Biol. Life Sci. Forum, 2023, IECT 2023

The growing interest in herbal and alternative medicines demands information on the toxicity risk assessment of the various plant extracts used in traditional medicines. The rich presence of sesquiterpene lactone, a potentially toxic phytochemical, in Tithonia diversifolia necessitates the toxicological evaluation of its biologically active constituents. This study evaluated the in vivo sub-chronic toxicity of the moderately polar fractions of T. diversifolia in a rat model. The ethyl acetate soluble portion from the methanol extract was separated using the vacuum liquid chromatographic method. Three dose levels—an observed adverse effect level (OAEL) of 2000 mg/kg, a no-observed adverse effect level (NOAEL) of 80 mg/kg, and an intermediate dose of 500 mg per kg body weight of rats per day—were selected for a 28-day period of repeated dosing for the sub-chronic toxicological evaluation. The LC-MS dereplication of the active fractions showed the presence of sesquiterpene lactones such as diversifolin, diversifolin methylether, tagitinin A, tagitinin C–F, woodhousin, and orizatin, as well as many unidentified peaks. There was a significant reduction (p < 0.05) in the weights of the rats dosed with OAEL and their food consumption of the fraction during week 1, which normalized during the subsequent weeks of the study. The histopathological examination showed mild necrosis and degeneration of hepatocytes in the centrilobular areas of the rats treated with OAEL of the active VLC fraction. There were no T. diversifolia-related adverse toxicological events in rats receiving 2000 mg/kg/day when dosed orally for 28 days. Full article

In this work, we studied the effects of various chemicals on P. physalis nematocyst discharge and the composition of the released venom. The exposure of nematocyst to K⁺ and Na⁺ induced a massive discharge in a short time of exposure. Conversely, the Ca²⁺ ions apparently resulted in an inhibitory effect. The electric stimulation was shown to be a reproducible and effective way to induce nematocyst discharge in a few seconds. The SDS-PAGE profile of the venom proteins released revealed a similar pattern, having a broad MW distribution and wide bands with 40, 25 and 20 kDa. The released venom exhibited proteolytic activities that are inhibited by PMSF and EDTA. The present study provides an overview of discharge sensation and venom released of P. physalis nematocysts and could contribute for future venom proteomics research efforts. Full article

In several cultures, black cumin, also known as Nigella sativa, has long been used medicinally. Recent research has revealed that this plant has potent anti-inflammatory and antioxidant qualities, making it a possible treatment for several medical conditions. Additionally, because of its capacity to detoxify the liver and protect it from harm, Nigella sativa has demonstrated positive results as an antidote for poisoning. Consumption of hazardous substances by accident or planned poisoning are two prevalent causes of poisoning. The liver is the primary organ in detoxification. According to studies, Nigella sativa can help the liver operate better and defend it from toxins’ harmful effects. Additionally, it has been demonstrated that Nigella sativa protects against heavy metal toxicity. Thymoquinone and thymohydroquinone, the plant’s active components, have been demonstrated to bond with heavy metals and stop the body from absorbing them. Further studies are needed to evaluate the efficacy and safety of Nigella sativa as an antidote for poisoning cases. Nigella sativa presents an interesting natural alternative for treating poisoning cases, potentially complementing traditional medical approaches. The main goal of this review is to explore the potential application of Nigella sativa as an antidote for poisoning cases. The article discusses the plant’s strong ability to detoxify and protect the liver. The review highlights preclinical studies that have shown promising results but also emphasizes the need for further clinical trials to determine the efficacy and safety of Nigella sativa as a natural alternative for treating poisoning cases. Full article

In Portugal, there are four venomous species, the horned viper (Vipera latastei), seoane (Vipera seoanei), rat snake (Malpolon monspessulanus), and the hooded snake (Macroprotodon brevis ibericus), and in the UK, there is one: the common European adder (Vipera berus). Snake venom is a complex mixture of toxins whose composition varies depending on the family, genera, species, and even subspecies. In Europe, particularly Portugal, there are no published data on the frequency of these types of incidents, but it is estimated to be high, mainly in dogs. Thus, to characterize the injuries caused by the bite of venomous snakes in domestic animals, the authors describe cases in dogs, cats, and goats with a suspected snakebite. Animals presented wounds compatible with snakebites, with two points 1 to 1.8 cm apart that could be noted on the head or limbs. The main clinical signs observed included pain, oedema, and necrosis. From the animals that died, a post-mortem examination revealed subcutaneous and muscular necrosis and hemorrhages of surrounding tissues, including muscles and organic hemorrhages. The severity of envenomation depends on the quantity of inoculated venom; the species, age, size, and previous state of health of the bitten animal; the bite location; and post-bite excitability. With this study, the authors hope to help improve the knowledge regarding snakebites in Europe. Full article

Macroalgae and microalgae are two sources of natural ingredients that are of interest for industrial sectors because of their multiple potential applications. Algal extracts are known to be rich in valuable compounds such as fatty acids, minerals, and secondary metabolites, among others. Bioactivities disclosed for these biocompounds include their antifungal capacity via the growth inhibition of foodborne pathogens and spoilage microorganisms such as Aspergillus sp., Fusarium sp., or Penicillium sp. Apart from their antifungal activity, algal extracts can minimize the toxicity of mycotoxins via two molecular mechanisms: their antioxidant and their chelating properties. Antioxidants may reduce the toxic effect of mycotoxins whereas algae bioadsorption, due to their rich composition in macromolecules (polysaccharides or proteins), may have the capacity to bind mycotoxins, prevent their metabolism, and facilitate their systemic release. These three mechanisms involved in the antifungal activity of micro- and macroalgal extracts will be critically assessed along this review in order to disclose their potential application as key tools in the food industry and public health. The use of algae extracts as antifungals and detoxifiers may underline their use of natural additives with no side effects associated that may represent an alternative to extend foodstuffs′ shelf-life and prevent the occurrence of mycotoxins, especially aflatoxin B1, aflatoxin M1, and ochratoxin A. Therefore, this review is aimed to evaluate the multiple benefits that algal extracts may provide to reinforce food safety, reduce food disposal, and prompt the utilization of underused biomass such as algae. Full article

Patulin (PAT) is one of the most common mycotoxins produced by Penicillium and Aspergillus species and is often associated with fruits and fruit by-products, mostly apple derivatives, although it has been detected in infant food and cereals. This toxin has shown a mutagenic and carcinogenic effect. Thus, the development of rapid and accurate methods for PAT detection is of utmost importance. Currently, the most widely used methods for the analysis and detection of mycotoxins are based on chromatography, including liquid chromatography (HPLC) and gas chromatography coupled to a mass detector (GC–MS), since these techniques provide high precision, selectivity, and sensitivity. In this work we show the preliminary results in the development of a GC–MS method for the detection of PAT without derivatization. Usually, the detection of mycotoxins by GC–MS needs a derivatization of all non-volatile and polar compounds. This is one drawback of gas chromatography compared to the liquid chromatographic technique for the determination of mycotoxins. In this sense, the PAT monitoring method by GC–MS proposed here is an alternative and useful technique to maintain high-quality foodstuffs and to ensure food safety. Full article

Fumonisins are secondary metabolites of mold whose presence has been proven in water. Since fumonisin B₁ (FB₁) is highly toxic and has dangerous effects on the health of living organisms, in this study, the influence of various water matrices on the effectiveness of UV and UV/H₂O₂ treatments for its removal was investigated. Different types of water (Danube River, tap and ground water) were simulated by addition of humic acid and the main ions for each type of water into ultrapure water (UPW). The results showed lower FB₁ removal efficiency in simulated water samples compared to that of UPW. Full article

Pteridium aquilinum (L.) Kuhn, commonly known as bracken fern, is a cosmopolitan plant species absent only from polar and desert regions. Worldwide, Pteridium aquilinum has been rapidly spreading due to the neglect of agricultural fields and the growing wave of forest fires. This plant has several toxic components such as thiaminases, brains, active glucosides, beta-glucopyranosides and ptaquloside in its constitution that can be lethal to animals. The animals do not normally choose it as food, except in situations of food shortage, in periods of drought, when the ferns are still green or when the animals graze in places with large amounts of the plant in question. In order to revisit the major syndromes caused by P. aquilinum’s major toxins, the authors present several cases of acute and chronic ruminant poisoning associated with ingesting P. aquilinum in Portugal in sheep and cattle. The primary associated syndromes observed in animals that consume this plant were thiamine deficiency, blindness in sheep, acute or subacute poisoning with bone marrow depression, and consequently, leucopenia and bladder tumors in cattle. Given the economic impact of the diseases caused by the toxins of this plant, and the possible public health implications, it is imperative to minimize these syndromes in animals and establish measures for their prevention. Full article

Bacillus thuringiensis (Bt) produces, during its vegetative growth, some insecticidal proteins that are secreted and diluted into the culture medium. These proteins are commonly known as vegetative insecticidal proteins (Vips), and include binary Vpb/Vpa proteins (formerly known as Vip1/Vip2) with coleopteran activity; Vip (formerly Vip3) with activity against lepidopterans; and Vpb4 proteins (formerly Vip4), also with coleopteran activity. Vip proteins are highly toxic to different species of lepidopteran pests; however, the difficulty in producing them in a concentrated form has not allowed their development as formulated biopesticides, and they are relegated to only being produced in transgenic crops. In this work, we demonstrated that the gene encoding the Vip protein Vip3Ag4 could be successfully expressed in an asporogenic strain of Bacillus megaterium using (D)-xylose as a low-cost inductor. Under certain conditions (37 °C and induction with 0.5% w/v xylose), active Vip3Ag4 protein was primarily produced in soluble form, remaining encapsulated within the cell wall of B. megaterium. After treatment with lugol (1% for 4 h), induced cells were completely killed (fixed) but maintained the functional Vip3Ag4 protein, which resulted in above 95% mortality against first-instar larvae of Chrysodeixis chalcites, Helicoverpa armigera, Spodoptera exigua, S. frugiperda, S. littoralis and Trichoplusia ni. The fact that the recombinant Vip3Ag4 protein was successfully produced in a soluble and an active form in this bacterium (with a low-cost inductor) suggests that B. megaterium is one of the hosts of choice for the production of sprayable formulations in “killed-microbial pesticides”, based on Vip3 proteins from Bt. Full article

The focus of this study is to investigate the gene expression related to the formation of neutrophil extracellular traps (NETs) stimulated by Calloselasma rhodostoma L-amino acid oxidase (Cr-LAAO). LAAOs found in snake venom have been shown to activate human neutrophils, leading to the production of reactive oxygen species (ROS), chemotaxis, phagocytosis, and the release of pro-inflammatory cytokines and lipid mediators. Additionally, it has been found that Cr-LAAO activates NADPH oxidase, which is responsible for the release of ROS. Neutrophils are known to release NETs to combat pathogens, and this process involves the migration of DNA from the nucleus to the cytoplasm, where it merges with the contents of the granules to produce NETs. Initially, the formation of NETs was associated with cell death, and this process was known as NETosis. However, two forms of NETosis have now been identified: classical or suicidal NETosis, which results in cell death, and vital NETosis, in which the cell retains its viability and many of its effector functions. To evaluate the gene expression related to the formation of NETs, a microarray assay was performed on human neutrophils stimulated with Cr-LAAO. The results show that Cr-LAAO stimulates the expression of important genes for the formation of NETs, such as TXNIP, FOXO3, PPARA, ELANE, CXCL8, and PADI4. This is the first report that shows the transcriptome of neutrophils related to Cr-LAAO-stimulated NETosis, which may lead to the development of local inflammatory effects observed in snakebite victims. Full article

Russell’s viper (Daboia siamensis) is a medically important snake in Myanmar due to its high morbidity and mortality. The genome of Myanmar Russell’s viper had not been sequenced until recently. Hence, RNA sequencing has been used to predict genes that encode this snake’s toxins. This can lead to deeper insights into the pathogenesis of envenoming and potential drug discovery. Venom glands were dissected from four adult D. siamensis specimens (two males and two females) provided by a local Myanmar snake farm. The mRNA was extracted and sequenced on the Illumina HiSeq platform, then assembled de novo using the Trinity software. Candidate toxin genes were identified using the Venomix pipeline, and their expression levels were calculated using RSEM software. The identified toxin candidates were aligned with previously described venom proteins using Clustal Omega. Candidate venom transcripts were classified into 23 toxin gene families, which included 53 unique transcripts identified as full-length sequences. Among them, 28 full-length sequences represented the eight newly identified toxin gene families in D. siamensis, including neprilysin (2), cystatin (5), waprin (1), vipericidin (1), veficolin (1), endothelial lipases (9), vespryn (ohanin) (8), and three-finger toxins (1). Their expression levels were found to be moderate to low (TPM = 1.49 to 213.37). The majority of the toxin candidates resembled typical elapid toxins, which usually exhibit neurotoxic activities and tissue damage. A smaller proportion of candidate toxin transcripts were predicted to display antimicrobial activity and anti-metastatic effects. Our results suggest their functional activities. They should be studied further for potential therapeutic applications. Full article

Scorpion venom possesses a lethal sting and potential medicinal properties, making it a captivating natural elixir. Our study aimed to unravel the composition of Androctonus scorpionvenoms in Morocco. Using electrospray mass spectrometry and high-performance liquid chromatography (HPLC), we conducted a thorough analysis to gain detailed insights into venom composition. The data unveiled a wide range of molecular weights, influenced by factors such as species, genus, location, age, sex, and diet. Short toxins (2000–4000 Da) predominated in the venoms, effectively blocking K⁺ channels, while larger molecular weights (>4000 Da) corresponded to long toxins that modulate Na⁺ channels. Furthermore, we made intriguing discoveries of previously unidentified peptides (<2000 Da). This study provides valuable knowledge, shedding light on the intricate composition of scorpion venoms. Full article

Inoserp^TM PAN-AFRICA is a polyvalent F(ab’)₂ antivenom that has been specifically developed for the management of snakebite envenomation in sub-Saharan Africa. The antivenom provides a very large coverage of medically important species in sub-Saharan Africa, with at least 24 species covered. This review presents all clinical data available on the use of Inoserp^TM PAN-AFRICA in sub-Saharan Africa since it was introduced in 2012. The antivenom has been used in more than 20 countries from west to east Africa, with approximately 200,000 vials distributed through marketing approvals, special import permits, and organizations such as armies or NGOs. Four clinical studies have been performed in five countries of West and Central Africa, encompassing 22 clinical sites and involving 676 patients exposed to Inoserp^TM PAN-AFRICA. Patients were rather young, with a median age ranging from 18 to 38 years, and a great majority were males, with a sex ratio (M/F) ranging from 2.7 to 4.5, according to the study. Snakebite envenomation was representative of the sub-Saharan African region with mostly hemorrhagic and cytotoxic but also neurotoxic syndromes. Overall, patients received an average dose of two to three vials, which was enough to obtain a rapid control of symptoms in the great majority of cases. The observed lethality rate was 0% to 4.4%, depending on the study. Adverse events were mostly of mild or moderate intensity and have been reported in 5 to 11% of patients. Other available data, such as published reports of patient cases, as well as the most updated pharmacovigilance surveillance report in 2022, have been used to complete this review. Overall, Inoserp^TM PAN-AFRICA benefits from a large experience in sub-Saharan Africa. Clinical data available consistently show a very good efficacy and safety profile of the antivenom. Full article

The EU Rapid Alert System for Food and Feed (RASFF) is a tool for the rapid exchange of information on food and feed safety issues between EU member states and the European Commission, destined to notify food safety authorities about products posing a risk to human health. Emerging marine and freshwater toxins and their impact on human health and aquatic ecosystems have become a growing concern in the recent years. This is also reflected in the RASFF notifications shared by European countries during the last decade, with the occasional appearance of relevant records. In this work, RASFF notifications related to emerging marine and freshwater toxins from 2012 to date were retrospectively analyzed to discover the patterns of their appearance, as well as to explore their relationship with concurrent occurrence data and/or risk assessment advancements in the field. A total of fifteen notifications involving emerging marine and freshwater toxins were found, which included: ten on ciguatoxins in fish, three on tetrodotoxins in bivalve molluscs, one on microcystins in algae powder, and one on pinnatoxins in bivalve molluscs. This study contributes to a better understanding of the reasons behind the RASFF’s provision of notifications on emerging toxins in EU countries. Full article

Using degenerated primers (LC1–LC2c) and two novel primer pairs, namely (KSLB–LC6) for Aspergillus niger and (AFl1F–LC2) for Aspergillus tubingensis, created for the acyl transferase (AT) and the KS sequences of fungal PKSs genes, a 700 pb PCR-derived DNA fragment was isolated from Aspergillus carbonarius, Aspergillus niger, and Aspergillus tubingensis. Testing was performed on DNA from most of the black Aspergillus species currently known to exist. This article describes the identification and characterisation of a portion of a novel putative OTA-polyketide synthase gene in A. tubingensis “AT Pks,” A. niger “AN Pks,” and A. carbonarius “AC Pks”. Phylogenetic methods were used to align and evaluate the sequences. The study’s primers demonstrated broad application, and several Aspergillus species from the section Nigri, particularly A. niger and A. tubingensis, were amplified satisfactorily. Predicted amino acid sequences known as “AC Pks” showed 66–81% similarity to several polyketide synthase genes, whereas “AN Pks” and “AT Pks” showed 68–71% and 81–97% similarity, respectively. The AT and KS sequences were linked to PKSs engaged in various mycotoxin production routes, including ochratoxin A, and they seemed to be specific for a specific kind of fungal PKSs. The sequences that have been reported in this paper are particularly useful in finding new fungal PKS gene clusters. Full article