Toxins

20 pages, 2054 KiB

Open AccessArticle

Changes in Growth, Photosynthesis Performance, Pigments, and Toxin Contents of Bloom-Forming Cyanobacteria after Exposure to Macroalgal Allelochemicals

by Gracjana Budzałek, Sylwia Śliwińska-Wilczewska, Marek Klin, Kinga Wiśniewska, Adam Latała and Józef Maria Wiktor

Toxins 2021, 13(8), 589; https://doi.org/10.3390/toxins13080589 - 23 Aug 2021

Cited by 3 | Viewed by 3640

Abstract

Macroalgae can directly restrict the growth of various phytoplankton species by releasing allelopathic compounds; therefore, considerable attention should be paid to the allelopathic potential of these organisms against harmful and bloom-forming cyanobacteria. The main aim of this study was to demonstrate for the [...] Read more.

Macroalgae can directly restrict the growth of various phytoplankton species by releasing allelopathic compounds; therefore, considerable attention should be paid to the allelopathic potential of these organisms against harmful and bloom-forming cyanobacteria. The main aim of this study was to demonstrate for the first time the allelopathic activity of Ulva intestinalis on the growth, the fluorescence parameters: the maximum PSII quantum efficiency (F_v/F_m) and the effective quantum yield of PSII photochemistry (ΦPSII), the chlorophyll a (Chl a) and carotenoid (Car) content, and the microcystin-LR (MC-LR) and phenol content of three bloom-forming cyanobacteria, Aphanizomenon sp., Nodularia spumigena, and Nostoc sp. We found both negative and positive allelopathic effects of U. intestinalis on tested cyanobacteria. The study clearly showed that the addition of the filtrate of U. intestinalis significantly inhibited growth, decreased pigment content and F_v/F_m and ΦPSII values of N. spumigena and Nostoc sp., and stimulated Aphanizomenon sp. The addition of different concentrations of aqueous extract also stimulated the cyanobacterial growth. It was also shown that the addition of extract obtained from U. intestinalis caused a significant decrease in the MC-LR content in Nostoc sp. cells. Moreover, it the phenol content in N. spumigena cells was increased. On the other hand, the cell-specific phenol content for Aphanizomenon sp. decreased due to the addition of the filtrate. In this work, we demonstrated that the allelopathic effect of U. intestinalis depends on the target species’ identity as well as the type of allelopathic method used. The study of the allelopathic Baltic macroalgae may help to identify their possible role as a significant biological factor influencing harmful cyanobacterial blooms in brackish ecosystems. Full article

(This article belongs to the Special Issue Cyanobacterial Toxins: Genotoxic and Cytotoxic Activity, Molecular Targets and Chemical Interactions)

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Number of cells (N = 105 cell mL−1) for Aphanizomenon sp. BA69 (A), N. spumigena BA15 (B), and Nostoc sp. BA81 (C) for controls and treatments with different concentrations of extract and cell-free filtrate additions obtained from macroalgae U. intestinalis after 7 (a) and 14 (b) days of the expositions (n = 3). Different letters indicate significant differences between the means of the treatments (p < 0.05, one-way ANOVA). Error bars display the standard deviation. Full article ">Figure 2
Chl a (pg cell−1; a) and Car (pg cell−1; b) content for Aphanizomenon sp. BA69 (A), N. spumigena BA15 (B), and Nostoc sp. BA81 (C) for controls and treatments with different concentrations of extract and cell-free filtrate additions obtained from macroalgae U. intestinalis after 7 days of the expositions (n = 3). Different letters indicate significant differences between the means of the treatments (p < 0.05, one-way ANOVA). Error bars display the standard deviation. Full article ">Figure 3
Fv/Fm (a) and ΦPSII (b) parameters for Aphanizomenon sp. BA69 (A), N. spumigena BA15 (B), and Nostoc sp. BA81 (C) for controls and treatments with different concentrations of extract and cell-free filtrate additions obtained from macroalgae U. intestinalis after 7 days of the expositions (n = 3). Different letters indicate significant differences between the means of the treatments (p < 0.05, one-way ANOVA). Error bars display the standard deviation. Full article ">Figure 4
Light microscope photographs of Aphanizomenon sp. BA69 (A), N. spumigena BA15 (B), and Nostoc sp. BA81 (C). ((a); scale = 10 μm). photographs of the cyanobacterial culture in 25-mL glass flasks from the ex-perimental phase (b); and PAR absorption spectra determined for this strain at an optical density (OD750) = 0.1 (c). Full article ">

18 pages, 5424 KiB

Open AccessArticle

Evaluation of Inner Exposure of Horses to Zearalenone (ZEN), Deoxynivalenol (DON) and Their Metabolites in Relation to Colic and Health-Related Clinical-Chemical Traits

by Sven Dänicke, Janine Saltzmann, Wendy Liermann, Maren Glatter, Liane Hüther, Susanne Kersten, Annette Zeyner, Karsten Feige and Tobias Warnken

Toxins 2021, 13(8), 588; https://doi.org/10.3390/toxins13080588 - 23 Aug 2021

Cited by 2 | Viewed by 3280

Abstract

Mycotoxin contaminated feed has been associated with colic of horses caused by intestinal disorders. Whether such disease conditions alter the intestinal toxin metabolism and transfer across a compromised mucosal barrier is unknown. A screening approach was used to relate blood residue levels of [...] Read more.

Mycotoxin contaminated feed has been associated with colic of horses caused by intestinal disorders. Whether such disease conditions alter the intestinal toxin metabolism and transfer across a compromised mucosal barrier is unknown. A screening approach was used to relate blood residue levels of DON, ZEN and their metabolites to the status of the horses (sick vs. healthy). A total of 55 clinically healthy horses from 6 different farms with varying feeding background served as control for sick horses (N = 102) hospitalized due to colic. ZEN, alpha-zearalenol (ZEL), beta-ZEL and DON were detectable in peripheral blood as indicators for the inner exposure with significant farm effects for alpha- and beta-ZEL. However, the levels in sick horses were similar to all farms. Moreover, the proportion of beta-ZEL of all detected ZEN metabolites as an indicator for the degree of metabolism of ZEN was not different for sick horses but differed amongst the control farms. Although the incidence of DON in blood was generally low and not significantly different amongst healthy and sick horses, the positive samples were nearly exclusively found in sick horses suggesting either a higher toxin transfer, an association of DON with the development of colic or a different feeding background. Full article

(This article belongs to the Special Issue Biomonitoring of Mycotoxins)

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14 pages, 3124 KiB

Open AccessEditor’s ChoiceArticle

In Vitro and In Vivo Analysis of Ochratoxin A-Derived Glucuronides and Mercapturic Acids as Biomarkers of Exposure

by Raphael Dekant, Michael Langer, Maria Lupp, Cynthia Adaku Chilaka and Angela Mally

Toxins 2021, 13(8), 587; https://doi.org/10.3390/toxins13080587 - 23 Aug 2021

Cited by 9 | Viewed by 3668

Abstract

Ochratoxin A (OTA) is a widespread food contaminant, with exposure estimated to range from 0.64 to 17.79 ng/kg body weight (bw) for average consumers and from 2.40 to 51.69 ng/kg bw per day for high consumers. Current exposure estimates are, however, associated with [...] Read more.

Ochratoxin A (OTA) is a widespread food contaminant, with exposure estimated to range from 0.64 to 17.79 ng/kg body weight (bw) for average consumers and from 2.40 to 51.69 ng/kg bw per day for high consumers. Current exposure estimates are, however, associated with considerable uncertainty. While biomarker-based approaches may contribute to improved exposure assessment, there is yet insufficient data on urinary metabolites of OTA and their relation to external dose to allow reliable estimates of daily intake. This study was designed to assess potential species differences in phase II biotransformation in vitro and to establish a correlation between urinary OTA-derived glucuronides and mercapturic acids and external exposure in rats in vivo. In vitro analyses of OTA metabolism using the liver S9 of rats, humans, rabbits and minipigs confirmed formation of an OTA glucuronide but provided no evidence for the formation of OTA-derived mercapturic acids to support their use as biomarkers. Similarly, OTA-derived mercapturic acids were not detected in urine of rats repeatedly dosed with OTA, while indirect analysis using enzymatic hydrolysis of the urine samples prior to LC–MS/MS established a linear relationship between urinary glucuronide excretion and OTA exposure. These results support OTA-derived glucuronides but not mercapturic acids as metabolites suitable for biomonitoring. Full article

(This article belongs to the Special Issue Toxicological Effects of Mycotoxins)

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Structures of Ochratoxin A (OTA)-derived glucuronides proposed by [<a href="#B10-toxins-13-00587" class="html-bibr">10</a>] (acyl-glucuronide (1), phenol-glucuronide (2) and amino-glucuronide (3)) and structure of OTB-NAC (4) reported to be present in human urine. Full article ">Figure 2
Representative LC–MS chromatograms of urine obtained from rats treated with OTA at 2 mg/kg bw for 2 weeks (a) and 210 µg/kg bw for 13 weeks (b) obtained by direct (a) and indirect analysis with and without β-glucuronidase treatment (b). Multiple reaction monitoring of m/z-transitions 578/402 and 578/358 demonstrating formation of an OTA-glucuronide in in vitro incubations of OTA with liver S9 from rats, rabbits, minipigs and humans (c). Enhanced product ion spectrum [M-H]− showing typical mass fragments of m/z 402, 358, 193, 175 and 113, consistent with a previously reported OTA-acyl-glucuronide [<a href="#B10-toxins-13-00587" class="html-bibr">10</a>] (d). Full article ">Figure 3
Urinary excretion of OTA-glucuronides within 24 h in rats treated with OTA at 0, 21, 70 or 210 μg/kg per bw (5 days/week) for 2, 4 and 13 weeks. Data are presented as mean ± standard deviation (n = 3–5 animals per group). The Pearson correlation coefficients (R2) and linear equations for the linear trend lines are displayed. Full article ">Figure 4
(a) LC–MS analysis of the photoreaction of OTA in the presence of NAC, yielding OTB-NAC and OTHQ-NAC. EPI spectra of the NAC conjugates OTHQ-NAC (b) and OTB-NAC (c). Full article ">Figure 5
(a) LC–MS analysis of urine of a rat treated with OTA at 210 µg/kg bw for 90 days, showing the total ion chromatogram (TIC) and extracted ion chromatograms of m/z transitions corresponding to potential OTA-derived metabolites. Besides OTA (RT 18.15), the TIC reveals the presence of the two previously identified glycosides, hydroxy-OTA, traces of OP-OTA and a further signal corresponding to one of the mass transitions of OTB-NAC (m/z 529 → 400). (b) LC–MS analysis of a spiking experiment in which rat urine was spiked with a solution of OTB-NAC generated by photosynthesis, demonstrating that the unknown compound (m/z 529 → 400) present in rat urine independent of OTA treatment is not identical to OTB-NAC. Full article ">Figure 6
LC–MS analysis of potential OTA metabolites formed in in vitro incubations of OTA with S9 mix of rat, rabbit, minipig and human in the presence of N-acetyl-cysteine (a) or GSH (b). Full article ">

30 pages, 1380 KiB

Open AccessReview

Mycotoxin Biomarkers in Pigs—Current State of Knowledge and Analytics

by Agnieszka Tkaczyk and Piotr Jedziniak

Toxins 2021, 13(8), 586; https://doi.org/10.3390/toxins13080586 - 23 Aug 2021

Cited by 20 | Viewed by 7496

Abstract

Farm animals are frequently exposed to mycotoxins, which have many adverse effects on their health and become a significant food safety issue. Pigs are highly exposed and particularly susceptible to mycotoxins, which can cause many adverse effects. For the above reasons, an appropriate [...] Read more.

Farm animals are frequently exposed to mycotoxins, which have many adverse effects on their health and become a significant food safety issue. Pigs are highly exposed and particularly susceptible to mycotoxins, which can cause many adverse effects. For the above reasons, an appropriate diagnostic tool is needed to monitor pig’ exposure to mycotoxins. The most popular tool is feed analysis, which has some disadvantages, e.g., it does not include individual exposure. In recent years, the determination of biomarkers as a method to assess the exposure to mycotoxins by using concentrations of the parent compounds and/or metabolites in biological matrices is becoming more and more popular. This review provides a comprehensive overview of reported in vivo mycotoxin absorption, distribution, metabolism and excretion (ADME) and toxicokinetic studies on pigs. Biomarkers of exposure for aflatoxins, deoxynivalenol, ochratoxin A, fumonisins, T-2 toxin and zearalenone are described to select the most promising compound for analysis of porcine plasma, urine and faeces. Biomarkers occur in biological matrices at trace levels, so a very sensitive technique—tandem mass spectrometry—is commonly used for multiple biomarkers quantification. However, the sample preparation for multi-mycotoxin methods remains a challenge. Therefore, a summary of different biological samples preparation strategies is included in that paper. Full article

(This article belongs to the Special Issue Reduction and Control of Mycotoxins along Entire Food and Feed Chain)

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16 pages, 2193 KiB

Open AccessEditor’s ChoiceArticle

Human-Relevant Sensitivity of iPSC-Derived Human Motor Neurons to BoNT/A1 and B1

by Maren Schenke, Hélène-Christine Prause, Wiebke Bergforth, Adina Przykopanski, Andreas Rummel, Frank Klawonn and Bettina Seeger

Toxins 2021, 13(8), 585; https://doi.org/10.3390/toxins13080585 - 22 Aug 2021

Cited by 5 | Viewed by 4473

Abstract

The application of botulinum neurotoxins (BoNTs) for medical treatments necessitates a potency quantification of these lethal bacterial toxins, resulting in the use of a large number of test animals. Available alternative methods are limited in their relevance, as they are based on rodent [...] Read more.

The application of botulinum neurotoxins (BoNTs) for medical treatments necessitates a potency quantification of these lethal bacterial toxins, resulting in the use of a large number of test animals. Available alternative methods are limited in their relevance, as they are based on rodent cells or neuroblastoma cell lines or applicable for single toxin serotypes only. Here, human motor neurons (MNs), which are the physiological target of BoNTs, were generated from induced pluripotent stem cells (iPSCs) and compared to the neuroblastoma cell line SiMa, which is often used in cell-based assays for BoNT potency determination. In comparison with the mouse bioassay, human MNs exhibit a superior sensitivity to the BoNT serotypes A1 and B1 at levels that are reflective of human sensitivity. SiMa cells were able to detect BoNT/A1, but with much lower sensitivity than human MNs and appear unsuitable to detect any BoNT/B1 activity. The MNs used for these experiments were generated according to three differentiation protocols, which resulted in distinct sensitivity levels. Molecular parameters such as receptor protein concentration and electrical activity of the MNs were analyzed, but are not predictive for BoNT sensitivity. These results show that human MNs from several sources should be considered in BoNT testing and that human MNs are a physiologically relevant model, which could be used to optimize current BoNT potency testing. Full article

(This article belongs to the Section Bacterial Toxins)

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9 pages, 258 KiB

Open AccessCommunication

Botulinum Toxin Services for Neurorehabiliation: Recommendations for Challenges and Opportunities during the COVID-19 Pandemic

by Ganesh Bavikatte, Jorge Jacinto, Thierry Deltombe and Joerg Wissel

Toxins 2021, 13(8), 584; https://doi.org/10.3390/toxins13080584 - 22 Aug 2021

Cited by 3 | Viewed by 3431

Abstract

The COVID-19 pandemic severely impacted the function of medical facilities and rehabilitation services worldwide, including toxin services delivering Botulinum toxin treatments for neuromuscular conditions such as spasticity, dystonia, and sialorrhea. The aim of this paper is to understand how toxin services have dealt [...] Read more.

The COVID-19 pandemic severely impacted the function of medical facilities and rehabilitation services worldwide, including toxin services delivering Botulinum toxin treatments for neuromuscular conditions such as spasticity, dystonia, and sialorrhea. The aim of this paper is to understand how toxin services have dealt with the situation and what strategies have been adopted to continue services. The recommendations are based on a virtual round table held with toxin services experts from different European countries who shared their experiences and discussed the best practices. The challenges for toxin services were reviewed based on the experts’ experiences and on relevant literature from 2020 and 2021. A set of recommendations and best practices were compiled, focusing firstly on guidance for clinical practice, including assessing patients’ health and risk status and the urgency of their treatment. Secondly, it was discussed how patients on botulinum toxin therapy can be cared for and supported during the pandemic, and how modern technology and tele-medicine platforms can be generally used to optimize effectiveness and safety of toxin treatments. The technological advances prompted by the COVID-19 crisis can result in better and more modern patient care in the future. Full article

(This article belongs to the Section Bacterial Toxins)

15 pages, 2898 KiB

Open AccessEditor’s ChoiceArticle

Metaproteomics Reveals Alteration of the Gut Microbiome in Weaned Piglets Due to the Ingestion of the Mycotoxins Deoxynivalenol and Zearalenone

by Johan S. Saenz, Alina Kurz, Ursula Ruczizka, Moritz Bünger, Maximiliane Dippel, Veronika Nagl, Bertrand Grenier, Andrea Ladinig, Jana Seifert and Evelyne Selberherr

Toxins 2021, 13(8), 583; https://doi.org/10.3390/toxins13080583 - 21 Aug 2021

Cited by 12 | Viewed by 4176

Abstract

The ingestion of mycotoxins can cause adverse health effects and represents a severe health risk to humans and livestock. Even though several acute and chronic effects have been described, the effect on the gut metaproteome is scarcely known. For that reason, we used [...] Read more.

The ingestion of mycotoxins can cause adverse health effects and represents a severe health risk to humans and livestock. Even though several acute and chronic effects have been described, the effect on the gut metaproteome is scarcely known. For that reason, we used metaproteomics to evaluate the effect of the mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) on the gut microbiome of 15 weaned piglets. Animals were fed for 28 days with feed contaminated with different concentrations of DON (DONlow: 870 μg DON/kg feed, DONhigh: 2493 μg DON/kg feed) or ZEN (ZENlow: 679 μg ZEN/kg feed, ZENhigh: 1623 μg ZEN/kg feed). Animals in the control group received uncontaminated feed. The gut metaproteome composition in the high toxin groups shifted compared to the control and low mycotoxin groups, and it was also more similar among high toxin groups. These changes were accompanied by the increase in peptides belonging to Actinobacteria and a decrease in peptides belonging to Firmicutes. Additionally, DONhigh and ZENhigh increased the abundance of proteins associated with the ribosomes and pentose-phosphate pathways, while decreasing glycolysis and other carbohydrate metabolism pathways. Moreover, DONhigh and ZENhigh increased the abundance of the antioxidant enzyme thioredoxin-dependent peroxiredoxin. In summary, the ingestion of DON and ZEN altered the abundance of different proteins associated with microbial metabolism, genetic processing, and oxidative stress response, triggering a disruption in the gut microbiome structure. Full article

(This article belongs to the Special Issue The Mutual Interaction between Mycotoxins and Gut Microbiome)

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Timeline of the animal experiment. After arrival at the Vetmeduni Vienna on day 7, all weaned piglets started an 8-day acclimation phase, in which the feed administered to the control group was fed to all piglets. Mycotoxin exposition started on day 1, and weaned piglets were fed with mycotoxin-contaminated feed according to their groups. Control weaned piglets received control feed without mycotoxins during this period. Weaned piglets feed 1 and weaned piglets feed 2 were used as base diets for all groups. Body weight (BW) was measured weekly. Full article ">Figure 2
The gut metaproteomic composition is altered by DONhigh and ZENhigh. (A) Unsupervised dimensionality reduction analysis for the digesta samples (Perplexity = 10, maximum iteration = 1200). Clustering by treatment (p = 0.001, PERMANOVA permutations = 999) and intestinal section (p = 0.076, permutations = 999). (B) Bray–Curtis dissimilarity (Control vs. Treatment) of peptides identified at the family level. * p < 0.05 Kruskal–Wallis and Wilcoxon rank sum test, p values were corrected by Benjamini–Hochberg for multiple comparisons. Full article ">Figure 3
DONhigh and ZENhigh increased the relative abundance of Actinobacteria and decreased the abundance of Firmicutes. (A) Relative abundances of the most abundant bacterial families across all treatments. * Significant difference between the treatment and control group. 95% confidence interval. (B) Ratio between the relative abundance of the phyla Actinobacteria and Firmicutes. p < * 0.05, ** 0.005, Kruskall-Wallis and Wilcoxon rank sum test, p values were corrected by Benjamini-Hochberg for multiple comparisons. Full article ">Figure 4
Bacterial protein profiles were more similar between DONhigh and ZENhigh. (A) Clustering of the 190 significantly different proteins group across the treatments. p < 0.05, Log2 fold change > 2, and >1 peptides identified. (B) The number of proteins identified by COG categories for each cluster of protein groups. (J) Translation, ribosomal structure and biogenesis, (G) carbohydrate transport and metabolism, (M) cell wall/membrane/envelope biogenesis, (S) function unknown, (E) amino acid transport and metabolism, (C) energy production and conversion, (F) nucleotide transport and metabolism, (H) coenzyme transport and metabolism, (O) post-translational modification, protein turnover, and chaperones, (K) transcription, (D) cell cycle control, cell division, chromosome partitioning, (P) inorganic ion transport and metabolism, (L) replication, recombination and repair, (I) lipid transport and metabolism. Full article ">Figure 5
DONhigh significantly increased the abundance of bacterial proteins associated with genetic processing. Log2 fold change between each treatment and the control group. Only significantly different proteins are shown. * Differentially abundant proteins between DONhigh and control group (p < 0.05). Full article ">Figure 6
Mycotoxins reduced the abundance of key enzymes of the upper glycolysis and increased proteins of the pentose phosphate pathway. Colored rectangles represent the Log2 fold change between the treatments and the control group for each identified enzyme. KEGG orthologs (KO) identifiers are shown over each square. Full article ">

21 pages, 25493 KiB

Open AccessArticle

Biological and Biochemical Characterization of Coronado Island Rattlesnake (Crotalus helleri caliginis) Venom and Antivenom Neutralization

by Cristian Franco-Servín, Edgar Neri-Castro, Melisa Bénard-Valle, Alejandro Alagón, Ramsés Alejandro Rosales-García, Raquel Guerrero-Alba, José Emanuel Poblano-Sánchez, Marcelo Silva-Briano, Alma Lilián Guerrero-Barrera and José Jesús Sigala-Rodríguez

Toxins 2021, 13(8), 582; https://doi.org/10.3390/toxins13080582 - 21 Aug 2021

Cited by 5 | Viewed by 4771

Abstract

The Baja California Peninsula has over 250 islands and islets with many endemic species. Among them, rattlesnakes are the most numerous but also one of the least studied groups. The study of island rattlesnake venom could guide us to a better understanding of [...] Read more.

The Baja California Peninsula has over 250 islands and islets with many endemic species. Among them, rattlesnakes are the most numerous but also one of the least studied groups. The study of island rattlesnake venom could guide us to a better understanding of evolutionary processes and the description of novel toxins. Crotalus helleri caliginis venom samples were analyzed to determine possible ontogenetic variation with SDS-PAGE in one and two dimensions and with RP-HPLC. Western Blot, ELISA, and amino-terminal sequencing were used to determine the main components of the venom. The biological and biochemical activities demonstrate the similarity of C. helleri caliginis venom to the continental species C. helleri helleri, with both having low proteolytic and phospholipase A₂ (PLA₂) activity but differing due to the absence of neurotoxin (crotoxin-like) in the insular species. The main components of the snake venom were metalloproteases, serine proteases, and crotamine, which was the most abundant toxin group (30–35% of full venom). The crotamine was isolated using size-exclusion chromatography where its functional effects were tested on mouse phrenic nerve–hemidiaphragm preparations in which a significant reduction in muscle twitch contractions were observed. The two Mexican antivenoms could neutralize the lethality of C. helleri caliginis venom but not the crotamine effects. Full article

(This article belongs to the Section Animal Venoms)

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13 pages, 1325 KiB

Open AccessEditor’s ChoiceArticle

Cytotoxicity of Mycotoxins Frequently Present in Aquafeeds to the Fish Cell Line RTGill-W1

by Elena Bernal-Algaba, Marta Pulgarín-Alfaro and María Luisa Fernández-Cruz

Toxins 2021, 13(8), 581; https://doi.org/10.3390/toxins13080581 - 20 Aug 2021

Cited by 13 | Viewed by 3785

Abstract

In the last decades, the aquaculture industry has introduced plant-based ingredients as a source of protein in aquafeeds. This has led to mycotoxin contaminations, representing an ecological, health and economic problem. The aim of this study was to determine in the RTgill-W1 fish [...] Read more.

In the last decades, the aquaculture industry has introduced plant-based ingredients as a source of protein in aquafeeds. This has led to mycotoxin contaminations, representing an ecological, health and economic problem. The aim of this study was to determine in the RTgill-W1 fish cell line the toxicity of fifteen mycotoxins of common occurrence in aquafeeds. To identify the most sensitive endpoint of toxicity, the triple assay was used. It consisted of three assays: alamarBlue, Neutral Red Uptake and CFDA-AM, which revealed the mitochondrial activity, the lysosomal integrity and the plasma membrane integrity, respectively. Most of the assayed mycotoxins were toxic predominantly at lysosomal level (enniatins, beauvericin, zearalenone, ochratoxin A, deoxynivalenol (DON) and its acetylated metabolites 15-O-acetyl-DON and 3-acetyl-DON). Aflatoxins B1 and B2 exerted the greatest effects at mitochondrial level, while fumonisins B1 and B2 and nivalenol were not toxic up to 100 µg/mL. In general, low toxicity was observed at plasma membrane level. The vast majority of the mycotoxins assayed exerted a pronounced acute effect in the fish RTgill-W1 cell line, emphasizing the need for further studies to ascertain the impact of mycotoxin contamination of fish feeds in the aquaculture industry and to establish safe limits in aquafeeds. Full article

(This article belongs to the Special Issue Selected Papers from the V Workshop of the Spanish National Network on Mycotoxins and Toxigenic Fungi and Their Decontamination Processes (MICOFOOD), 10–11 December 2020)

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12 pages, 1415 KiB

Open AccessArticle

An Update on Ciguatoxins and CTX-like Toxicity in Fish from Different Trophic Levels of the Selvagens Islands (NE Atlantic, Madeira, Portugal)

by Pedro Reis Costa, Pablo Estévez, Lucía Soliño, David Castro, Susana Margarida Rodrigues, Viriato Timoteo, José Manuel Leao-Martins, Carolina Santos, Neide Gouveia, Jorge Diogène and Ana Gago-Martínez

Toxins 2021, 13(8), 580; https://doi.org/10.3390/toxins13080580 - 20 Aug 2021

Cited by 27 | Viewed by 4255

Abstract

The Selvagens Islands, which are a marine protected area located at the southernmost point of the Portuguese maritime zone, have been associated with fish harboring ciguatoxins (CTX) and linked to ciguatera fish poisonings. This study reports the results of a field sampling campaign [...] Read more.

The Selvagens Islands, which are a marine protected area located at the southernmost point of the Portuguese maritime zone, have been associated with fish harboring ciguatoxins (CTX) and linked to ciguatera fish poisonings. This study reports the results of a field sampling campaign carried out in September 2018 in these remote and rarely surveyed islands. Fifty-six fish specimens from different trophic levels were caught for CTX-like toxicity determination by cell-based assay (CBA) and toxin content analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Notably, high toxicity levels were found in fish with an intermediate position in the food web, such as zebra seabream (Diplodus cervinus) and barred hogfish (Bodianus scrofa), reaching levels up to 0.75 µg CTX1B equivalent kg⁻¹. The LC-MS/MS analysis confirmed that C-CTX1 was the main toxin, but discrepancies between CBA and LC-MS/MS in D. cervinus and top predator species, such as the yellowmouth barracuda (Sphyraena viridis) and amberjacks (Seriola spp.), suggest the presence of fish metabolic products, which need to be further elucidated. This study confirms that fish from coastal food webs of the Selvagens Islands represent a high risk of ciguatera, raising important issues for fisheries and environmental management of the Selvagens Islands. Full article

(This article belongs to the Special Issue Ciguatoxins)

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Location of the Selvagens Islands, NE Atlantic, Madeira, Portugal. Full article ">Figure 2
CTX-like toxicity (µg CTX1B equivalent kg−1) determined by CBA in fish species collected from the marine food web of the Selvagens Islands during 5–7 September 2018 (median, 25 and 75 quartiles, minimum and maximum, total n = 55). See Materials and Methods section for details on samples. Full article ">Figure 3
LC-MS/MS chromatogram in MRM mode of: (A) Laboratory reference material containing C-CTX1 (8.79 min) and C-CTX1-Me (10.84 min), (A.1) Zoom of C-CTX1 less intense ion transitions: m/z 1123.6 [M+H-H2O]+ -> m/z 1069.6 [M+H-3H2O]+ (purple line), m/z 1123.6 [M+H-H2O]+ -> m/z 191.1 (blue line), m/z 1123.6 [M+H-H2O]+ -> m/z 108.9 (green line), (A.2) Zoom of C-CTX1-Me less intense ion transitions: m/z 1123.6 [M+H-CH3-H2O]+ -> m/z 1069.6 [M+H-CH3-3H2O]+ (purple line), m/z 1123.6 [M+H-CH3-H2O]+ -> m/z 191.1 (blue line), m/z 1123.6 [M+H-CH3-H2O]+ -> m/z 108.9 (green line); (B) C-CTX1 (8.79 min) and an unknown compound (4.94 min) detected in a Bodianus scrofa; (C) C-CTX-1157 (7.64 min) detected in the Diplodus cervinus. Full article ">

16 pages, 3346 KiB

Open AccessArticle

Origin and Characterization of Extracellular Vesicles Present in the Spider Venom of Ornithoctonus hainana

by Chengfeng Xun, Lu Wang, Hailin Yang, Zixuan Xiao, Min Deng, Rongfang Xu, Xi Zhou, Ping Chen and Zhonghua Liu

Toxins 2021, 13(8), 579; https://doi.org/10.3390/toxins13080579 - 20 Aug 2021

Cited by 6 | Viewed by 4702

Abstract

Extracellular vesicles (EVs), including exosomes and microvesicles, are membranous vesicles released from nearly all cellular types. They contain various bioactive molecules, and their molecular composition varies depending on their cellular origin. As research into venomous animals has progressed, EVs have been discovered in [...] Read more.

Extracellular vesicles (EVs), including exosomes and microvesicles, are membranous vesicles released from nearly all cellular types. They contain various bioactive molecules, and their molecular composition varies depending on their cellular origin. As research into venomous animals has progressed, EVs have been discovered in the venom of snakes and parasitic wasps. Although vesicle secretion in spider venom glands has been observed, these secretory vesicles’ origin and biological properties are unknown. In this study, the origin of the EVs from Ornithoctonus hainana venom was observed using transmission electron microscopy (TEM). The Ornithoctonus hainana venom extracellular vesicles (HN-EVs) were isolated and purified by density gradient centrifugation. HN-EVs possess classic membranous vesicles with a size distribution ranging from 50 to 150 nm and express the arthropod EV marker Tsp29Fb. The LC-MS/MS analysis identified a total of 150 proteins, which were divided into three groups according to their potential function: conservative vesicle transport-related proteins, virulence-related proteins, and other proteins of unknown function. Functionally, HN-EVs have hyaluronidase activity and inhibit the proliferation of human umbilical vein endothelial cells (HUVECs) by affecting the cytoskeleton and cell cycle. Overall, this study investigates the biological characteristics of HN-EVs for the first time and sheds new light on the envenomation process of spider venom. Full article

(This article belongs to the Section Animal Venoms)

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Ultrastructure of glandular epithelium cells from Ornithoctonus hainana venom gland. The adult Ornithoctonus hainana is about 15 cm long (leg diameter), with a 2 cm venom gland. Transmission electron microscopy was used to observe a cross-sectional view of the gland and the secretion of EVs by glandular epithelium cells. Secretory epithelium (Se), lumen (Lu), extracellular vesicles (EVs), multi-vesicular bodies (MVBs). Scale bar: 100 nm. Full article ">Figure 2
Isolation and characterization of secreted EVs from the venom of Ornithoctonus hainana. (A) Schematic of the HN-EVs isolation protocol from spider venom. Asterisk mark near the middle of the centrifuge tube with a Nycodenz gradient indicates level of the light-scattering band. (B) TEM analysis showed that the purified HN-EVs have a classic exosomal cup-shaped and lipid bilayer membrane morphology. Scale bar: 100 nm. (C) Statistical analysis of the particle size distribution of HN-EVs in TEM images shows that the particle size of HN-EVs is mainly distributed between 50 and 150 nm (61.7%) (n = 102). (D) Western blotting detected the expression of arthropod EV marker Tsp29Fb in HN-EVs. Ornithoctonus hainana venom extracellular vesicles (HN-EVs), venom centrifugal supernatant (HN-Sup). Full article ">Figure 3
Analysis and identification of HN-EV proteins. (A) SDS-PAGE analysis of HN-EVs, venom, and HN-Sup. (B) Tricine-PAGE analysis of HN-EVs, venom, and HN-Sup. (C) Evaluation of molecular weight distribution of 150 proteins identified by LC-MS/MS. (D) The cellular component analysis showed that HN-EV proteins were found mainly in the cytoplasm, extracellular, nucleus, and mitochondria. Full article ">Figure 4
Functional classification of HN-EV proteins. After a database search, 150 proteins were identified with high confidence. Select HN-EV proteins, many of which are expected to be vesicular transport-associated and virulence-associated proteins, are displayed by their proteomic classes (1–3; bulleted within descriptive subclasses). Full article ">Figure 5
Hyaluronidase activity of HN-EVs. Hyaluronic acid (HA) was incubated with HN-EVs, HN-Sup, venom, and hyaluronidase at 40 °C overnight and separated on a 1% agarose gel at 50 mA for 3 h. Degradation was revealed by sequential toluidine blue and Stains-All staining. Full article ">Figure 6
HN-EVs inhibit the viability of HUVECs. (A) Fluorescence microscopy analysis of PKH67-labeled HN-EV internalization by HUVECs. The green-labeled EVs were visible in the perinuclear region of recipient cells, scale bar: 50 μm. (B) HUVECs were treated with several concentrations of HN-EVs, HN-Sup, and whole venom, and cell viability was assessed by MTT assay. The half-inhibitory concentration (IC50) was determined to be 575 μg/mL (HN-EVs) and 100 μg/mL (whole venom). Results are shown as mean ± SD (n = 3). (C,D) Crystal violet staining was used to determine cell viability and count the number of cells in each treatment group. Results are shown as mean ± SD (n = 3). *** p < 0.001. Full article ">Figure 7
The influence of HN-EVs on the cell morphology and cell cycle of HUVECs. (A) Fluorescein-phalloidin staining was used to observe cell morphology and changes in actin filament organization (green) and nucleus (blue) of HUVECs. Scale bar: 50 μm. (B,C) The cell cycle distribution was detected using flow cytometric analysis and was quantified. Results are shown as mean ± SD (n = 3). No significance (ns). * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">

11 pages, 1428 KiB

Open AccessArticle

Inhibition of Diarrheal Shellfish Toxins Accumulation in the Mussel Perna viridis by Curcumin and Underlying Mechanisms

by Kuan-Kuan Yuan, Guo-Fang Duan, Qing-Yuan Liu, Hong-Ye Li and Wei-Dong Yang

Toxins 2021, 13(8), 578; https://doi.org/10.3390/toxins13080578 - 20 Aug 2021

Cited by 7 | Viewed by 2914

Abstract

Diarrheal shellfish toxins (DSTs) are among the most widely distributed phytotoxins, and are associated with diarrheal shellfish poisoning (DSP) events in human beings all over the world. Therefore, it is urgent and necessary to identify an effective method for toxin removal in bivalves. [...] Read more.

Diarrheal shellfish toxins (DSTs) are among the most widely distributed phytotoxins, and are associated with diarrheal shellfish poisoning (DSP) events in human beings all over the world. Therefore, it is urgent and necessary to identify an effective method for toxin removal in bivalves. In this paper, we found that curcumin (CUR), a phytopolylphenol pigment, can inhibit the accumulation of DSTs (okadaic acid-eq) in the digestive gland of Perna viridis after Prorocentrum lima exposure. qPCR results demonstrated that CUR inhibited the induction of DSTs on the aryl hydrocarbon receptor (AhR), hormone receptor 96 (HR96) and CYP3A4 mRNA, indicating that the CUR-induced reduction in DSTs may be correlated with the inhibition of transcriptional induction of AhR, HR96 and CYP3A4. The histological examination showed that P. lima cells caused severe damage to the digestive gland of P. viridis, and the addition of curcumin effectively alleviated the damage induced by P. lima. In conclusion, our findings provide a potential method for the effective removal of toxins from DST-contaminated shellfish. Full article

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

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19 pages, 2985 KiB

Open AccessArticle

Interactions between Filter-Feeding Bivalves and Toxic Diatoms: Influence on the Feeding Behavior of Crassostrea gigas and Pecten maximus and on Toxin Production by Pseudo-nitzschia

by Aurore Sauvey, Françoise Denis, Hélène Hégaret, Bertrand Le Roy, Christophe Lelong, Orianne Jolly, Marie Pavie and Juliette Fauchot

Toxins 2021, 13(8), 577; https://doi.org/10.3390/toxins13080577 - 19 Aug 2021

Cited by 6 | Viewed by 3713

Abstract

Among Pseudo-nitzschia species, some produce the neurotoxin domoic acid (DA), a source of serious health problems for marine organisms. Filter-feeding organisms—e.g., bivalves feeding on toxigenic Pseudo-nitzschia spp.—are the main vector of DA in humans. However, little is known about the interactions between bivalves [...] Read more.

Among Pseudo-nitzschia species, some produce the neurotoxin domoic acid (DA), a source of serious health problems for marine organisms. Filter-feeding organisms—e.g., bivalves feeding on toxigenic Pseudo-nitzschia spp.—are the main vector of DA in humans. However, little is known about the interactions between bivalves and Pseudo-nitzschia. In this study, we examined the interactions between two juvenile bivalve species—oyster (Crassostrea gigas) and scallop (Pecten maximus)—and two toxic Pseudo-nitzschia species—P. australis and P. fraudulenta. We characterized the influence of (1) diet composition and the Pseudo-nitzschia DA content on the feeding rates of oysters and scallops, and (2) the presence of bivalves on Pseudo-nitzschia toxin production. Both bivalve species fed on P. australis and P. fraudulenta. However, they preferentially filtered the non-toxic Isochrysis galbana compared to Pseudo-nitzschia. The presence of the most toxic P. australis species resulted in a decreased clearance rate in C. gigas. The two bivalve species accumulated DA in their tissues (up to 0.35 × 10⁻³ and 5.1 × 10⁻³ µg g⁻¹ for C. gigas and P. maximus, respectively). Most importantly, the presence of bivalves induced an increase in the cellular DA contents of both Pseudo-nitzschia species (up to 58-fold in P. fraudulenta in the presence of C. gigas). This is the first evidence of DA production by Pseudo-nitzschia species stimulated in the presence of filter-feeding bivalves. The results of this study highlight complex interactions that can influence toxin production by Pseudo-nitzschia and accumulation in bivalves. These results will help to better understand the biotic factors that drive DA production by Pseudo-nitzschia and bivalve contamination during Pseudo-nitzschia blooms. Full article

(This article belongs to the Special Issue Phycotoxins: From Producers to Transfer in the Food Chain)

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Cell concentrations (A,E), clearance rates (CRs: B,F), and filtration rates (FRs: C,D,G,H) over the 5 days of exposure of C. gigas to P. fraudulenta and I. galbana—condition 1 (A–D)—or P. australis and I. galbana—condition 2 (E–H). Full article ">Figure 2
Cell concentrations (A), clearance rates (CRs: B,C), and filtration rates (FRs: B,C) over the 5 days of exposure of C. gigas to P. fraudulenta—condition 3 (A,B)—or P. australis—condition 4 (A,C). Full article ">Figure 3
Cell concentrations (A,E), clearance rates (CRs: B,F), and filtration rates (FRs: C,D,G,H) throughout the 6 h of exposure of P. maximus to P. fraudulenta and I. galbana—condition 1 (A–D)—or P. australis and I. galbana—condition 2 (E–H). Full article ">Figure 4
DA concentrations in C. gigas (A) and P. maximus (B) at the end of the exposure experiments. An asterisk (*) indicates a significant difference at p < 0.05. Full article ">Figure 5
cDA concentrations (fg cell−1) in P. fraudulenta (A,C) and P. australis (B,D) at the end of the experiments with C. gigas (A,C) and P. maximus (B,D). The asterisks indicate significant difference at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). Full article ">Figure 6
Linear regression for all data between P. australis cDA concentrations (fg cell−1) and the average clearance rates (CR, mL h−1 ind−1, A) or the average filtration rates (FR, cells mL−1 ind−1, B) of C. gigas throughout the 5 days of the experiment. Full article ">Figure 7
DA concentrations in C. gigas (10−6 µg DA g−1) as a function of cDA concentrations in P. australis or P. fraudulenta (fg cell−1). The linear regression only applies to P. australis. Crossed circles: C. gigas exposed to mixed cultures of I. galbana and P. australis or P. fraudulenta. Full article ">

10 pages, 1271 KiB

Open AccessArticle

Changes of DNA Damage Effect of T-2 or Deoxynivalenol Toxins during Three Weeks Exposure in Common Carp (Cyprinus carpio L.) Revealed by LORD-Q PCR

by Rubina Tünde Szabó, Mária Kovács-Weber, Krisztián Milán Balogh, Miklós Mézes and Balázs Kovács

Toxins 2021, 13(8), 576; https://doi.org/10.3390/toxins13080576 - 19 Aug 2021

Cited by 4 | Viewed by 2476

Abstract

The present study aimed to adapt a Long-run Real-time DNA Damage Quantification (LORD-Q) qPCR-based method for the analysis of the mitochondrial genome of Common carp (Cyprinus carpio L.) and detect the DNA damaging effect of T-2 (4.11 mg kg⁻¹) and [...] Read more.

The present study aimed to adapt a Long-run Real-time DNA Damage Quantification (LORD-Q) qPCR-based method for the analysis of the mitochondrial genome of Common carp (Cyprinus carpio L.) and detect the DNA damaging effect of T-2 (4.11 mg kg⁻¹) and deoxynivalenol (5.96 mg kg⁻¹) mycotoxins in a 3-week feeding period. One-year-old Common carp were treated in groups (control, T-2 and DON). The mycotoxins were sprayed over the complete pelleted feed, and samples were taken weekly. Following the adaptation of LORD-Q PCR method for the Common carp species, the number of lesions were calculated to determine the amount of DNA damage. In the first and second weeks, the T-2 and the DON treated groups differed significantly from each other; however these differences disappeared in the third week. There was a significant difference in the DNA lesion values between weeks 1 and 3 in the deoxynivalenol-contaminated groups. While in the T-2 treated groups, the DNA lesion values were significantly reduced on weeks 2 and 3 compared to week 1. The results suggested that the trichothecene mycotoxins have a relevant DNA damaging effect. Full article

(This article belongs to the Section Mycotoxins)

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Specificity of the short (a) and long (b) fragments by agarose gel electrophoresis. Agarose gel (1.5%) electrophoresis of real-time PCR products with ethidium bromide staining; template DNS is 10 ng of template. (a) aAmplified products of the short fragment, the expected product size was 150 bp. Loading well 1–2 from T-2, loading well 3–4 from DON treated group (week 3). Loading well 5 is the negative control sample, loading well 6 is the size marker (GeneRulerTM 100 bp DNA ladder). (b) Amplified products of the long fragment, the expected product size was 3400 bp. Loading well 1 is the size marker (GeneRuler TM 100 bp DNA ladder). Loading well 2–3 is from T-2, loading well 4–5 is from the DON treated group. Loading well 6 is the negative control sample. Full article ">Figure 2
Amplification plot and melt curve of the short (a) and long (b) fragments by StepOne™/StepOnePlus™ Software (a) amplification plot, (b) melt curve of short fragment: two samples from T-2 group (2 blue line), two samples from the DON group (purple, pink line), one control sample (red, yellow, two green line) in the quadruplicate from week 3. The negative control is the magenta line. (c) Amplification plot of the long fragment: one control (yellow, two green, red) and one treated (two purple, two blue) samples in quadruplicate. (d) Melt curve of the long fragment: two samples from T-2 group (2 blue line), two samples from DON group (magenta, pink line), one control sample (red, yellow, two green line) in quadruplicate from week 3. The negative control is the purple line. Full article ">Figure 2 Cont.
Amplification plot and melt curve of the short (a) and long (b) fragments by StepOne™/StepOnePlus™ Software (a) amplification plot, (b) melt curve of short fragment: two samples from T-2 group (2 blue line), two samples from the DON group (purple, pink line), one control sample (red, yellow, two green line) in the quadruplicate from week 3. The negative control is the magenta line. (c) Amplification plot of the long fragment: one control (yellow, two green, red) and one treated (two purple, two blue) samples in quadruplicate. (d) Melt curve of the long fragment: two samples from T-2 group (2 blue line), two samples from DON group (magenta, pink line), one control sample (red, yellow, two green line) in quadruplicate from week 3. The negative control is the purple line. Full article ">

11 pages, 2329 KiB

Open AccessArticle

A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery

by Tim Lüddecke, Anne Paas, Lea Talmann, Kim N. Kirchhoff, Björn M. von Reumont, André Billion, Thomas Timm, Günter Lochnit and Andreas Vilcinskas

Toxins 2021, 13(8), 575; https://doi.org/10.3390/toxins13080575 - 18 Aug 2021

Cited by 7 | Viewed by 3453

Abstract

Arthropod venoms offer a promising resource for the discovery of novel bioactive peptides and proteins, but the limited size of most species translates into minuscule venom yields. Bioactivity studies based on traditional fractionation are therefore challenging, so alternative strategies are needed. Cell-free synthesis [...] Read more.

Arthropod venoms offer a promising resource for the discovery of novel bioactive peptides and proteins, but the limited size of most species translates into minuscule venom yields. Bioactivity studies based on traditional fractionation are therefore challenging, so alternative strategies are needed. Cell-free synthesis based on synthetic gene fragments is one of the most promising emerging technologies, theoretically allowing the rapid, laboratory-scale production of specific venom components, but this approach has yet to be applied in venom biodiscovery. Here, we tested the ability of three commercially available cell-free protein expression systems to produce venom components from small arthropods, using U₂-sicaritoxin-Sdo1a from the six-eyed sand spider Hexophtalma dolichocephala as a case study. We found that only one of the systems was able to produce an active product in low amounts, as demonstrated by SDS-PAGE, mass spectrometry, and bioactivity screening on murine neuroblasts. We discuss our findings in relation to the promises and limitations of cell-free synthesis for venom biodiscovery programs in smaller invertebrates. Full article

(This article belongs to the Section Animal Venoms)

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Architecture of the linear gene construct F120. The construct comprises a 49-bp 5′ UTR, including the T7 promoter (T7) and ribosome binding site (RBS), followed by the gene of interest (ATG start codon and the codon-optimized USCTX sequence) and a 35-bp 3′ UTR, including a TAA stop codon and T7 terminator (Ter). Functionally corresponding parts are shown in matching colors, with the UTRs in light gray, the RBS in orange, start/stop codons in dark gray, and promoter/terminator sequences in blue. Spacer nucleotides are indicated by noncontiguous boxes. Full article ">Figure 2
Production and purification of USCTX. (a) Commercially available cell-free synthesis systems differ in their ability to produce USCTX (anticipated band size = 4.3 kDa). No such band was produced by the S30 Extract System (left) or the TnT T7 Insect Cell Extract Protein Expression System (right), but a band of the expected size was produced by the NEB PURExpress In Vitro Protein Synthesis System (middle, in duplicate to highlight reproducibility). (b) Purification of USCTX, showing the elution fractions E1–E3 from the His-Spin column. The red box indicates the area in which USCTX bands should appear. Full article ">Figure 3
Effect of the recombinant USCTX produced by cell-free expression on the viability of mouse neuroblasts (N2a cells). We tested all three IMAC elution fractions, namely the purification flow-through (E1), water elution (E2), and washing buffer elution (E3). The inhibitory effect was measured relative to the positive control (HPC; maximum inhibition induced by Triton X-100, lowest viability) and negative control (HNC; untreated cells in growth medium, zero inhibition, highest viability). Full article ">

8 pages, 1105 KiB

Open AccessEditor’s ChoiceReview

Secretion of Pertussis Toxin from Bordetella pertussis

by Drusilla L. Burns

Toxins 2021, 13(8), 574; https://doi.org/10.3390/toxins13080574 - 18 Aug 2021

Cited by 7 | Viewed by 4307

Abstract

Production and secretion of pertussis toxin (PT) is essential for the virulence of Bordetella pertussis. Due to the large oligomeric structure of PT, transport of the toxin across bacterial membrane barriers represents a significant hurdle that the bacteria must overcome in order [...] Read more.

Production and secretion of pertussis toxin (PT) is essential for the virulence of Bordetella pertussis. Due to the large oligomeric structure of PT, transport of the toxin across bacterial membrane barriers represents a significant hurdle that the bacteria must overcome in order to maintain pathogenicity. During the secretion process, PT undergoes a two-step transport process. The first step involves transport of the individual polypeptide chains of PT across the inner membrane utilizing a generalized secretion pathway, most likely the bacterial Sec system. The second step involves the use of a specialized apparatus to transport the toxin across the outer membrane of the bacterial cell. This apparatus, which has been termed the Ptl transporter and which is unique to the PT secretion pathway, is a member of the type IV family of bacterial transporters. Here, the current understanding of the PT secretion process is reviewed including a description of the Ptl proteins that assemble to form the transporter, the general structure of type IV transporters, the known similarities and differences between canonical type IV substrate transport and Ptl-mediated transport of PT, as well as the known sequence of events in the assembly and secretion of PT. Full article

(This article belongs to the Special Issue Pertussis Toxin and Research on Pertussis Vaccine)

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25 pages, 4324 KiB

Open AccessReview

The Therapeutic Strategies for Uremic Toxins Control in Chronic Kidney Disease

by Ping-Hsun Lu, Min-Chien Yu, Meng-Jiun Wei and Ko-Lin Kuo

Toxins 2021, 13(8), 573; https://doi.org/10.3390/toxins13080573 - 17 Aug 2021

Cited by 18 | Viewed by 8525

Abstract

Uremic toxins (UTs) are mainly produced by protein metabolized by the intestinal microbiota and converted in the liver or by mitochondria or other enzymes. The accumulation of UTs can damage the intestinal barrier integrity and cause vascular damage and progressive kidney damage. Together, [...] Read more.

Uremic toxins (UTs) are mainly produced by protein metabolized by the intestinal microbiota and converted in the liver or by mitochondria or other enzymes. The accumulation of UTs can damage the intestinal barrier integrity and cause vascular damage and progressive kidney damage. Together, these factors lead to metabolic imbalances, which in turn increase oxidative stress and inflammation and then produce uremia that affects many organs and causes diseases including renal fibrosis, vascular disease, and renal osteodystrophy. This article is based on the theory of the intestinal–renal axis, from bench to bedside, and it discusses nonextracorporeal therapies for UTs, which are classified into three categories: medication, diet and supplement therapy, and complementary and alternative medicine (CAM) and other therapies. The effects of medications such as AST-120 and meclofenamate are described. Diet and supplement therapies include plant-based diet, very low-protein diet, probiotics, prebiotics, synbiotics, and nutraceuticals. The research status of Chinese herbal medicine is discussed for CAM and other therapies. This review can provide some treatment recommendations for the reduction of UTs in patients with chronic kidney disease. Full article

(This article belongs to the Section Uremic Toxins)

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35 pages, 2122 KiB

Open AccessReview

Integrated Mycotoxin Management System in the Feed Supply Chain: Innovative Approaches

by Francesca Fumagalli, Matteo Ottoboni, Luciano Pinotti and Federica Cheli

Toxins 2021, 13(8), 572; https://doi.org/10.3390/toxins13080572 - 16 Aug 2021

Cited by 46 | Viewed by 8410

Abstract

Exposure to mycotoxins is a worldwide concern as their occurrence is unavoidable and varies among geographical regions. Mycotoxins can affect the performance and quality of livestock production and act as carriers putting human health at risk. Feed can be contaminated by various fungal [...] Read more.

Exposure to mycotoxins is a worldwide concern as their occurrence is unavoidable and varies among geographical regions. Mycotoxins can affect the performance and quality of livestock production and act as carriers putting human health at risk. Feed can be contaminated by various fungal species, and mycotoxins co-occurrence, and modified and emerging mycotoxins are at the centre of modern mycotoxin research. Preventing mould and mycotoxin contamination is almost impossible; it is necessary for producers to implement a comprehensive mycotoxin management program to moderate these risks along the animal feed supply chain in an HACCP perspective. The objective of this paper is to suggest an innovative integrated system for handling mycotoxins in the feed chain, with an emphasis on novel strategies for mycotoxin control. Specific and selected technologies, such as nanotechnologies, and management protocols are reported as promising and sustainable options for implementing mycotoxins control, prevention, and management. Further research should be concentrated on methods to determine multi-contaminated samples, and emerging and modified mycotoxins. Full article

(This article belongs to the Special Issue Improved and Innovative Actions for Mycotoxin Management in the Food and Feed Chain)

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13 pages, 1661 KiB

Open AccessArticle

Evaluation of Salivary Indoxyl Sulfate with Proteinuria for Predicting Graft Deterioration in Kidney Transplant Recipients

by Natalia Korytowska, Aleksandra Wyczałkowska-Tomasik, Leszek Pączek and Joanna Giebułtowicz

Toxins 2021, 13(8), 571; https://doi.org/10.3390/toxins13080571 - 16 Aug 2021

Cited by 7 | Viewed by 3090

Abstract

Acute kidney injury (AKI) is a significant risk factor for developing chronic kidney disease and progression to end-stage renal disease in elderly patients. AKI is also a relatively common complication after kidney transplantation (KTx) associated with graft failure. Since the lifespan of a [...] Read more.

Acute kidney injury (AKI) is a significant risk factor for developing chronic kidney disease and progression to end-stage renal disease in elderly patients. AKI is also a relatively common complication after kidney transplantation (KTx) associated with graft failure. Since the lifespan of a transplanted kidney is limited, the risk of the loss/deterioration of graft function (DoGF) should be estimated to apply the preventive treatment. The collection of saliva and urine is more convenient than collecting blood and can be performed at home. The study aimed to verify whether non-invasive biomarkers, determined in saliva and urine, may be useful in the prediction of DoGF in kidney transplant recipients (KTRs) (n = 92). Salivary and serum toxins (p-cresol sulfate, pCS; indoxyl sulfate, IS) concentrations were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urinary proteins, hemoglobin, and glucose were measured using a semi-quantitative strip test. Salivary IS (odds ratio (OR) = 1.19), and proteinuria (OR = 3.69) were demonstrated as independent factors for the prediction of DoGF. Satisfactory discriminatory power (area under the receiver operating characteristic curve (AUC) = 0.71 ± 0.07) and calibration of the model were obtained. The model showed that categories of the increasing probability of the risk of DoGF are associated with the decreased risk of graft survival. The non-invasive diagnostic biomarkers are a useful screening tool to identify high-risk patients for DoGF. Full article

(This article belongs to the Special Issue Uremic Toxins and Urinary Acute Kidney Injury Biomarkers)

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The correlation between eGFR and (a) serum pCS (rs = −0.50); (b) salivary pCS (rs = −0.55); (c) serum IS (rs = −0.78); and (d) salivary IS (rs = −0.76) at M1 for all subjects. eGFR: estimated glomerular filtration rate; IS: indoxyl sulfate; pCS: p-cresol sulfate. Full article ">Figure 2
The correlation between (a) salivary and serum pCS (rs = 0.92); (b) salivary and serum IS (rs = 0.81) at M1 for all subjects. IS: indoxyl sulfate; pCS: p-cresol sulfate. Full article ">Figure 3
The area under the receiver operating characteristic curve (AUC) for the model of deterioration of graft function included salivary IS and proteinuria at M1. Full article ">Figure 4
Graft survival according to the (a) categories of predicted probabilities of deterioration of graft function: calculated risk of deterioration of graft function lower than 10%, between 10% and 40%, and above 40% (p < 0.00001); (b) sub-groups: low IS and no proteinuria, high IS and no proteinuria, low IS with proteinuria, and high IS with proteinuria. * The only statistically significant difference was observed between the group with low IS and no proteinuria and the group with high IS with proteinuria (p = 0.04250). Full article ">

11 pages, 2836 KiB

Open AccessArticle

Analysis of Aflatoxin Biomarkers in the Hair of Experimental Animals

by Innocent Mupunga, Ilse Janse van Rensburg, Nokuthula Luthuli, Ovokeroye A. Abafe, Leshweni J. Shai and David R. Katerere

Toxins 2021, 13(8), 570; https://doi.org/10.3390/toxins13080570 - 16 Aug 2021

Cited by 3 | Viewed by 2980

Abstract

Analysis of body fluids and tissues of aflatoxin exposed individuals for the presence of aflatoxins and aflatoxin metabolites has emerged as a reliable indicator of exposure and metabolism of aflatoxins. However, current aflatoxin biomarkers are not appropriate for investigating the long-term effects of [...] Read more.

Analysis of body fluids and tissues of aflatoxin exposed individuals for the presence of aflatoxins and aflatoxin metabolites has emerged as a reliable indicator of exposure and metabolism of aflatoxins. However, current aflatoxin biomarkers are not appropriate for investigating the long-term effects of aflatoxin exposure. In this explorative study, we investigated the analysis of hair as a complementary or alternative matrix for the assessment of biomarkers of long-term aflatoxin exposure. Three groups of guinea pigs were orally dosed with 5 ugkg⁻¹bw⁻¹, 50 ugkg⁻¹bw⁻¹, and 100 ugkg⁻¹bw⁻¹ of AFB1. Urine and hair samples were collected on days 0, 1, 2, 3, 7, 30, 60, and 90 and analysed for AFB1 and AFM1 using UHPLC-MS/MS. AFB1 and AFM1 were detected in 75% and 13.6%, respectively, of the day 1 to day 7 urine samples. AFB1 was detected in hair samples collected from day 3 up to day 60. This is the first report to confirm the deposition of AFB1 in the hair of experimental animals. These findings indicate that hair analysis has the potential to provide an accurate long-term historical record of aflatoxin exposure with potentially important implications for the field of aflatoxin biomarkers. Full article

(This article belongs to the Special Issue Human Biomonitoring and Risk Assessment of Mycotoxins)

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14 pages, 2809 KiB

Open AccessEditor’s ChoiceArticle

Association of Polygenic Risk Score and Bacterial Toxins at Screening Colonoscopy with Colorectal Cancer Progression: A Multicenter Case-Control Study

by Alfonso Piciocchi, Elena Angela Pia Germinario, Koldo Garcia Etxebarria, Silvia Rossi, Lupe Sanchez-Mete, Barbara Porowska, Vittoria Stigliano, Paolo Trentino, Andrea Oddi, Fabio Accarpio, Gian Luca Grazi, Giovanni Bruno, Massimo Bonucci, Massimo Giambenedetti, Patrizia Spigaglia, Fabrizio Barbanti, Slawomir Owczarek, Ida Luzzi, Elisabetta Delibato, Zaira Maroccia, Lorenza Nisticò, Carla Fiorentini, Mauro D’Amato, Roberta De Angelis and Alessia Fabbri Show full author list Hide full author list

Toxins 2021, 13(8), 569; https://doi.org/10.3390/toxins13080569 - 16 Aug 2021

Cited by 16 | Viewed by 5502

Abstract

Colorectal cancer (CRC) is a leading cause of cancer death worldwide, and its incidence is correlated with infections, chronic inflammation, diet, and genetic factors. An emerging aspect is that microbial dysbiosis and chronic infections triggered by certain bacteria can be risk factors for [...] Read more.

Colorectal cancer (CRC) is a leading cause of cancer death worldwide, and its incidence is correlated with infections, chronic inflammation, diet, and genetic factors. An emerging aspect is that microbial dysbiosis and chronic infections triggered by certain bacteria can be risk factors for tumor progression. Recent data suggest that certain bacterial toxins implicated in DNA attack or in proliferation, replication, and death can be risk factors for insurgence and progression of CRC. In this study, we recruited more than 300 biopsy specimens from people undergoing colonoscopy, and we analyzed to determine whether a correlation exists between the presence of bacterial genes coding for toxins possibly involved in CRC onset and progression and the different stages of CRC. We also analyzed to determine whether CRC-predisposing genetic factors could contribute to bacterial toxins response. Our results showed that CIF toxin is associated with polyps or adenomas, whereas pks+ seems to be a predisposing factor for CRC. Toxins from Escherichia coli as a whole have a higher incidence rate in adenocarcinoma patients compared to controls, whereas Bacteroides fragilis toxin does not seem to be associated with pre-cancerous nor with cancerous lesions. These results have been obtained irrespectively of the presence of CRC-risk loci. Full article

(This article belongs to the Special Issue Bacterial Toxins – the Key Causal and Preventive Markers of Bacterial Disease)

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14 pages, 4850 KiB

Open AccessArticle

Reduced Expression of Metallothionein-I/II in Renal Proximal Tubules Is Associated with Advanced Chronic Kidney Disease

by Yi-Jhu Lu, Ya-Ju Wu, Lu-Jen Chen, Bor-Sheng Ko, Tzu-Ching Chang, Yi-Ju Wu, Shu-Man Liang, Yee-Jee Jan and Jun-Yang Liou

Toxins 2021, 13(8), 568; https://doi.org/10.3390/toxins13080568 - 15 Aug 2021

Cited by 6 | Viewed by 3515

Abstract

Chronic kidney disease (CKD) is a commonly occurring complex renal syndrome that causes overall mortality in many diseases. The clinical manifestations of CKD include renal tubulointerstitial fibrosis and loss of renal function. Metallothionein-I/II (MT-I/II) is potentially expressed in the liver and kidney, and [...] Read more.

Chronic kidney disease (CKD) is a commonly occurring complex renal syndrome that causes overall mortality in many diseases. The clinical manifestations of CKD include renal tubulointerstitial fibrosis and loss of renal function. Metallothionein-I/II (MT-I/II) is potentially expressed in the liver and kidney, and possesses antioxidant and metal detoxification properties. However, whether MT-I/II expression is associated with the prognosis of nephropathy remains unknown. In this study, we investigated the MT-I/II level in human CKD, using immunohistochemistry. MT-I/II is located on the proximal tubules and is notably reduced in patients with CKD. MT-I/II expression was significantly correlated with the functional and histological grades of CKD. In an aristolochic acid (AAI)-induced nephropathy mouse model, MT-I/II was abundantly increased after AAI injection for 7 days, but decreased subsequently compared to that induced in the acute phase when injected with AAI for 28 days. Furthermore, we found that ammonium pyrrolidinedithiocarbamate (PDTC) restored AAI-induced MT-I/II reduction in HK2 cells. The injection of PDTC ameliorated AAI-induced renal tubulointerstitial fibrosis and reduced the concentrations of blood urea nitrogen and creatinine in mouse sera. Taken together, our results indicate that MT-I/II reduction is associated with advanced CKD, and the retention of renal MT-I/II is a potential therapeutic strategy for CKD. Full article

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MT-I/II located within proximal tubule (PT) in normal and CKD renal parenchyma. The histopathological images of human kidney slides stained with hematoxylin and eosin (H&E) (Left panel). The location and morphology of PT displayed with alpha-methyl CoA racemase (AMACR) using immunohistochemistry (Middle Panel). Histological images revealed the location and changes of MT-I/II by their specific antibody (Right panel). Bar = 500 μm. Full article ">Figure 2
MT-I/II levels in kidney of aristolochic acid (AAI)-induced nephropathy (AAN) mice. (A) Histological images of MT-I/II stained using immunohistochemistry in mice without (CTR) or with AAI (5 mg/kg bw) injection for 7 days. (B) Histological images of MT-I/II in mice without (CTR) or with AAI injection (2.5 and 5 mg/kg) for 4 weeks. Bar = 200 μm in low magnification image; Bar = 50 μm in high magnification image. (C) The statistic figure showing the raw values of distribution area times stain intensity of MT-I/II in (A) and (B). n ≥ 6 in each group. Full article ">Figure 3
The amounts of serum BUN and CRE in AAN mice. Serum concentrations of (A) BUN and (B) CRE in mice without (CTR) or with AAI (2.5 and 5 mg/kg) injection detected using ELISA analysis at 7 days and 4 weeks as indicated. n ≥ 6 in each group. P values of the t-test were denoted as indications. Full article ">Figure 4
PDTC restored AAI-reduced MT-I/II expression in the HK2 cells. Representative images of (A) Expression of MT-I/II treated with 25 µM AAI (DMSO as control) for 0–6 days in HK2 cells. (B) Expression of MT-I/II treated with 0–30 µM of PDTC (DMSO as control) for 2 days in HK2 cells. (C) MT-I/II expression treated with 25 µM AAI or/and 30 µM PDTC for 2 days in HK2 cells. MT-I/II levels as determined using Western blot analysis. Actin or tubulin was used as the internal loading control. Full article ">Figure 5
Level of serum BUN and CRE in PDTC treated AAN mice. Serum concentrations of (A) BUN and (B) CRE in AAN mice injected with 2.5 mg/kg AAI or/and 10 mg/kg PDTC for 4 weeks as determined using ELISA analysis. n ≥ 6 in each group. P values of the t-test were denoted as indications. Full article ">Figure 6
Effect of PDTC for kidney injury in AAN mice. (A) The histological analysis of renal fibrosis in AAN (2.5 mg/kg) mice injected with/without PDTC (10 mg/kg) for 4 weeks examined using Masson’s Trichrome stain. Bar = 200 μm. (B) The statistical analysis (the raw values of distribution area times stain intensity) of renal fibrosis was performed using image J analysis. n ≥ 6 in each group. P values of the t-test were denoted as indications. Full article ">Figure 6 Cont.
Effect of PDTC for kidney injury in AAN mice. (A) The histological analysis of renal fibrosis in AAN (2.5 mg/kg) mice injected with/without PDTC (10 mg/kg) for 4 weeks examined using Masson’s Trichrome stain. Bar = 200 μm. (B) The statistical analysis (the raw values of distribution area times stain intensity) of renal fibrosis was performed using image J analysis. n ≥ 6 in each group. P values of the t-test were denoted as indications. Full article ">

19 pages, 1309 KiB

Open AccessReview

Panorama of the Intracellular Molecular Concert Orchestrated by Actinoporins, Pore-Forming Toxins from Sea Anemones

by Carlos Alvarez, Carmen Soto, Sheila Cabezas, Javier Alvarado-Mesén, Rady Laborde, Fabiola Pazos, Uris Ros, Ana María Hernández and María Eliana Lanio

Toxins 2021, 13(8), 567; https://doi.org/10.3390/toxins13080567 - 13 Aug 2021

Cited by 8 | Viewed by 3505

Abstract

Actinoporins (APs) are soluble pore-forming proteins secreted by sea anemones that experience conformational changes originating in pores in the membranes that can lead to cell death. The processes involved in the binding and pore-formation of members of this protein family have been deeply [...] Read more.

Actinoporins (APs) are soluble pore-forming proteins secreted by sea anemones that experience conformational changes originating in pores in the membranes that can lead to cell death. The processes involved in the binding and pore-formation of members of this protein family have been deeply examined in recent years; however, the intracellular responses to APs are only beginning to be understood. Unlike pore formers of bacterial origin, whose intracellular impact has been studied in more detail, currently, we only have knowledge of a few poorly integrated elements of the APs’ intracellular action. In this review, we present and discuss an updated landscape of the studies aimed at understanding the intracellular pathways triggered in response to APs attack with particular reference to sticholysin II, the most active isoform produced by the Caribbean Sea anemone Stichodactyla helianthus. To achieve this, we first describe the major alterations these cytolysins elicit on simpler cells, such as non-nucleated mammalian erythrocytes, and then onto more complex eukaryotic cells, including tumor cells. This understanding has provided the basis for the development of novel applications of sticholysins such as the construction of immunotoxins directed against undesirable cells, such as tumor cells, and the design of a cancer vaccine platform. These are among the most interesting potential uses for the members of this toxin family that have been carried out in our laboratory. Full article

(This article belongs to the Special Issue New Frontiers in Pore-Forming Toxins and Related Proteins: Molecular Mechanisms, Functions and Applications)

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6 pages, 524 KiB

Open AccessArticle

Humoral Immune Response Evaluation in Horses Vaccinated with Recombinant Clostridium perfringens Toxoids Alpha and Beta for 12 Months

by Nayra F. Q. R. Freitas, Denis Y. Otaka, Cleideanny C. Galvão, Dayane M. de Almeida, Marcos R. A. Ferreira, Clóvis Moreira Júnior, Marina M. M. H. Hidalgo, Fabricio R. Conceição and Felipe M. Salvarani

Toxins 2021, 13(8), 566; https://doi.org/10.3390/toxins13080566 - 13 Aug 2021

Cited by 2 | Viewed by 2781

Abstract

In horses, Clostridium perfringens is associated with acute and fatal enterocolitis, which is caused by a beta toxin (CPB), and myonecrosis, which is caused by an alpha toxin (CPA). Although the most effective way to prevent these diseases is through vaccination, specific clostridial [...] Read more.

In horses, Clostridium perfringens is associated with acute and fatal enterocolitis, which is caused by a beta toxin (CPB), and myonecrosis, which is caused by an alpha toxin (CPA). Although the most effective way to prevent these diseases is through vaccination, specific clostridial vaccines for horses against C. perfringens are not widely available. The aim of this study was to pioneer the immunization of horses with three different concentrations (100, 200 and 400 µg) of C. perfringens recombinant alpha (rCPA) and beta (rCPB) proteins, as well as to evaluate the humoral immune response over 360 days. Recombinant toxoids were developed and applied to 50 horses on days 0 and 30. Those vaccines attempted to stimulate the production of alpha antitoxin (anti-CPA) and beta antitoxin (anti-CPB), in addition to becoming innocuous, stable and sterile. There was a reduction in the level of neutralizing anti-CPA and anti-CPB antibodies following the 60th day; therefore, the concentrations of 200 and 400 µg capable of inducing a detectable humoral immune response were not determined until day 180. In practical terms, 200 µg is possibly the ideal concentration for use in the veterinary industry’s production of vaccines against the action of C. perfringens in equine species. Full article

(This article belongs to the Collection Clostridium difficile/Clostridium sordellii and Clostridium perfringens Toxins)

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27 pages, 2859 KiB

Open AccessEditor’s ChoiceReview

In Vivo Models and In Vitro Assays for the Assessment of Pertussis Toxin Activity

by Marieke Esther Hoonakker

Toxins 2021, 13(8), 565; https://doi.org/10.3390/toxins13080565 - 12 Aug 2021

Cited by 5 | Viewed by 7630

Abstract

One of the main virulence factors produced by Bordetella pertussis is pertussis toxin (PTx) which, in its inactivated form, is the major component of all marketed acellular pertussis vaccines. PTx ADP ribosylates Gα_i proteins, thereby affecting the inhibition of adenylate cyclases and [...] Read more.

One of the main virulence factors produced by Bordetella pertussis is pertussis toxin (PTx) which, in its inactivated form, is the major component of all marketed acellular pertussis vaccines. PTx ADP ribosylates Gα_i proteins, thereby affecting the inhibition of adenylate cyclases and resulting in the accumulation of cAMP. Apart from this classical model, PTx also activates some receptors and can affect various ADP ribosylation- and adenylate cyclase-independent signalling pathways. Due to its potent ADP-ribosylation properties, PTx has been used in many research areas. Initially the research primarily focussed on the in vivo effects of the toxin, including histamine sensitization, insulin secretion and leukocytosis. Nowadays, PTx is also used in toxicology research, cell signalling, research involving the blood–brain barrier, and testing of neutralizing antibodies. However, the most important area of use is testing of acellular pertussis vaccines for the presence of residual PTx. In vivo models and in vitro assays for PTx often reflect one of the toxin’s properties or details of its mechanism. Here, the established and novel in vivo and in vitro methods used to evaluate PTx are reviewed, their mechanisms, characteristics and limitations are described, and their application for regulatory and research purposes are considered. Full article

(This article belongs to the Special Issue Pertussis Toxin and Research on Pertussis Vaccine)

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The classical models for PTx binding, internalisation, ADP ribosylation, and its effect on cell signalling. The B oligomer of PTx binds to glycoconjugate proteins on the cell surface, upon which the holotoxin enters the cell by endocytosis, followed by retrograde transport through the endosome, Golgi and the endoplasmatic reticulum. Subsequently, the S1 subunit is released into the cytosol. Within the cytosol, the S1 subunit catalyses the transfer of ADP-ribose from NAD+ to the α-subunit of Gαi/o/t proteins, thereby preventing interaction of these proteins with their cognate receptors. ADP ribosylation fixes the α-subunit of the G-proteins in their inactive (GDP-bound) form, thereby rendering it unable to inhibit its target enzymes; ACs. ACs catalyse the conversion of ATP into cAMP. This second messenger binds to and activates protein kinase A (PKA), which is involved in a range of pathways, one of which is the phosphorylation of the cAMP response element-binding protein (CREB). CREB binds to the DNA sequence cAMP response elements (CRE) and thereby increases the transcription of CRE responsive genes. PTx-induced cAMP might also directly bind to “exchange protein directly activated by cAMP” (EPAC), which are guanine nucleotide exchange factors for Rap molecules. Full article ">Figure 2
The classical in vivo methods for PTx and models for the mechanisms. (A). The histamine sensitization test is based on the sensitizing effects of PTx in mice to histamine doses which would normally cause no effect. Under “normal” conditions, vascular permeability and blood pressure is maintained by the vascular endothelial cells and vascular smooth muscle cells. Administration of histamine results in the reorganization of actin filament structure and adherence junctions, increased endothelial permeability and vascular leakiness. Contraction of vascular smooth muscle cells can (partly) compensate for the blood volume loss, but this compensatory mechanism is inhibited by PTx, causing a significantly reduction in blood pressure. (B). In the leukocytosis promotion test, the effect of PTx on leukocyte numbers is measured. PTx inhibits lymphocyte extravasation and restores lymphocyte egress from lymph nodes to the lymph, resulting in a rise in circulating leukocyte number. Migration involves rolling mediated by attachment to selectins and arrest mediated by integrins. The binding of integrins and endothelial adhesion molecules triggers the opening of endothelial junctions, allowing leukocytes to transmigrate. PTx does not affect selectins and rolling, but does inhibit integrin mediated arrest. Integrins can only bind to adhesion molecules on the high endothelial venule cells upon activation by Gαi-coupled chemokine receptors. These Gαi-coupled receptors might be the target of PTx, although additional research will be necessary to confirm whether these receptors are responsible for the leukocytosis. (C). The mouse weight gain test is based upon the negative effect of PTx on the weight gain of mice throughout a period of seven days. Although considered a general toxicity test, its mechanism is unknown. (D). In the islet activation protein test, PTx enhances the glucose-induced release of insulin. In the test, PTx is responsible for ADP ribosylation of Gαi/o proteins in β-cell, resulting in accumulation of cAMP. Although not studied directly in relation to PTx, enhanced cAMP levels can activate PKA and EPAC and stimulate the glucose pathway that induces the release of insulin. The other pathway shown to be involved in the IAP test is mediated by ghrelin. Ghrelin is an endogenous ligand for the Gαi-coupled growth hormone secretagogue receptor, which can suppress K+ exflux. As a result of the ADP ribosylation of this Gαi-coupled receptor, K+ exflux is inhibited. The resulting depolarization causes Ca2+ influx and enhanced levels of Ca2+ are essential for the release of insulin. Full article ">Figure 3
The cellular in vitro assays for PTx and their mechanisms. (A) The CHO cell clustering assay is based on the clustered growth pattern of CHO cells in response to PTx and requires an active S1 subunit. The morphological changes require rearrangement the cytoskeletal structures, but the underlying responsible mechanisms have not been fully elucidated. The first proposed mechanism (A1) involves the RhoA pathway, which normally results in polymerization of actin filaments. cAMP-dependent activation of PKA might result in an inactive state of RhoA, thereby diminishing or preventing actin polymerisation. Alternatively (A2), PTx-induced uncoupling of αi proteins might directly affect the functioning of Rap1 and thereby reduce polymerization of cytoskeletal structures. In the third proposed mechanism (A3), uncoupling of Gαi/o protein causes cGMP accumulation which also suppresses actin polymerization, affecting both cell shape and motility. Additional studies will be required to determine the involvement these three mechanisms in the CHO cell clustering assay. Clustering can be analysed manually and quantified by electrical impedance, continuous phase-contrast imaging or by measuring the distance between nearest neighbouring nuclei. (B) The ATP and cAMP PTx assays are based on the effects of PTx on the conversion of ATP to cAMP by the ACs. In the ATP-PTx assay (B1), the effect of PTx on ATP levels in peripheral blood mononuclear cells is measured using an ATP-luminescence assay. The cAMP-PTx assay is based on the rise in cAMP upon exposure of cells to PTx. Initially, A10 cells (B2) were used to monitor for PTx in combination with isoprenaline as an activator of AC using and a commercially available ELISA kits to measure cAMP levels. Subsequently, a CHO cell line stably expressing a CRE controlled NanoLuc construct was developed (B3). Upon stimulation with forskolin, PTx enhances the production of cAMP, leading to the activation of PKA, resulting in enhanced transcription of NanoLuc. The iGIST assay (C) is based upon HEK293 cells that co-express the Gαi-coupled SSTR2 receptor and a luminescent cAMP probe. In combination with octreotide and forskolin, these cells allow for real time assessment of cellular cAMP levels. PTx ADP ribosylates the αi subunit of SSTR2, diminishing the effect of octreotide and results in enhanced levels of cAMP. cAMP binding to the probe causes a conformational change and increases the luminescence signal. In the MoDC assay (D), the effect of PTx on the production of the cytokines IL-2 and IFN-γ by MoDC is assessed. As PTx is described to stimulate the TLR4 receptor, this pathway may be involved in MoDC activation. Full article ">

19 pages, 1711 KiB

Open AccessArticle

Experimental Evidence of Ciguatoxin Accumulation and Depuration in Carnivorous Lionfish

by Isabel do Prado Leite, Khalil Sdiri, Angus Taylor, Jérôme Viallon, Hela Ben Gharbia, Luiz Laureno Mafra Júnior, Peter Swarzenski, François Oberhaensli, Hélène Taiana Darius, Mireille Chinain and Marie-Yasmine Dechraoui Bottein

Toxins 2021, 13(8), 564; https://doi.org/10.3390/toxins13080564 - 11 Aug 2021

Cited by 13 | Viewed by 3834

Abstract

Ciguatera poisoning is a food intoxication associated with the consumption of fish or shellfish contaminated, through trophic transfer, with ciguatoxins (CTXs). In this study, we developed an experimental model to assess the trophic transfer of CTXs from herbivorous parrotfish, Chlorurus microrhinos, to [...] Read more.

Ciguatera poisoning is a food intoxication associated with the consumption of fish or shellfish contaminated, through trophic transfer, with ciguatoxins (CTXs). In this study, we developed an experimental model to assess the trophic transfer of CTXs from herbivorous parrotfish, Chlorurus microrhinos, to carnivorous lionfish, Pterois volitans. During a 6-week period, juvenile lionfish were fed naturally contaminated parrotfish fillets at a daily dose of 0.11 or 0.035 ng CTX3C equiv. g⁻¹, as measured by the radioligand-receptor binding assay (r-RBA) or neuroblastoma cell-based assay (CBA-N2a), respectively. During an additional 6-week depuration period, the remaining fish were fed a CTX-free diet. Using r-RBA, no CTXs were detectable in muscular tissues, whereas CTXs were measured in the livers of two out of nine fish sampled during exposure, and in four out of eight fish sampled during depuration. Timepoint pooled liver samples, as analyzed by CBA-N2a, confirmed the accumulation of CTXs in liver tissues, reaching 0.89 ng CTX3C equiv. g⁻¹ after 41 days of exposure, followed by slow toxin elimination, with 0.37 ng CTX3C equiv. g⁻¹ measured after the 6-week depuration. These preliminary results, which need to be pursued in adult lionfish, strengthen our knowledge on CTX transfer and kinetics along the food web. Full article

(This article belongs to the Special Issue Ciguatoxins)

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Proportion of control and CTX-exposed lionfish rejecting the food in relation to the number of remaining individuals at a given time during both the exposure and depuration periods. Full article ">Figure 2
Quantification of CTX in fish liver during the exposure phase of the experiment. Dose-response curves of N2a cells when exposed to increasing concentrations of fish liver extracts, obtained from the exposure phase of the experiment, in OV− (open symbols) and OV+ (solid symbols) conditions; (A) CTX3C (◆), (B) non-exposed control fish at day 42 (pool of 3 specimens) (∆/▲), (C) exposed fish at day 30 (pool of 4 specimens) (○/●), and (D) exposed fish at day 41 (pool of 5 specimens) (□/■). Data represent the mean ± SD of 1 assay, with each point run in triplicate. The dotted vertical line corresponds to the maximum concentration of liver tissue (MCE) that does not induce a matrix effect on the assay, which was estimated at 20 mg mL−1 of fish liver extracts. Full article ">Figure 3
Quantification of CTX in fish liver during the depuration phase of the experiment. Dose-response curves of N2a cells when exposed to increasing concentrations of pooled fish liver extracts, obtained from the depuration stage of the experiment, in OV− (open symbols) and OV+ (solid symbols); (A) 8 days of depuration (pool of 3 specimens) (○/●), (B) 29 days of depuration (pool of three specimens) (□/■), and (C) 43 days of depuration (pool of two specimens) (∆/▲). Data represent the mean ± SD of one assay, with each point run in triplicate. The dotted vertical line corresponds to the maximum concentration of liver tissue (MCE) that does not induce the matrix effect, which was established at 20 mg mL−1 of fish liver extracts. Full article ">Figure 4
Design of the experiment, with 5 tanks initially containing 6 or 8 fish (n) assigned to the CTX-exposed treatment (T) and 2 tanks for control (C) containing 6 fish and 10 fish (n). The image represents a picture of the juvenile lionfish (Pterois volitans) individuals and their shelters made of dark tubes in one of the experimental tanks. Full article ">

23 pages, 2967 KiB

Open AccessArticle

A Comparative Analysis of Methods (LC-MS/MS, LC-MS and Rapid Test Kits) for the Determination of Diarrhetic Shellfish Toxins in Oysters, Mussels and Pipis

by Penelope A. Ajani, Chowdhury Sarowar, Alison Turnbull, Hazel Farrell, Anthony Zammit, Stuart Helleren, Gustaaf Hallegraeff and Shauna A. Murray

Toxins 2021, 13(8), 563; https://doi.org/10.3390/toxins13080563 - 11 Aug 2021

Cited by 4 | Viewed by 4371

Abstract

Rapid methods for the detection of biotoxins in shellfish can assist the seafood industry and safeguard public health. Diarrhetic Shellfish Toxins (DSTs) are produced by species of the dinoflagellate genus Dinophysis, yet the comparative efficacy of their detection methods has not been [...] Read more.

Rapid methods for the detection of biotoxins in shellfish can assist the seafood industry and safeguard public health. Diarrhetic Shellfish Toxins (DSTs) are produced by species of the dinoflagellate genus Dinophysis, yet the comparative efficacy of their detection methods has not been systematically determined. Here, we examined DSTs in spiked and naturally contaminated shellfish–Sydney Rock Oysters (Saccostrea glomerata), Pacific Oysters (Magallana gigas/Crassostrea gigas), Blue Mussels (Mytilus galloprovincialis) and Pipis (Plebidonax deltoides/Donax deltoides), using LC-MS/MS and LC-MS in 4 laboratories, and 5 rapid test kits (quantitative Enzyme-Linked Immunosorbent Assay (ELISA) and Protein Phosphatase Inhibition Assay (PP2A), and qualitative Lateral Flow Assay (LFA)). We found all toxins in all species could be recovered by all laboratories using LC-MS/MS (Liquid Chromatography—tandem Mass Spectrometry) and LC-MS (Liquid Chromatography—Mass Spectrometry); however, DST recovery at low and mid-level concentrations (<0.1 mg/kg) was variable (0–150%), while recovery at high-level concentrations (>0.86 mg/kg) was higher (60–262%). While no clear differences were observed between shellfish, all kits delivered an unacceptably high level (25–100%) of falsely compliant results for spiked samples. The LFA and the PP2A kits performed satisfactorily for naturally contaminated pipis (0%, 5% falsely compliant, respectively). There were correlations between spiked DSTs and quantitative methods was highest for LC-MS (r² = 0.86) and the PP2A kit (r² = 0.72). Overall, our results do not support the use of any DST rapid test kit as a stand-alone quality assurance measure at this time. Full article

(This article belongs to the Special Issue Marine Toxins from Harmful Algae and Seafood Safety)

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15 pages, 1084 KiB

Open AccessArticle

Proteo-Transcriptomic Characterization of Sirex nitobei (Hymenoptera: Siricidae) Venom

by Chenglong Gao, Lili Ren, Ming Wang, Zhengtong Wang, Ningning Fu, Huiying Wang, Xiaochen Wang, Tegen Ao, Wensheng Du, Zijin Zheng, Huadong Li and Juan Shi

Toxins 2021, 13(8), 562; https://doi.org/10.3390/toxins13080562 - 11 Aug 2021

Cited by 10 | Viewed by 3053

Abstract

The wood-boring woodwasp Sirex nitobei is a native pest in Asia, infecting and weakening the host trees in numerous ecological and commercial coniferous forest plantations. In China, hosts of S. nitobei are diverse, so the pest has spread to several provinces of China, [...] Read more.

The wood-boring woodwasp Sirex nitobei is a native pest in Asia, infecting and weakening the host trees in numerous ecological and commercial coniferous forest plantations. In China, hosts of S. nitobei are diverse, so the pest has spread to several provinces of China, resulting in considerable economic and ecological damage. During female oviposition, S. nitobei venom along with arthrospores of the symbiotic fungus Amylostereum areolatum or A. chaetica is injected into host trees, and the combination of these two biological factors causes the death of xylem host trees. The presence of venom alone causes only the yellowing and wilting of needles. In this study, we constructed the venom gland transcriptome of S. nitobei for the first time and a total of 15,036 unigenes were acquired. From the unigenes, 11,560 ORFs were identified and 537 encoding protein sequences with signal peptides at the N-terminus. Then, we used the venomics approach to characterize the venom composition of female S. nitobei and predicted 1095 proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. We focused on seven proteins that were both highly expressed in the venom gland transcriptome and predicted in the crude venom proteome. These seven proteins are laccase-2, laccase-3, a protein belonging to the Kazal family, chitooligosaccharidolytic β-N-acetylglucosaminidase, beta-galactosidase, icarapin-like protein, and waprin-Thr1-like protein. Using quantitative real-time PCR (qRT-PCR), we also proved that the genes related to these seven proteins are specifically expressed in the venom glands. Finally, we revealed the functional role of S. nitobei venom in the physiological response of host trees. It can not only promote the colonization of symbiotic fungus but contribute to the development of eggs and larvae. This study provides a deeper understanding of the molecular mechanism of the woodwasp–pine interaction. Full article

(This article belongs to the Special Issue Evolution, Genomics and Proteomics of Venom)

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20 pages, 770 KiB

Open AccessEditor’s ChoiceReview

Deoxynivalenol and Zearalenone—Synergistic or Antagonistic Agri-Food Chain Co-Contaminants?

by Asmita Thapa, Karina A. Horgan, Blánaid White and Dermot Walls

Toxins 2021, 13(8), 561; https://doi.org/10.3390/toxins13080561 - 11 Aug 2021

Cited by 35 | Viewed by 6029

Abstract

Deoxynivalenol (DON) and Zearalenone (ZEN) are two commonly co-occurring mycotoxins produced by members of the genus Fusarium. As important food chain contaminants, these can adversely affect both human and animal health. Critically, as they are formed prior to harvesting, their occurrence cannot [...] Read more.

Deoxynivalenol (DON) and Zearalenone (ZEN) are two commonly co-occurring mycotoxins produced by members of the genus Fusarium. As important food chain contaminants, these can adversely affect both human and animal health. Critically, as they are formed prior to harvesting, their occurrence cannot be eliminated during food production, leading to ongoing contamination challenges. DON is one of the most commonly occurring mycotoxins and is found as a contaminant of cereal grains that are consumed by humans and animals. Consumption of DON-contaminated feed can result in vomiting, diarrhoea, refusal of feed, and reduced weight gain in animals. ZEN is an oestrogenic mycotoxin that has been shown to have a negative effect on the reproductive function of animals. Individually, their mode of action and impacts have been well-studied; however, their co-occurrence is less well understood. This common co-occurrence of DON and ZEN makes it a critical issue for the Agri-Food industry, with a fundamental understanding required to develop mitigation strategies. To address this issue, in this targeted review, we appraise what is known of the mechanisms of action of DON and ZEN with particular attention to studies that have assessed their toxic effects when present together. We demonstrate that parameters that impact toxicity include species and cell type, relative concentration, exposure time and administration methods, and we highlight additional research required to further elucidate mechanisms of action and mitigation strategies. Full article

(This article belongs to the Special Issue Mycotoxins, Food Safety and Metrology)

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15 pages, 5249 KiB

Open AccessArticle

Evaluation of the Therapeutic Effects of Protocatechuic Aldehyde in Diabetic Nephropathy

by Yu-Teng Chang, Mu-Chi Chung, Chang-Chi Hsieh, Jeng-Jer Shieh and Ming-Ju Wu

Toxins 2021, 13(8), 560; https://doi.org/10.3390/toxins13080560 - 10 Aug 2021

Cited by 11 | Viewed by 3718

Abstract

Diabetic nephropathy (DN) is one of the most severe chronic kidney diseases in diabetes and is the main cause of end-stage renal disease (ESRD). Protocatechuic aldehyde (PCA) is a natural product with a variety of effects on pulmonary fibrosis. In this study, we [...] Read more.

Diabetic nephropathy (DN) is one of the most severe chronic kidney diseases in diabetes and is the main cause of end-stage renal disease (ESRD). Protocatechuic aldehyde (PCA) is a natural product with a variety of effects on pulmonary fibrosis. In this study, we examined the effects of PCA in C57BL/KS db/db male mice. Kidney morphology, renal function indicators, and Western blot, immunohistochemistry, and hematoxylin and eosin (H&E) staining data were analyzed. The results revealed that treatment with PCA could reduce diabetic-induced renal dysfunction, as indicated by the urine albumin-to-creatinine ratio (db/m: 120.1 ± 46.1μg/mg, db/db: 453.8 ± 78.7 µg/mg, db/db + 30 mg/kg PCA: 196.6 ± 52.9 µg/mg, db/db + 60 mg/kg PCA: 163.3 ± 24.6 μg/mg, p < 0.001). However, PCA did not decrease body weight, fasting plasma glucose, or food and water intake in db/db mice. H&E staining data revealed that PCA reduced glomerular size in db/db mice (db/m: 3506.3 ± 789.3 μm², db/db: 6538.5 ± 1818.6 μm², db/db + 30 mg/kg PCA: 4916.9 ± 1149.6 μm², db/db + 60 mg/kg PCA: 4160.4 ± 1186.5 μm²p < 0.001). Western blot and immunohistochemistry staining indicated that PCA restored the normal levels of diabetes-induced fibrosis markers, such as transforming growth factor-beta (TGF-β) and type IV collagen. Similar results were observed for epithelial–mesenchymal transition-related markers, including fibronectin, E-cadherin, and α-smooth muscle actin (α-SMA). PCA also decreased oxidative stress and inflammation in the kidney of db/db mice. This research provides a foundation for using PCA as an alternative therapy for DN in the future. Full article

(This article belongs to the Special Issue Chronic Kidney Disease (CKD) Studies on Humans and Animals)

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Toxins, Volume 13, Issue 8 (August 2021) – 83 articles

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