Toxins

28 pages, 1684 KB

Open AccessReview

Identification and Detection of Aflatoxigenic Aspergillus flavus and Aflatoxins: Current Technologies and Future Perspectives

by Md Mostafa Masud, Sumyya Waliullah, Abdus Sobhan and Emran Ali

Toxins 2026, 18(8), 343; https://doi.org/10.3390/toxins18080343 - 4 Aug 2026

Abstract

Aspergillus flavus is a major fungal species that contaminates a wide range of crops and produces aflatoxins under favorable environmental conditions. Several approaches have been developed for aflatoxigenic A. flavus identification and aflatoxins detection. This review summarizes the existing methods for the identification [...] Read more.

Aspergillus flavus is a major fungal species that contaminates a wide range of crops and produces aflatoxins under favorable environmental conditions. Several approaches have been developed for aflatoxigenic A. flavus identification and aflatoxins detection. This review summarizes the existing methods for the identification of aflatoxigenic A. flavus, along with recent progress in aflatoxins detection methods. It also highlights limitations and possible factors associated with the accurate identification of aflatoxigenic isolates. Particularly, this review underscores the utilization of advanced analytical or detection methods along with existing identification techniques to accurately identify aflatoxigenic A. flavus. Full article

(This article belongs to the Special Issue Advances in Detection and Removal of Mycotoxins in Food)

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14 pages, 1898 KB

Open AccessArticle

Effectiveness of Onabotulinumtoxin-A in Patients with Chronic Migraine and Previous CGRP-mAb Treatment Failure: A Multicentre 6-Month Real-World Experience

by Marcello Silvestro, Maria Albanese, Ilaria Orologio, Luigi Francesco Iannone, Francesca Pistoia, Stefania Battistini, Gianluca Avino, Martina Petracca, Marina Romozzi, Raffaele Ornello, Diego Centonze, Simona Sacco, Antonio Russo and on behalf of the Italian Headache Registry (RICe) Study Group

Toxins 2026, 18(8), 342; https://doi.org/10.3390/toxins18080342 - 4 Aug 2026

Abstract

Background: A substantial proportion of patients with chronic migraine (CM) with previous failures among oral standard-of-care migraine medications do not respond to monoclonal antibodies targeting the calcitonine gene related peptide pathway (CGRP-mAbs), leaving limited evidence-based options for subsequent preventive strategies. The present multicentre [...] Read more.

Background: A substantial proportion of patients with chronic migraine (CM) with previous failures among oral standard-of-care migraine medications do not respond to monoclonal antibodies targeting the calcitonine gene related peptide pathway (CGRP-mAbs), leaving limited evidence-based options for subsequent preventive strategies. The present multicentre real-world study evaluated the effectiveness of onabotulinumtoxin-A (BoNT-A) in CM patients with previous CGRP-mAb failure and multiple prior oral preventive failures. Methods: This prospective, observational, non-randomized multicentre study enrolled patients with CM who had failed ≥3 classes of oral preventive treatments and ≥1 CGRP-mAb due to lack of efficacy or intolerance. Participants received BoNT-A according to the PREEMPT “follow-the-pain” paradigm every three months and were followed for 6 months. Co-primary outcomes were change in monthly headache days (MHD) after three and six months and ≥50% MHD responder rates at both time points. Results: Fifty-three patients were analysed (85% female, aged 48.5 ± 15.2 years, range 20–73). Compared to the baseline, at the third month and sixth month, MHD decreased from 22.83 (SD 6.74) to 15.38 (SD 7.91; p < 0.001) and 14.55 (SD 9.35; p < 0.001), respectively. The percentages of participants achieving the response status in MMD at the third month and sixth month were 30% and 45%, respectively. In the third and sixth months, 22 patients (41.5%) and 27 patients (50.9%), respectively, converted from chronic to episodic migraine, while the prevalence of medication-overuse headache decreased from 81.1% of patients at baseline to 45.3% and 43.4%, respectively. Migraine-related impact and disability scores improved over follow-up, alongside improvements in anxiety and depressive symptoms and in migraine-specific quality of life. Conclusions: The present findings support the use of BoNT-A in difficult-to-treat patients with CM following CGRP-mAb failure and provide further evidence that its therapeutic effects may rely on mechanisms that are at least partially distinct from, and potentially complementary to, those of CGRP-targeted therapies. Full article

(This article belongs to the Section Bacterial Toxins)

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18 pages, 5795 KB

Open AccessArticle

Supplementation with Yellow Mealworm (Tenebrio molitor) Larvae Grown on Deoxynivalenol-Contaminated Substrate Improves Growth and Gut Integrity in Broilers

by Revathi Shanmugasundaram, Klint W. McCafferty, Laharika Kappari, Xin Ye, Kelsy Robinson and Anthony E. Glenn

Toxins 2026, 18(8), 341; https://doi.org/10.3390/toxins18080341 - 4 Aug 2026

Abstract

Deoxynivalenol (DON), a Fusarium mycotoxin frequently detected in poultry feeds, impairs broiler health by damaging gut integrity, altering immune response, and compromising growth performance. Insect-derived proteins, such as those from yellow mealworm (Tenebrio molitor) larvae meal (YMW), are gaining interest as [...] Read more.

Deoxynivalenol (DON), a Fusarium mycotoxin frequently detected in poultry feeds, impairs broiler health by damaging gut integrity, altering immune response, and compromising growth performance. Insect-derived proteins, such as those from yellow mealworm (Tenebrio molitor) larvae meal (YMW), are gaining interest as an alternate protein source for broiler chickens. Hence, this study evaluated whether defatted YMW produced from larvae reared on DON-contaminated substrates can be safely included in broiler diets. A total of 400 one-day-old Cobb 700 mixed-sex broilers were assigned to four dietary treatments—(1) the control, (2) DON (15 mg/kg), (3) 2% YMW, and (4) 4% YMW—in ten replicates with 10 birds per replicate for 15 days. On day 14, performance parameters, intestinal morphology, tight-junction protein (TJP) gene expression, and CD4+ and CD8+ T-cell populations in cecal tonsils were evaluated. Data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. DON exposure reduced body weight by 1% and body weight gain (BWG) by 1.2%, decreased the CD4+: CD8+ T-cell ratio by 80%, and reduced the villus height in the jejunum by 19.8% and ileum by 16%. DON altered the TJP expression by upregulating claudin-2 mRNA expression by 2.1-fold in the jejunum and 5.2-fold in the ileum, and it downregulated claudin-4 expression by 6.4-fold in the ileum (p < 0.05). Dietary inclusion of 2% or 4% YMW from DON-contaminated substrates had no negative effect on growth performance or immune parameters and maintained intestinal morphology and TJP gene expression, comparable to the control groups. In conclusion, defatted YMW produced from larvae reared on DON-contaminated substrates can be safely used as a sustainable alternative protein ingredient in broiler diets. Full article

(This article belongs to the Special Issue Mycotoxin Contamination in Animal Nutrition and Its Mitigation Strategies)

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22 pages, 5078 KB

Open AccessArticle

Toxicity and DNA Adduct Formation Reinforce AI-Guided Prediction of Aflatoxin B1 Bioactivation in VERO E6 Cells

by Bharti Sangwan, Ugochukwu Okoro, Isabella Atteck, Pawel Jaruga, Chinwe Ekenna and Michael Fasullo

Toxins 2026, 18(8), 339; https://doi.org/10.3390/toxins18080339 - 4 Aug 2026

Abstract

VERO cells, derived from the kidney epithelium of the African green monkey, are widely used in virology, but their ability to metabolize xenobiotics is not fully understood. Since cytochrome P450 (CYP) enzymes participate in xenobiotic metabolism, we investigated which CYP genes are expressed [...] Read more.

VERO cells, derived from the kidney epithelium of the African green monkey, are widely used in virology, but their ability to metabolize xenobiotics is not fully understood. Since cytochrome P450 (CYP) enzymes participate in xenobiotic metabolism, we investigated which CYP genes are expressed in VERO-E6 cells. Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) showed that VERO-E6 cells express CYP3A4, CYP3A5, and CYP3A7. In contrast, CYP1A1, CYP1A2, CYP1B1, CYP2E1, CYP2D6, and CYP2C9 transcripts were either not detected or at a low detection level. To determine whether the encoded enzymes have the potential to activate aflatoxin B1 (AFB1), we used artificial intelligence (AI)-based structural modeling along with molecular docking. AI modeling suggested that CYP3A enzymes can position AFB1 in an orientation compatible with the formation of the reactive intermediate, and CYP3A4 showed the most favorable predicted interaction (docking score: −16.3 kcal/mol). To demonstrate AFB1 bioactivation, we exposed VERO-E6 cells to 200 nmol/L AFB1. After 10 days, we observed about 40% cell death. Liquid chromatography–tandem mass spectroscopy (LC–MS/MS) analysis confirmed the presence of AFB1-derived DNA adducts, indicating that metabolic activation occurred in these cells. These findings support the presence of CYP-dependent AFB1 bioactivation in VERO-E6 cells. Thus, combining computational and experimental approaches elucidates xenobiotic metabolism in cells where biochemical data are limited. Full article

(This article belongs to the Special Issue Recent Advances and Future Perspectives on Genotoxicity of Toxins)

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18 pages, 7157 KB

Open AccessArticle

Integrated Gut Microbiota and Metabolome Analysis Reveals Dysbiosis and Metabolic Disturbances in Diarrhea of the Complex-Toothed Flying Squirrel (Trogopterus xanthipes): Possible Mycotoxin-Induction?

by Lifeng Che, Bichen Miao, Lijuan Suo and Jie Tang

Toxins 2026, 18(8), 340; https://doi.org/10.3390/toxins18080340 - 3 Aug 2026

Abstract

The gut microbiota and its metabolic functions are critical for intestinal homeostasis, but their integrated responses to diarrhea remain poorly understood in non-model mammals, such as the complex-toothed flying squirrel (Trogopterus xanthipes). Here, we collected fecal samples from six captive animals [...] Read more.

The gut microbiota and its metabolic functions are critical for intestinal homeostasis, but their integrated responses to diarrhea remain poorly understood in non-model mammals, such as the complex-toothed flying squirrel (Trogopterus xanthipes). Here, we collected fecal samples from six captive animals with diarrhea and six healthy controls; the diarrheal animals had been provided with moldy corn, but mycotoxin levels were not measured. Fecal microbiota was profiled by full-length 16S rRNA sequencing and untargeted metabolomics with integrative analyses. Diarrheal animals showed significantly decreased fecal microbial alpha-diversity and distinct community separation. LEfSe analysis identified Lawsonia intracellularis and Streptococcus macedonicus as the most enriched taxa in the diarrhea group. Untargeted metabolomics revealed 2216 differential metabolites (1931 downregulated), primarily enriched in steroid hormone biosynthesis, arachidonic acid metabolism, and bile secretion pathways. Procrustes and Mantel tests confirmed significant microbiome–metabolome concordance (M² = 0.422, p < 0.001). Spearman correlation networks showed that L. intracellularis was positively correlated with multiple differential metabolites, while S. macedonicus exhibited predominantly negative correlations. These findings suggest that diarrhea in T. xanthipes may be associated with moldy corn exposure and is accompanied by gut dysbiosis and metabolic disturbances. L. intracellularis and S. macedonicus may be associated with disease progression through pathways linked to lipid and bile acid metabolism and inflammation. This study provides novel microbial–metabolic insights into diarrhea in a medicinal mammal, supporting disease prevention and healthy breeding. Full article

(This article belongs to the Special Issue Mycotoxin Occurrence, Toxicology, and Effects on Animals, Animal Feeds and Detoxification Methods)

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22 pages, 23882 KB

Open AccessArticle

Recombinant BaMtx Preserves the Biological Properties of a Lys49 Phospholipase A₂ from Bothrops atrox and Reveals Differential Responses in Tumor and Non-Tumor Cell Models

by Daniel Torrejón, Angie Regalado, Alex Proleón, Víctor Otárola, Fanny Lazo, Edith Rodríguez, Erasmo Colona-Vallejos, Libertad Alzamora-Gonzales, Jherson Cisneros-Gutierrez, Jacquelyne Zarria-Romero, Miryam Paola Alvarez Flores, Renata Nascimento Gomes, Thatiana Corrêa de Melo, Ronnie G. Gavilan, Javier Cárdenas Tenorio, Félix A. Urra, Dan E. Vivas-Ruiz and Armando Yarlequé

Toxins 2026, 18(8), 338; https://doi.org/10.3390/toxins18080338 - 2 Aug 2026

Abstract

Lys49 phospholipase A2 (Lys49-PLA₂) homologues are catalytically inactive snake venom toxins with diverse biological activities, but their functional characterization requires recombinant production strategies to overcome the limitations inherent to native venom-derived proteins. Here, we produced recombinant BaMtx (rBaMtx), a Lys49-PLA₂ [...] Read more.

Lys49 phospholipase A2 (Lys49-PLA₂) homologues are catalytically inactive snake venom toxins with diverse biological activities, but their functional characterization requires recombinant production strategies to overcome the limitations inherent to native venom-derived proteins. Here, we produced recombinant BaMtx (rBaMtx), a Lys49-PLA₂ homologue from Bothrops atrox, in the Pichia pastoris KM71 expression system and evaluated whether its biochemical and biological properties were preserved. rBaMtx was secreted into the culture medium, purified by cation-exchange chromatography, and characterized by SDS-PAGE, Western blotting, RP-HPLC (85.9% purity), and MALDI-TOF mass spectrometry, confirming a molecular mass of 13,821 Da. Despite lacking PLA₂ activity, rBaMtx retained pronounced myotoxicity in vivo, inducing dose-dependent plasma creatine kinase release and skeletal muscle damage comparable to or greater than native BaMtx. In vitro, rBaMtx reduced viability of murine 4T1 breast carcinoma cells (IC₅₀ = 60.94 µg/mL), altered cell morphology, modestly increased IL-1β release, and produced context-dependent effects in paired human tumor and non-tumor cell models, differing from native BaMtx and crude venom. These findings establish rBaMtx as a reproducible model for investigating the mechanisms and biomedical potential of catalytically inactive Lys49-PLA₂ homologues. Full article

(This article belongs to the Special Issue Biochemistry, Pathology and Applications of Venoms)

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17 pages, 3998 KB

Open AccessArticle

Sphingopyxis sp. IM-1 Reduces Intact Microcystin-LR and Attenuates MC-LR-Associated Inflammatory Gene Expression in Hep3B Hepatocytes

by Apurva Lad, Mudit Bhatia, Johnna A. Birbeck, Alex Kuang, Elliot Furr, Judy Westrick, Youngwoo Seo, Dae-Wook Kang, David J. Kennedy and Steven T. Haller

Toxins 2026, 18(8), 337; https://doi.org/10.3390/toxins18080337 - 1 Aug 2026

Abstract

Excessive cyanobacterial proliferation and toxin production increasingly threaten freshwater ecosystems, drinking water systems, and public health. Among cyanotoxins, microcystin-LR (MC-LR) is one of the most prevalent variants and is recognized for its hepatotoxicity, with evidence showing it can also alter gut microbiota composition. [...] Read more.

Excessive cyanobacterial proliferation and toxin production increasingly threaten freshwater ecosystems, drinking water systems, and public health. Among cyanotoxins, microcystin-LR (MC-LR) is one of the most prevalent variants and is recognized for its hepatotoxicity, with evidence showing it can also alter gut microbiota composition. Previous studies, including our own, demonstrate that MC exposure can induce inflammation, oxidative stress, and changes in gene expression associated with immune responses, even at concentrations below guideline limits. This study investigated the protective effect of an MC-degrading bacterium, Sphingopyxis sp. IM1 (IM1) with a known enzymatic MC degradation pathway, against MC-LR-induced hepatotoxicity under in vitro conditions. Human Hep3B hepatocytes were pretreated with varying ratios of IM1 bacteria and subsequently exposed to 9.95 ppm of MC-LR for 24 h. RT-qPCR analysis demonstrated that MC-LR exposure strongly increased the expression of the inflammatory markers TNFα and TGF-β1, whereas pretreatment with IM1 significantly attenuated these responses. Mass spectrometric analysis of cell pellets and spent culture media demonstrated reduced concentrations of intact MC-LR in IM1-pretreated samples compared to MC-LR-only controls, accompanied by increased detection of tetrapeptide degradation products generated during mlr-mediated MC degradation. These results demonstrate that IM1-mediated degradation of MC-LR attenuates toxin-induced hepatotoxic and inflammatory responses, highlighting the potential of a novel microbial-based therapeutic approach to mitigate MC-induced toxicity. Full article

(This article belongs to the Special Issue Unveiling the Toxic Effects of Harmful Algal Blooms: 2nd Edition)

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9 pages, 320 KB

Open AccessArticle

Acute Toxicity and No Observable Adverse Effect Level of Nodularin-R in Mice

by Sarah C. Finch, Craig Waugh, D. Tim Harwood, Alison R. Turnbull, Andrew I. Selwood, Emillie M. F. Passfield and Jonathan Puddick

Toxins 2026, 18(8), 336; https://doi.org/10.3390/toxins18080336 - 1 Aug 2026

Abstract

Nodularin is a cyanobacterial toxin predominantly produced by Nodularia spumigena Mertens. This cyanobacterium has been associated with fish, bird, and animal deaths, and the toxin can contaminate fish and shellfish intended for human consumption. With climate change, blooms of Nodularia spumigena are becoming [...] Read more.

Nodularin is a cyanobacterial toxin predominantly produced by Nodularia spumigena Mertens. This cyanobacterium has been associated with fish, bird, and animal deaths, and the toxin can contaminate fish and shellfish intended for human consumption. With climate change, blooms of Nodularia spumigena are becoming more frequent and more widespread, meaning that the risk this cyanobacterium poses to human health must be evaluated. However, toxicity information is scarce, meaning that data from the closely related toxin class, microcystins, are currently used instead. In the current study, the LD₅₀ of nodularin-R by intraperitoneal injection was found to be 50 µg/kg, consistent with previous reports. For the first time, the LD₅₀ of nodularin-R by oral administration was determined using the voluntary consumption of laced cream cheese (7.5 mg/kg), which is similar to that reported for microcystin-LR (10.9 mg/kg). The use of microcystin toxicology data in food safety assessments for nodularin, therefore, seems reasonable—but it is critical that an oral repeated-dose study is performed on nodularin to better understand the risk posed by this toxin through chronic exposure. Full article

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

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22 pages, 1083 KB

Open AccessPerspective

A Framework for Monitoring Contaminants in Decentralized Agricultural Systems: Leveraging Stakeholder Incentives in Low-Resource Settings

by Lee E. Voth-Gaeddert, Hannah Glesener and Gabriela Montenegro-Bethancourt

Toxins 2026, 18(8), 335; https://doi.org/10.3390/toxins18080335 - 1 Aug 2026

Abstract

Decentralized agricultural systems (DAS) are essential to food security for millions of people in low-resource settings, yet most existing contaminant monitoring frameworks have been designed for centralized supply chains. This paper proposes a structured framework for integrating contaminant monitoring into DAS by (1) [...] Read more.

Decentralized agricultural systems (DAS) are essential to food security for millions of people in low-resource settings, yet most existing contaminant monitoring frameworks have been designed for centralized supply chains. This paper proposes a structured framework for integrating contaminant monitoring into DAS by (1) mapping the diverse supply chain structures and stakeholders that constitute these systems; (2) analyzing stakeholder motivations, constraints, and value propositions for monitoring adoption; and (3) identifying efficient monitoring points where these value propositions align with product quality improvement. The framework is organized around four core principles: tiered monitoring matched to supply chain position and stakeholder capacity, monitoring at stakeholder handoff points rather than within individual segments, incentive structures that make monitoring self-sustaining, and capacity building and governance as durable infrastructure. The framework is illustrated through the case of maize and aflatoxin contamination in Guatemala. It remains a conceptual framework, however, and its principles are intended to be generalizable across crops, contaminants, and geographies rather than demonstrated to be so. Empirical validation across these dimensions remains a necessary next step. The central argument is that through this framework, small, strategically placed incentives at high-leverage points in decentralized supply chains can shift system-wide quality outcomes. This can occur without requiring top-down regulatory enforcement, which is often neither feasible nor effective in these contexts. Full article

(This article belongs to the Special Issue Strategies for Mitigating Mycotoxin Contamination in Food and Feed)

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19 pages, 1958 KB

Open AccessArticle

Indoxyl Sulfate-Mediated Blood–Brain Barrier Damage in Chronic Kidney Disease

by Leah Hernandez, Camillo Tancredi Strizzi, Miriam Rosina, Angelina Schwarz, Nina Kronqvist, Samsul Arefin, Peter Stenvinkel and Karolina Kublickiene

Toxins 2026, 18(8), 334; https://doi.org/10.3390/toxins18080334 - 1 Aug 2026

Abstract

Chronic kidney disease is associated with neurovascular complications including cognitive impairment, potentially involving blood–brain barrier (BBB) impairment. The protein-bound uremic toxin indoxyl sulfate (IS) promotes endothelial injury, but its effects on human brain microvascular endothelial cells remain incompletely understood. Human cerebral microvascular endothelial [...] Read more.

Chronic kidney disease is associated with neurovascular complications including cognitive impairment, potentially involving blood–brain barrier (BBB) impairment. The protein-bound uremic toxin indoxyl sulfate (IS) promotes endothelial injury, but its effects on human brain microvascular endothelial cells remain incompletely understood. Human cerebral microvascular endothelial cells (hCMEC/D3) were exposed to IS 200 and 900 μM. BBB integrity was assessed by the FITC-dextran (4 kDa) transwell permeability assay and claudin-5 immunofluorescence. Transcriptional responses were quantified by qPCR for aryl hydrocarbon (AhR) target genes, oxidative stress-associated and inflammatory markers, senescence, and junction-associated genes. Senescence-associated phenotypic changes were evaluated by SA-β-galactosidase staining and cytokine array profiling. IS increased endothelial permeability at 24 and 48 h (~1.5-fold relative to control) without evidence of cytotoxicity and reduced claudin-5 staining intensity. IS strongly upregulated AhR target genes, including CYP1A1, CYP1B1, and CYP1A2. NFE2L2 and IDO1 increased, while NFKB1 remained unchanged. SA-β-gal positivity increased, accompanied by elevated GM-CSF and G-CSF secretion, while CDKN1A decreased at IS 900 µM and CDKN2A remained unchanged. CDH5 was downregulated, TJP1 increased at 900 µM, and CLDN5 remained unchanged. These findings indicate that IS exposure is associated with impaired BBB integrity, AhR-related transcriptional responses, oxidative stress-associated transcriptional changes, junctional remodeling, and senescence-like endothelial features. However, causal attribution to individual pathways requires inhibition or knockdown studies. Full article

(This article belongs to the Special Issue Uremic Toxins and Chronic Kidney Disease)

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16 pages, 1059 KB

Open AccessArticle

Nasal Exposure to Microcystin Can Trigger Immune Responses in the Lung and Gut Microbiome Dysbiosis via the Lung–Gut Axis: A Pilot Acute Mouse Study

by Minseung Kim, Sangwoon Chung, John W. Christman and Jiyoung Lee

Toxins 2026, 18(8), 333; https://doi.org/10.3390/toxins18080333 - 1 Aug 2026

Abstract

Cyanotoxin events are among the most serious consequences of cyanobacterial harmful algal blooms. Microcystins (MCs), among the most prevalent cyanotoxins, are known to adversely affect human health. Recently, respiratory exposure has emerged as an important exposure route for MCs, with potential downstream effects [...] Read more.

Cyanotoxin events are among the most serious consequences of cyanobacterial harmful algal blooms. Microcystins (MCs), among the most prevalent cyanotoxins, are known to adversely affect human health. Recently, respiratory exposure has emerged as an important exposure route for MCs, with potential downstream effects on the gut microbiome through the lung–gut axis. In this pilot study, we investigated acute respiratory immune responses and gut microbiome alterations following MC inhalation using female C57BL/6J mice. Mice were assigned to three dose groups: control (0 µg/kg body weight), medium (25 µg/kg), and high (50 µg/kg). MC-LR was administered intranasally once daily for 3 days. Fecal samples were collected daily for 16S rRNA sequencing, and bronchoalveolar lavage (BAL) fluids were collected following sacrifice for immune cell analysis. MC exposure resulted in significantly increased monocyte counts (p < 0.1), while neutrophil, macrophage, and total cell counts did not significantly change (p > 0.1), suggesting selective lower respiratory tract inflammation. Functional prediction analysis of gut microbiota revealed significant increases (p < 0.1) in pathways associated with host health, including heme biosynthesis, sulfur oxidation, carbon metabolism, and antibiotic resistance. These findings suggest that inhaled MCs may induce respiratory inflammation and contribute to gut microbiome dysbiosis via the lung–gut axis. Full article

(This article belongs to the Special Issue Unveiling the Toxic Effects of Harmful Algal Blooms: 2nd Edition)

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22 pages, 3040 KB

Open AccessReview

Italian Consensus and Review in Lower Face Mimetic Muscle Treatment with Botulinum Toxin-A

by Salvatore Fundarò, Paola Molinari, Giovanni Brunelli, Maria Cazzulani, Guido Dalla Costa, Roberto Dell’Avanzato, Roberta D’Emilio, Carlo Di Gregorio, Antonella Franceschelli, Monica Renga, Giovanni Salti, Adriano Santorelli, Ugo Simone Urso and Ilaria Zollino

Toxins 2026, 18(8), 332; https://doi.org/10.3390/toxins18080332 - 31 Jul 2026

Abstract

Background: Treatment of the lower face with botulinum toxin type A (BoNT-A) is particularly challenging because of the important functional roles of the involved muscles, which increases the risk of adverse events and complications. Methods: To address this issue, a panel of 14 [...] Read more.

Background: Treatment of the lower face with botulinum toxin type A (BoNT-A) is particularly challenging because of the important functional roles of the involved muscles, which increases the risk of adverse events and complications. Methods: To address this issue, a panel of 14 experts conducted a three-round consensus study, including digital surveys and a formal meeting, on the aesthetic treatment of the mentalis (MT), depressor anguli oris (DAO), and platysma (PL) muscles using BoNT-A. The study was complemented by a literature review of 16 international consensus papers published between 2004 and 2024. Results: The primary indications for MT treatment that achieved strong consensus were “peau d’orange” (“golf ball”) chin appearance and hyperactivity. The Italian panel recommended a mean total dose of 8 U (distributed bilaterally), which was higher than the international mean dose of 6.5 U. The preferred injection technique consisted of a combination of intramuscular and intradermal injections. For the DAO, the strongest indication was correction of downturned oral commissures. Both the Italian panel and the international consensus reported a mean total dose of 5.6 U. Most experts (92.8%) preferred intramuscular injections, typically administered at a single injection point lateral to the marionette line in order to remain within a safe treatment zone and avoid diffusion to the depressor labii inferioris muscle. Indications for PL treatment included platysmal bands, jawline recontouring, and improvement of skin texture. The techniques discussed included treatment of both platysmal bands and the jawline. The consensus panel recommended a significantly higher mean total dose (48.4 U) than the international average (38.2 U). The paper also highlights the growing importance of ultrasound imaging as a tool for identifying individual anatomical variations in the lower facial musculature. Conclusions: As the demand for lower face rejuvenation continues to increase, standardized injection techniques and the integration of ultrasound imaging are essential for minimizing functional complications and optimizing aesthetic outcomes. Full article

(This article belongs to the Special Issue Anatomical, Experimental, and Clinical Studies of Botulinum Neurotoxin in Cosmetic Medicine)

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21 pages, 4631 KB

Open AccessArticle

Effects of Four Marine Toxins on Murine Hepatic Biotransformation Enzymes

by Joanna Soto de Jesus, Carmen González-Keelan, Peter A. Meléndez, Carmen L. Cadilla, Jasmine Contreras and Braulio D. Jiménez-Vélez

Toxins 2026, 18(8), 331; https://doi.org/10.3390/toxins18080331 - 30 Jul 2026

Abstract

This study assesses the impact of sublethal levels of four marine toxins ciguatoxin (CTX-1), maitotoxin-2 (MTX-2), saxitoxin (STX) and brevetoxin-2 (BTX-2) on murine hepatic detoxification enzymes expressed in mouse liver. CTX-1, BTX-2, and STX altered hepatic detoxification responses, but their effects were generally [...] Read more.

This study assesses the impact of sublethal levels of four marine toxins ciguatoxin (CTX-1), maitotoxin-2 (MTX-2), saxitoxin (STX) and brevetoxin-2 (BTX-2) on murine hepatic detoxification enzymes expressed in mouse liver. CTX-1, BTX-2, and STX altered hepatic detoxification responses, but their effects were generally more limited or temporally variable than those observed with MTX-2. CTX-1 produced time-dependent changes in cytochromes P450 (CYPs)-associated activities, BTX-2 induced early CYP1A2 and CYP3A11 responses followed by later suppression, and STX reduced CYP1A2 and CYP3A11 while increasing microsomal reductase activities. Of these toxins, MTX-2 exhibited the highest toxicity, notably decreasing key proteins such as CYPs, including CYP1A2 and CYP3A11. CYP enzymes are vital for metabolizing important endogenous and various xenobiotic substances, including therapeutic drugs. After MTX-2 exposure, both CYP1A2 and CYP3A11 levels dropped significantly at 12 h (p < 0.0001 for CYP3A11, p < 0.0001 for CYP1A2). Histopathological analysis revealed liver damage; however, albumin mRNA levels remained stable post-MTX-2 treatment, indicating that hepatotoxicity was not the sole cause of CYP3A11 reduction. Immunohistochemical analysis displayed uniform CYP3A11 distribution across liver regions after MTX-2 treatment. This suggests that MTX-2 exposure could augment the toxicity of drugs like Aldactone, Erythromycin, and Cyclosporine that utilize this metabolic pathway in humans. This is the first type of research performed of this nature, which could add to our understanding of marine toxin metabolism. These findings provide additional toxicological insights into the effects of four marine toxins on detoxification enzymes, with particular interest in MTX-2 toxicity, and potential implications for the treatment of fish poisoning, including ciguatera fish poisoning. Full article

(This article belongs to the Collection Ciguatoxin)

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11 pages, 893 KB

Open AccessFeature PaperArticle

Fascial Remodeling After Botulinum Toxin Injection in Post-Stroke Spasticity: A Retrospective Ultrasonographic Study

by Betül Aydın, Münire Nazlı Höbek Başer, İpek Midi, Naime Evrim Karadağ Saygı and Özge Keniş Coşkun

Toxins 2026, 18(8), 330; https://doi.org/10.3390/toxins18080330 - 30 Jul 2026

Abstract

Post-stroke spasticity involves both neural and non-neural mechanisms, including structural alterations of muscles and surrounding fascia. Although botulinum toxin type A (BoNT-A) is widely used to treat focal spasticity, its effects on fascial tissue remain unclear. This retrospective observational study investigated longitudinal changes [...] Read more.

Post-stroke spasticity involves both neural and non-neural mechanisms, including structural alterations of muscles and surrounding fascia. Although botulinum toxin type A (BoNT-A) is widely used to treat focal spasticity, its effects on fascial tissue remain unclear. This retrospective observational study investigated longitudinal changes in fascial thickness after BoNT-A injection using ultrasonography. Thirty-seven patients with post-stroke spasticity were included. Fascial thickness was measured at baseline, 3 weeks, and 3 months after treatment in the injected muscle regions. Clinical outcomes were assessed using the Modified Ashworth Scale (MAS), Fugl–Meyer Assessment (FMA), and Brunnstrom stages. Repeated-measures analysis demonstrated significant reductions in fascial thickness across all evaluated regions. Pairwise comparisons showed significant decreases from baseline to week 3 and month 3, whereas continued reductions between week 3 and month 3 were observed only in the flexor digitorum superficialis fascia, the flexor digitorum superficialis–flexor digitorum profundus interfascial layer, and the deep brachialis fascia. MAS scores and upper extremity Fugl–Meyer scores improved significantly, whereas Brunnstrom stages remained unchanged. These findings suggest that the therapeutic effects of BoNT-A may extend beyond neural inhibition to include fascial remodeling. Ultrasonographic assessment of fascial thickness may represent a potential imaging biomarker for monitoring structural treatment responses in post-stroke spasticity. Full article

(This article belongs to the Special Issue Botulinum Toxin in Neuro-Rehabilitation: Expanding Horizons in Spasticity and Beyond)

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27 pages, 1997 KB

Open AccessReview

Hemodiafiltration in Dialysis Practice: Global Adoption, Toxin Clearance, Inflammation Control, and Clinical Safety

by Thomas Lang, Adam M. Zawada, Xiaoling Wang, Jochen G. Raimann, Manuela Stauss-Grabo and Nadja Grobe

Toxins 2026, 18(8), 329; https://doi.org/10.3390/toxins18080329 - 28 Jul 2026

Abstract

Hemodiafiltration (HDF) combines diffusion and convection to enhance solute removal compared with hemodialysis (HD). While widely adopted in Europe and parts of Asia, uptake elsewhere remains limited due to regulatory, reimbursement, and infrastructural constraints. Recent randomized trials, large observational studies, and meta-analyses have [...] Read more.

Hemodiafiltration (HDF) combines diffusion and convection to enhance solute removal compared with hemodialysis (HD). While widely adopted in Europe and parts of Asia, uptake elsewhere remains limited due to regulatory, reimbursement, and infrastructural constraints. Recent randomized trials, large observational studies, and meta-analyses have renewed interest in HDF, particularly post-dilution HDF achieving convection volumes of ≥23 L/session, in which the most consistent clinical benefits have been observed. This review summarizes the mechanistic rationale, clinical effects, and safety profile of high-volume HDF. Compared with HD, HDF provides consistently superior removal of middle molecules, whereas clearance of protein-bound uremic toxins (PBUTs) remains constrained by albumin binding and compartmental redistribution, resulting in only modest or variable advantages. Emerging strategies aimed at releasing or capturing PBUTs may address this gap. Beyond solute removal, evidence suggests that HDF may lower micro-inflammation by enhanced clearance of inflammatory mediators and reduced immune activation. When delivered with adequate infrastructure, monitoring, modern equipment, ultrapure fluids, trained staff, appropriate vascular access, and anticoagulation, high-volume HDF has a favorable safety profile and is associated with fewer intradialytic hypotension (IDH) events and improved treatment tolerability. Available evidence has not identified a consistent excess risk of device- or infection-related complications, although comparative data remain limited. Overall, current evidence supports clinically relevant advantages of high-volume HDF under optimal treatment conditions. However, important challenges remain, particularly the limited removal of PBUTs, underscoring the need for further innovation in uremic toxin management. Full article

(This article belongs to the Special Issue Contemporary and Emerging Modalities of Kidney Assistance Therapy for Patients with Kidney Dysfunction Requiring Dialysis)

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12 pages, 3029 KB

Open AccessArticle

Single-Point Periorbital Botulinum Neurotoxin Injection and Its Potential Role in Hematoma Risk Reduction: A Technical Report

by Alexander Gardetto and Luis Thaler-Wolf

Toxins 2026, 18(8), 328; https://doi.org/10.3390/toxins18080328 - 28 Jul 2026

Abstract

Botulinum neurotoxin type A (BoNT-A) injection for lateral canthal rhytids is among the most frequently performed nonsurgical aesthetic procedures; however, procedure-related hematoma and ecchymosis remain frequent local adverse events associated with the multiple puncture sites required in the conventional three-point technique. This prospective [...] Read more.

Botulinum neurotoxin type A (BoNT-A) injection for lateral canthal rhytids is among the most frequently performed nonsurgical aesthetic procedures; however, procedure-related hematoma and ecchymosis remain frequent local adverse events associated with the multiple puncture sites required in the conventional three-point technique. This prospective single-center preliminary clinical observation evaluated a single-point intramuscular injection technique designed to reduce percutaneous vascular-layer penetrations while preserving treatment territory, dose, and intramuscular target localization. A total of 152 consecutive patients (104 female and 48 male) underwent bilateral treatment between May 2025 and May 2026 using a single skin entry point per side, positioned 10–15 mm caudal to and 10 mm temporal to the lateral canthus, followed by 18–20 mm intramuscular advancement within the lateral orbicularis oculi and continuous retrograde infusion of 12 U of onabotulinumtoxinA per side. Post-procedural assessment after injection demonstrated small unilateral ecchymosis in 2 of 304 treated sides (0.66%; Clopper–Pearson 95% Confidence Interval (CI), 0.08–2.36%). After the 10-day follow-up, no persistent bruising or other adverse events were reported, and all patients confirmed satisfaction with the treatment. At the structured one-year follow-up, the satisfactory reduction in dynamic lateral canthal rhytids and continued patient satisfaction with the treatment were confirmed, while a subset elected to return for a subsequent treatment session during the observation period. By reducing bilateral percutaneous venous-layer penetrations from six to two while maintaining dose and treatment-territory equivalence, the single-point intramuscular technique may represent a structurally coherent refinement of established BoNT-A treatment concepts for lateral canthal rhytids. Because this was an uncontrolled preliminary clinical observation, the observed rate should be interpreted as descriptive only; any potential reduction in hematoma risk remains hypothetical. Full article

(This article belongs to the Section Bacterial Toxins)

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14 pages, 2555 KB

Open AccessBrief Report

Multimodal Rehabilitation with Focal Vibration After Botulinum Toxin Injection in Ambulatory Children with Cerebral Palsy: A Proof-of-Concept Pilot Study

by Alessandro Picelli, Rita Di Censo, Ilaria Di Maria, Antonella Vangelista, Maria Vittoria Benetti, Nicola Smania, Valentina Varalta and Mirko Filippetti

Toxins 2026, 18(8), 327; https://doi.org/10.3390/toxins18080327 - 28 Jul 2026

Abstract

Cerebral palsy is a leading cause of childhood motor disability, and ambulatory children with spastic hemiplegia commonly present with dynamic equinus, calf muscle overactivity, and gait impairment. Evidence on focal vibration after botulinum toxin type A injection remains limited. This exploratory proof-of-concept pilot [...] Read more.

Cerebral palsy is a leading cause of childhood motor disability, and ambulatory children with spastic hemiplegia commonly present with dynamic equinus, calf muscle overactivity, and gait impairment. Evidence on focal vibration after botulinum toxin type A injection remains limited. This exploratory proof-of-concept pilot study assessed short-term changes following a four-week multimodal rehabilitation program incorporating focal vibration. In this uncontrolled single-group pre–post study, nine ambulatory children (mean age, 12.1 years), all classified as level II on the Gross Motor Function Classification System, received onabotulinumtoxinA injections into the affected gastrocnemius muscles, followed by twelve outpatient sessions combining focal vibration, conventional physiotherapy, and robotic gait training. Passive ankle dorsiflexion increased, calf muscle spasticity decreased, and selected gait parameters improved. Walking speed increased from 0.79 to 0.90 m per second, an absolute change of 0.11 m per second, comparable in magnitude to published estimates of clinically important change in ambulatory children with cerebral palsy. After Bonferroni correction, only walking speed and affected-limb stride length remained significant. No adverse events occurred. These findings describe outcomes associated with the combined program but do not establish the efficacy or independent contribution of focal vibration. Full article

(This article belongs to the Special Issue Botulinum Toxin in Neuro-Rehabilitation: Expanding Horizons in Spasticity and Beyond)

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28 pages, 4817 KB

Open AccessArticle

Discovery of Protein-Derived Candidate Anticancer Peptides from Toad Poison (ChanSu) Using an Integrated Proteomics and Bioinformatics-Guided Strategy

by Juan Chen, Bing Wang, Yingying Xie, Fei Xue, Li Shi, Yang Jiao and Yongqiang Lin

Toxins 2026, 18(8), 326; https://doi.org/10.3390/toxins18080326 - 27 Jul 2026

Abstract

Toad poison (ChanSu), a traditional animal-derived medicine, has long been used in East Asian medical systems for the treatment of inflammatory conditions and tumor-related disorders. While bufadienolides have been extensively investigated as its major bioactive constituents, the contribution of peptide components to its [...] Read more.

Toad poison (ChanSu), a traditional animal-derived medicine, has long been used in East Asian medical systems for the treatment of inflammatory conditions and tumor-related disorders. While bufadienolides have been extensively investigated as its major bioactive constituents, the contribution of peptide components to its antitumor effects remains largely unexplored. Here, we identified protein-derived candidate antiproliferative peptides from toad poison using an integrated proteomics and bioinformatics-guided strategy. Proteomic analysis identified 135 proteins, from which 2117 peptide sequences were generated via in silico digestion with trypsin and pepsin. Subsequent multi-step screening using PeptideRanker, AntiCP, iACP, and ACPred yielded twelve candidate peptides with predicted anticancer activity. Network pharmacology analysis suggested their potential involvement in cancer-related targets and pathways. ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) evaluation was subsequently used as a complementary assessment of drug-like and safety-related properties, further prioritizing four peptides for experimental validation, while molecular docking supported their interactions with key lung cancer-associated targets. In vitro assays demonstrated that three of the four prioritized peptides (WEAWN, NSQWG, and ACGVIGICQ) exhibited initial antiproliferative activity against human lung cancer A549 cells, though these findings should be interpreted with caution given the absence of a positive control in the MTT assay. This study provides preliminary evidence suggesting that protein-derived peptide candidates from toad poison may possess antiproliferative potential, representing an early systematic exploration of its previously unexplored peptidome as a source of candidate antiproliferative peptides warranting further pharmacological investigation. The integrated strategy presented here offers an efficient approach for the discovery of bioactive peptides from animal-derived traditional medicines. Full article

(This article belongs to the Section Animal Venoms)

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23 pages, 3754 KB

Open AccessReview

Anti-Mycotoxin Agents in Pig Production Through the Lens of Circular Economy and Life Cycle Assessment: A Comprehensive Review

by Georgios I. Papakonstantinou, Christos Eliopoulos, Dimitrios Arapoglou, Nikolaos Tsekouras, Katerina Manolakou, Labrini V. Athanasiou, Dimitrios Gougoulis, Soultanidis Aris and Vasileios G. Papatsiros

Toxins 2026, 18(8), 325; https://doi.org/10.3390/toxins18080325 - 27 Jul 2026

Abstract

Mycotoxin contamination of cereal-based pig feeds is a persistent threat to swine health and global pork safety. Pigs are uniquely susceptible to mycotoxicosis, and the principal toxins—aflatoxins (AFs), deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FUMs), and ochratoxin A (OTA)—cause impaired growth, reproductive failure, hepato- [...] Read more.

Mycotoxin contamination of cereal-based pig feeds is a persistent threat to swine health and global pork safety. Pigs are uniquely susceptible to mycotoxicosis, and the principal toxins—aflatoxins (AFs), deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FUMs), and ochratoxin A (OTA)—cause impaired growth, reproductive failure, hepato- and nephrotoxicity, immunosuppression, and carry-over residues in edible tissue. Anti-mycotoxin feed additives, including mineral adsorbents, biotransforming agents, and multicomponent mycotoxin-detoxifying agents (MMDAs) combining clays, phytogenic antioxidants (curcumin, silymarin), and postbiotics (yeast cell wall, hydrolyzed yeast), constitute the primary mitigation strategy. Beyond animal health, these agents play underappreciated roles in both the circular economy (CE)—enabling safe valorization of by-product feed ingredients and reducing feed waste—and in the life cycle assessment (LCA) sustainability profile of pork production, where feed conversion ratio (FCR) drives 22–95% of greenhouse gas emissions. Field-based in vivo studies in sows and weaned piglets, integrating plasma oxidative stress biomarkers (TBARS, protein carbonyls, total antioxidant capacity) with performance and reproductive endpoints, provide the most comprehensive real-world evidence for MMDA efficacy to date. This review synthesizes mycotoxin toxicology, anti-mycotoxin agent mechanisms, clinical evidence, and the CE/LCA sustainability framework, proposing integrated mycotoxin management as a One Health imperative in modern swine production. Full article

(This article belongs to the Section Mycotoxins)

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