfate (1606 UI/mL) or 2% triton X-100 (small checkered) or not (white bars). The same compounds were also added directly to the culture growth (large checkered) or not (white bars) and then centrifuged. The supernatants treated or not were tested for LT (A) or for ST (B) by cELISA. The error bars represent the absorbance means and standard errors of duplicates of three independent experiments. Full article ">Figure 3
LT (A) and ST (B) production in presence or absence of antibiotics. The ETEC H10407 strain was cultivated in EC broth containing lincomycin (black line), ciprofloxacin (green line), lincomycin plus ciprofloxacin (red line) or no antibiotic (blue line). The supernatants were tested for LT (A) by cELISA or for ST (B) by indirect ELISA. Full article ">Figure 4
In vitro effects of lincomycin and ciprofloxacin on enterotoxigenic Escherichia coli isolates. (A) LT-producing strains. (B) ST/LT-producing strains. The strains were cultivated in EC broth (○/white bars) or EC broth containing lincomycin and ciprofloxacin (●/crosshatched bars), and bacterial growth cultures were treated with 2% triton X-100. Each supernatant was tested for LT by cELISA. Full article ">Figure 5
In vitro effects of lincomycin and ciprofloxacin on enterotoxigenic Escherichia coli isolates. (A) ST-producing strains. (B) ST/LT-producing strains. The strains were cultivated in EC broth (○/white bars) or EC broth containing lincomycin and ciprofloxacin (●/crosshatched bars), and the bacterial growth cultures were treated with 2% triton X-100. Each supernatant was tested for ST by cELISA. Full article ">

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Open AccessReview

Biotoxin Detection Using Cell-Based Sensors

by Pratik Banerjee, Spyridon Kintzios and Balabhaskar Prabhakarpandian

Toxins 2013, 5(12), 2366-2383; https://doi.org/10.3390/toxins5122366 - 29 Nov 2013

Cited by 49 | Viewed by 15189

Abstract

Cell-based biosensors (CBBs) utilize the principles of cell-based assays (CBAs) by employing living cells for detection of different analytes from environment, food, clinical, or other sources. For toxin detection, CBBs are emerging as unique alternatives to other analytical methods. The main advantage of [...] Read more.

Cell-based biosensors (CBBs) utilize the principles of cell-based assays (CBAs) by employing living cells for detection of different analytes from environment, food, clinical, or other sources. For toxin detection, CBBs are emerging as unique alternatives to other analytical methods. The main advantage of using CBBs for probing biotoxins and toxic agents is that CBBs respond to the toxic exposures in the manner related to actual physiologic responses of the vulnerable subjects. The results obtained from CBBs are based on the toxin-cell interactions, and therefore, reveal functional information (such as mode of action, toxic potency, bioavailability, target tissue or organ, etc.) about the toxin. CBBs incorporate both prokaryotic (bacteria) and eukaryotic (yeast, invertebrate and vertebrate) cells. To create CBB devices, living cells are directly integrated onto the biosensor platform. The sensors report the cellular responses upon exposures to toxins and the resulting cellular signals are transduced by secondary transducers generating optical or electrical signals outputs followed by appropriate read-outs. Examples of the layout and operation of cellular biosensors for detection of selected biotoxins are summarized. Full article

(This article belongs to the Special Issue Advances in Toxin Detection)

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1414 KiB

Open AccessArticle

An Extract of Rhodobacter sphaeroides Reduces Cisplatin-Induced Nephrotoxicity in Mice

by Wen-Wei Chang, Jau-Jin Liu, Chi-Fan Liu, Wen-Sheng Liu, Yun-Ping Lim, Yu-Jung Cheng and Che-Hsin Lee

Toxins 2013, 5(12), 2353-2365; https://doi.org/10.3390/toxins5122353 - 29 Nov 2013

Cited by 22 | Viewed by 6461

Abstract

Cisplatin is used as a treatment for various types of solid tumors. Renal injury severely limits the use of cisplatin. Renal cell apoptosis, oxidative stress, and inflammation contribute to cisplatin-induced nephrotoxicity. Previously, we found that an extract of Rhodobacter sphaeroides (Lycogen™) inhibited proinflammatory [...] Read more.

Cisplatin is used as a treatment for various types of solid tumors. Renal injury severely limits the use of cisplatin. Renal cell apoptosis, oxidative stress, and inflammation contribute to cisplatin-induced nephrotoxicity. Previously, we found that an extract of Rhodobacter sphaeroides (Lycogen™) inhibited proinflammatory cytokines and the production of nitric oxide in activated macrophages in a dextran sodium sulfate (DSS)-induced colitis model. Here, we evaluated the effect of Lycogen™, a potent anti-inflammatory agent, in mice with cisplatin-induced renal injury. We found that attenuated renal injury correlated with decreased apoptosis due to a reduction in caspase-3 expression in renal cells. Oral administration of Lycogen™ significantly reduced the expression of tumor necrosis factor-? and interleukin-1? in mice with renal injury. Lycogen™ reduces renal dysfunction in mice with cisplatin-induced renal injury. The protective effects of the treatment included blockage of the cisplatin-induced elevation in serum urea nitrogen and creatinine. Meanwhile, Lycogen™ attenuated body weight loss and significantly prolonged the survival of mice with renal injury. We propose that Lycogen™ exerts anti-inflammatory activities that represent a promising strategy for the treatment of cisplatin-induced renal injury. Full article

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The effects of Lycogen™ and cisplatin on the viability of mesangial cells (MES-13). MES-13 cells were treated with the indicated concentrations of Lycogen™ or cisplatin for 48 h. Cell viability was measured after (A) Lycogen™ treatment or (B) cisplatin treatment using a WST-1 assay. *** p < 0.001 (mean ± SD, n = 6). Each experiment was repeated three times with similar results. Full article ">Figure 2
Lycogen™ reduced cisplatin-induced cell apoptosis. MES-13 cells were pretreated with Lycogen™ at concentration of 0, 2, 4, 8 or 16 μM for 48 h. Next, cisplatin (5 μg/mL) was added to the cells for 48 h. (A) Cell viability was measured using a WST-1 assay; (B) The expression of nuclear p53 and caspase 3 was measured by Western blot analysis. Proliferating cell nuclear antigen (PCNA) and β-actin expression served as loading controls for nuclear proteins and total proteins, respectively. Inserted values indicate the relative protein expression when compared with pro-caspase 3. * p < 0.05, ** p < 0.01, *** p < 0.001 (mean ± SD, n = 6); (C) TNF-α secreted by MES-13 cells is mediated by TLR4 signaling. The cells were preincubated with anti-TLR4 or with control IgG, and then treated with or without Lycogen™ for 48 h. TNF-α was measured using an enzyme-linked immunosorbent assay. Each experiment was repeated three times with similar results. Full article ">Figure 3
Lycogen™ ameliorated cisplatin-induced renal dysfunction. Mice were treated with Lycogen™ (1 mg/kg) for three consecutive days, starting on day 0. Mice were given an intraperitoneal injection (i.p.) of cisplatin (30 mg/kg) on day 3. Control mice received PBS. The effect of Lycogen™ on (A) creatinine and (B) blood urine nitrogen (BUN) levels 72 h after cisplatin administration. * p < 0.05 (mean ± SD, n = 4). (C) Sections of kidney were stained with PAS or TUNEL 72 h after the cisplatin injection. The arrows indicate the location of positive TUNEL staining in the kidney. Each experiment was repeated three times with similar results. Full article ">Figure 4
Lycogen™ ameliorated cisplatin-induced renal inflammation. Mice were treated with Lycogen™ (1 mg/kg) for three consecutive days starting on day 0. Next, mice were injected i.p. with cisplatin (30 mg/kg) on day 3. Control mice received PBS. (A) TNF-α and (B) IL-1β levels in the sera as measured by ELISA 72 h after cisplatin treatment. * p < 0.05 (mean ± SD, n = 4). (C) Lycogen™ reduced cytokine expression in the kidney. A portion of the mice received Lycogen™ treatment. After three days, mice were killed, kidneys were collected, and the renal lysates were analyzed for TNF-α and IL-1β expression using immunoblot analysis. Each experiment was repeated three times with similar results. Full article ">Figure 5
The effect of Lycogen™ on cisplatin-induced renal injury in mice. (A) The mice were orally administered Lycogen™ (1 mg/kg) for three consecutive days after an i.p. injection of cisplatin (30 mg/kg) and the body weight of mice was recorded daily (mean ± SD, n = 8, * p < 0.01 for cisplatin-induced renal injury mice pretreated with Lycogen™ versus cisplatin-induced renal injury mice pretreated with PBS); (B) Kaplan-Meier survival curves up to day 14 are shown (n = 8). Significant differences between continuous variables was assessed using Student’s t-test. Mice survival analysis was performed using the Kaplan-Meier survival curve and log-rank test. * p < 0.05, ** p < 0.01; (C) The mice were injected i.p. with cisplatin (30 mg/kg) and then orally administered Lycogen™ (1 mg/kg) for three consecutive days beginning 16 h after the cisplatin administration. The body weights of mice were recorded (mean ± SD, n = 8); (D) Kaplan-Meier survival curves up to day 14 are shown (n = 8). Full article ">

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Open AccessArticle

Deoxynivanelol and Fumonisin, Alone or in Combination, Induce Changes on Intestinal Junction Complexes and in E-Cadherin Expression

by Karina Basso, Fernando Gomes and Ana Paula Loureiro Bracarense

Toxins 2013, 5(12), 2341-2352; https://doi.org/10.3390/toxins5122341 - 28 Nov 2013

Cited by 47 | Viewed by 7072

Abstract

Fusariotoxins such as fumonisin B1 (FB1) and deoxynivalenol (DON) cause deleterious effects on the intestine of pigs. The aim of this study was to evaluate the effect of these mycotoxins, alone and in combination, on jejunal explants from piglets, using histological, immunohistochemical and [...] Read more.

Fusariotoxins such as fumonisin B1 (FB1) and deoxynivalenol (DON) cause deleterious effects on the intestine of pigs. The aim of this study was to evaluate the effect of these mycotoxins, alone and in combination, on jejunal explants from piglets, using histological, immunohistochemical and ultrastructural assays. Five 24-day old pigs were used for sampling the explants. Forty-eight explants were sampled from each animal. Explants were incubated for 4 hours in culture medium and medium containing FB1 (100 µM), DON (10 µM) and both mycotoxins (100 µM FB1 plus 10 µM DON). Exposure to all treatments induced a significant decrease in the normal intestinal morphology and in the number of goblet cells, which were more severe in explants exposed to DON and both mycotoxins. A significant reduction in villus height occurred in groups treated with DON and with co-contamination. Expression of E-cadherin was significantly reduced in explants exposed to FB1 (40%), DON (93%) and FB1 plus DON (100%). The ultrastructural assay showed increased intercellular spaces and no junction complexes on enterocytes exposed to mycotoxins. The present data indicate that FB1 and DON induce changes in cell junction complexes that could contribute to increase paracellular permeability. The ex vivo model was adequate for assessing intestinal toxicity induced by exposure of isolated or associated concentrations of 100 µM of FB1 and 10 µM of DON. Full article

(This article belongs to the Special Issue Recent Advances and Perspectives in Deoxynivalenol Research)

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Effects of individual and combined exposition of jejunal explants to fumonisin and deoxynivalenol on histology. Explants were exposed to culture medium ( <img alt="Toxins 05 02341 i001" src="/toxins/toxins-05-02341/article_deploy/html/images/toxins-05-02341-i001.png"/>) or culture medium with fumonisin B1 (FB1) ( <img alt="Toxins 05 02341 i002" src="/toxins/toxins-05-02341/article_deploy/html/images/toxins-05-02341-i002.png"/>), deoxynivalenol (DON) ( <img alt="Toxins 05 02341 i003" src="/toxins/toxins-05-02341/article_deploy/html/images/toxins-05-02341-i003.png"/>) or FB1 + DON (■). (A) Control explants. Edema of the lamina propria and mild villi atrophy (arrow); (B) FB1-exposed explant. Moderate fusion (arrow) and villous atrophy; (C) DON-exposed explant. Severe loss of apical enterocytes, fusion and atrophy (arrow); (D) FB1 + DON-exposed explant. Lysis of intestinal epithelium, villi atrophy, fusion (arrow) and cell debris. HE. Bar 100 µm; (E) Control explant showing a strong and homogeneous E-cadherin expression. Bar 20 µm; (F) DON-exposed explant showing reduced expression of E-cadherin. Bar 20 µm; (G) Tissue scores of pig intestinal explants exposed to FB1, DON and both mycotoxins; (H) Villi height in pig intestinal explants treated with FB1, DON and FB1 + DON; (I) Number of goblet cells per villus of pig intestinal explants treated with FB1, DON and FB1 + DON. Values are means with their standard deviation of the mean represented by vertical bars (n 5 animals). Mean values with unlike letters were significantly different (p ≤ 0.05). AU = Arbitrary Units. Full article ">Figure 2
Effects of individual and combined exposition of jejunal explants to fumonisin and deoxynivalenol on ultrastructure. (A) Control explant. Enterocytes with normal morphology of microvilli and cytoplasm. Bar 500 nm; (B) Control explant. Enterocytes with junction complexes (arrow) and glycogen granules scattered in the cytoplasm. Bar 2 nm; (C) FB1-exposed explant. Focal loss of apical enterocytes (arrow head) and loss of microvilli (arrow). Bar 2 nm; (D) DON-exposed explant. Increased intercellular space (arrow) and loss of junction complexes. Bar 5 nm; (E) DON-exposed explant. Vacuoles within cytoplasm, membrane blebs and loss of apical enterocytes. Bar 10 nm. Full article ">

327 KiB

Open AccessArticle

Impact of pH on the Stability and the Cross-Reactivity of Ochratoxin A and Citrinin

by Ingrid Bazin, Virginie Faucet-Marquis, Marie-Carmen Monje, Micheline El Khoury, Jean-Louis Marty and Annie Pfohl-Leszkowicz

Toxins 2013, 5(12), 2324-2340; https://doi.org/10.3390/toxins5122324 - 28 Nov 2013

Cited by 41 | Viewed by 8999

Abstract

Mycotoxins are secondary metabolites produced by several fungi contaminating crops. In several countries, the maximum permitted levels of mycotoxins are found in foodstuffs and feedstuffs. The common strategy of mycotoxin analysis involves extraction, clean-up and quantification by chromatography. In this paper, we analyzed [...] Read more.

Mycotoxins are secondary metabolites produced by several fungi contaminating crops. In several countries, the maximum permitted levels of mycotoxins are found in foodstuffs and feedstuffs. The common strategy of mycotoxin analysis involves extraction, clean-up and quantification by chromatography. In this paper, we analyzed the reasons of underestimation of ochratoxin A (OTA) content in wine, and overestimation of OTA in wheat, depending on the pH of the clean-up step and the simultaneous presence of citrinin (CIT). We demonstrated that the increase of pH by adding polyethylene glycol (PEG) to wine led to an underestimation of OTA by conversion of OTA into open ring ochratoxin A OP-OA. In comparing three methods of extraction and clean-up for the determination of OTA and CIT in wheat—(i) an inter-laboratory validated method for OTA in cereals using immunoaffinity column clean-up (IAC) and extraction by acetonitrile/water; (ii) a validated method using IAC and extraction with 1% bicarbonate Na; and (iii) an in-house validated method based on acid liquid/liquid extraction—we observed an overestimation of OTA after immunoaffinity clean-up when CIT is also present in the sample, whereas an underestimation was observed when OTA was alone. Under neutral and alkaline conditions, CIT was partially recognized by OTA antibodies. Full article

(This article belongs to the Special Issue Recent Advances in Ochratoxins Research)

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Figure 1
Chemical structure of ochratoxin A (OTA). Full article ">Figure 2
Chemical structure of citrinin, existing in both forms. Full article ">Figure 3
UV-Spectra of ochratoxin A: Curve A represents the spectrum of OTA at pH 4. Curve B represents the spectrum of OTA at pH 7. Curve C represents the spectrum of OTA at pH 8. Curve D represents the spectrum of OTA at pH 12. Full article ">Figure 4
UV-Spectra of citrinin and ochratoxin A in mixture: Curve A represents the spectrum of CIT at four pH (4, 7, 8 and 12). Curve B represents the spectrum of CIT in mixture with OTA at pH 4. Curve C represents the spectrum of CIT in mixture with OTA at pH 7. Curve D represents the spectrum of CIT in mixture with OTA at pH 8. Full article ">Figure 5
Ring opening of OTA (OP-OA) under alkaline conditions adapted from [<a href="#B50-toxins-05-02324" class="html-bibr">50</a>,<a href="#B51-toxins-05-02324" class="html-bibr">51</a>,<a href="#B52-toxins-05-02324" class="html-bibr">52</a>]. Full article ">Figure 6
Ring opening of citrinin (citrinin H2) induced by increase of pH [<a href="#B56-toxins-05-02324" class="html-bibr">56</a>]. Full article ">Figure 7
HPLC separation of OTA metabolites from the mixture of OTA and citrinin in aqueous solution. Full article ">

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Open AccessArticle

Influence of Fermentation and Drying Materials on the Contamination of Cocoa Beans by Ochratoxin A

by Sébastien Djédjé Dano, Pierre Manda, Ardjourma Dembélé, Ange Marie-Joseph Kouassi Abla, Joel Henri Bibaud, Julien Zroh Gouet and Charles Bruno Ze Maria Sika

Toxins 2013, 5(12), 2310-2323; https://doi.org/10.3390/toxins5122310 - 28 Nov 2013

Cited by 20 | Viewed by 8768

Abstract

Ochratoxin A (OTA) is a mycotoxin produced mainly by species of Aspergillus and Penicillium. Contamination of food with OTA is a major consumer health hazard. In Cote D’Ivoire, preventing OTA contamination has been the subject of extensive study. The current study was [...] Read more.

Ochratoxin A (OTA) is a mycotoxin produced mainly by species of Aspergillus and Penicillium. Contamination of food with OTA is a major consumer health hazard. In Cote D’Ivoire, preventing OTA contamination has been the subject of extensive study. The current study was conducted to evaluate the influence of fermentation and drying materials on the OTA content in cocoa. For each test, 7000 intact cocoa pods were collected, split open to remove the beans, fermented using 1 of 3 different materials, sun-dried on 1 of 3 different platform types and stored for 30 days. A total of 22 samples were collected at each stage of post-harvesting operations. The OTA content in the extracted samples was then quantified by high-performance liquid chromatography. OTA was detected in beans at all stages of post-harvesting operations at varying levels: pod-opening (0.025 ± 0.02 mg/kg), fermentation (0.275 ± 0.2 mg/kg), drying (0.569 ± 0.015 mg/kg), and storage (0.558 ± 0.04 mg/kg). No significant relationships between the detected OTA level and the materials used in the fermentation and drying of cocoa were observed. Full article

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Open AccessArticle

Exposure Assessment for Italian Population Groups to Deoxynivalenol Deriving from Pasta Consumption

by Carlo Brera, Valentina Bertazzoni, Francesca Debegnach, Emanuela Gregori, Elisabetta Prantera and Barbara De Santis

Toxins 2013, 5(12), 2293-2309; https://doi.org/10.3390/toxins5122293 - 26 Nov 2013

Cited by 19 | Viewed by 6222

Abstract

Four hundred and seventy-two pasta samples were collected from long retail distribution chain sales points located in North, Central and South Italy. Representative criteria in the sample collection were followed in terms of number of samples collected, market share, and types of pasta. [...] Read more.

Four hundred and seventy-two pasta samples were collected from long retail distribution chain sales points located in North, Central and South Italy. Representative criteria in the sample collection were followed in terms of number of samples collected, market share, and types of pasta. Samples were analysed by an accredited HPLC-UV method of analysis. The mean contamination level (64.8 ?g/kg) of deoxynivalenol (DON) was in the 95th percentile (239 ?g/kg) and 99th percentile (337 ?g/kg), far below the legal limit (750 ?g/kg) set by Regulation EC/1126/2007, accounting for about one tenth, one third and half the legal limit, respectively. Ninety-nine percent of samples fell below half the legal limit. On the basis of the obtained occurrence levels and considering the consumption rates reported by the Italian official database, no health concern was assessed for all consumer groups, being that exposure was far below the Tolerable Daily Intake (TDI) of 1000 ng/kg b.w/day. Nevertheless, despite this, particular attention should be devoted to the exposure to DON by high consumers, such as children aged 3–5 years, who could reach the TDI even with very low levels of DON contamination. Full article

(This article belongs to the Special Issue Recent Advances and Perspectives in Deoxynivalenol Research)

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Toxins, Volume 5, Issue 12 (December 2013) – 20 articles , Pages 2293-2685

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