Effects of the cyclopeptide mycotoxin destruxin A on the Malpighian tubules of Rhodnius prolixus (Stål)

The production of peptide toxins by entomopathogenic fungi during the infection process plays critical roles in pathogenesis. To gain insight into the mechanism of action of these mycotoxins on insect internal organs, we have evaluated the effects of destruxin A, a cyclic peptide produced by Metarhizium anispliae, on Rhodnius prolixus Malpighian tubules measuring fluid secretion rate, transepithelial electrical potential (TEP), pH and ion composition of secreted fluid, and ATP content. Destruxin A dramatically inhibited fluid secretion rate on tubules stimulated by 5-Hydroxytryptamine (5-HT) or cAMP. The calculated IC₅₀ for destruxin A on 5-HT-stimulated tubules was 3 × 10⁻⁷ M. Fluid secretion rate by Malpighian tubules exposed for 20 min to 10⁻⁶ M destruxin A recovered completely when tubules were washed with saline; however, when tubules were exposed to 5 × 10⁻⁶ M destruxin A the fluid secretion rate only partially recovered upon wash off. The use of Ca²⁺-free saline or addition of the calcium channel blocker CoCl₂ to the bathing saline did not interfere with the effects of destruxin A, and neither did the modification of intracellular calcium by TMB-8. Measurement of TEP of tubules challenged with 5-HT after preincubation for 10 min in saline containing 10⁻⁶ M destruxin A showed that the second and third phases of the typical triphasic response to 5-HT were disrupted. Likewise, the positive shift in TEP in response to 5-HT in chloride-free bathing saline was significantly reduced when tubules were preincubated for 10 min in 10⁻⁶ M destruxin A. The pH of the secreted fluid, but not the Na⁺ or K⁺ concentration, increased significantly when 5-HT-stimulated tubules were exposed to 10⁻⁶ M destruxin A. The ATP content was not significantly different when tubules stimulated with 5-HT were exposed to destruxin A. Taken together, these results show that destruxin A, without interfering with the intracellular ATP production, strongly inhibits fluid secretion rate by the Malpighian tubules of R. prolixus. Changes in properties of the TEP suggest that one of the target sites for this peptide toxin might be associated with inhibition of the apical V-type H⁺ ATPase of tubule cells.     

Effects of the mycotoxin citrinin on micronucleus formation in a cytokinesis-block genotoxicity assay in cultured human lymphocytes

Some mycotoxins produced by microfungi are capable of causing disease and death in animals and humans. In the present study, the mycotoxin citrinin (CTN) was evaluated for its genotoxic effects to human peripheral blood lymphocytes from six different individuals. Lymphocyte cultures were treated for 48 h with CTN at six different concentrations between 10 and 100 microM. Lymphocyte cultures were also incubated with 0.1 microM mitomycin c (MMC) as a positive control, and 0.5% absolute ethanol as a vehicle control.CTN caused a significant concentration-dependent increase in micronucleus (MN) frequency in human lymphocytes. At the 60 microM, 80 microM and 100 microM concentrations, CTN was found to induce MN in cytokinesis-blocked lymphocytes in comparison with negative controls (P = 0.014). All the CTN concentrations also led to a clear decrease in the percentages of binucleated/mononucleated cells (P = 0.014). These results indicate that CTN at high concentrations is genotoxic in cultured human lymphocytes.     

Effects of the mycotoxin fumonisin B(1) on cell death in human kidney cells and human lung fibroblasts in primary culture

Fumonisins are mycotoxins produced by Fusarium verticillioides. The toxic effects of fumonisin B(1) (FB(1)) at the cellular level consist of a mixture of both necrosis and apoptosis. We studied the effect of FB(1) in human lung fibroblasts (NHLF) and human kidney epithelial cells (RPTEC) in primary culture. Apoptotic and necrotic cell death, collagen and fibronectin secretion were determined mainly after 14 days' exposure. The protein content of NHLF and RPTEC cells was slightly increased after 14 days' exposure to low FB(1) concentrations (0.1 or 1 microm). Caspase-3 activity tended to increase in NHLF and to decrease in RPTEC cells with higher FB(1) concentrations after 14 days' exposure. LDH release was slightly decreased in both cell types after 14 days. Collagen I and III secretion was enhanced in NHLF cells. Collagen III was decreased in RPTEC. Collagen IV was not changed in both cell types. Fibronectin secretion was uninfluenced in RPTEC and interim increased in NHLF. Furthermore LC-MS/MS studies did not give any hints for a metabolism of FB(1). Therefore, the main risk of prolonged FB(1) exposure seems to be altered collagen secretion pattern.     

Characterization of voltage-dependent calcium channel blocking peptides from the venom of the tarantula Grammostola rosea

Voltage-dependent calcium channel blocking peptides were purified and sequenced from the venom of the tarantula, Grammostola rosea. cDNAs encoding the peptide sequences were cloned from the venom gland cDNA library. The electrophysiological effects of the peptides on several types of voltage-dependent calcium channels were evaluated using a Xenopus laevis oocyte expression system. A peptide contained in one of the HPLC peak fractions inhibited P/Q type voltage-dependent calcium channels (Ca_v2.1). The amino acid sequence of this peptide is identical to that of ω-grammotoxin SIA. A peptide from another discrete peak, which is identical to GsAFII except for one tryptophan residue in the C-terminus, inhibited L-type voltage-dependent calcium channels (Ca_v1.2). A novel peptide, named GTx1-15 (Accession number, AB201016), shows 76.5% sequence homology with the sodium channel blocker phrixotoxin 3, however, GTx1-15 preferentially inhibited T-type voltage-dependent calcium channels (Ca_v3.1). In silico secondary and tertiary structure prediction revealed that GTx1-15 and sodium channel blockers such as hainantoxin-IV, phrixotoxin 3, and ceratotoxin 2 show very similar β-strand composition, distribution of Optimal Docking Areas (continuous surface patches likely to be involved in protein-protein interactions), and surface electrostatic potential. These findings suggest that these peptide toxins evolved from common ancestors by gene duplication to maintain surface atmospheres appropriate for interaction with low-voltage-dependent ion channels.     

Influence of mycotoxins and a mycotoxin adsorbing agent on the oral bioavailability of commonly used antibiotics in pigs

It is recognized that mycotoxins can cause a variety of adverse health effects in animals, including altered gastrointestinal barrier function. It is the aim of the present study to determine whether mycotoxin-contaminated diets can alter the oral bioavailability of the antibiotics doxycycline and paromomycin in pigs, and whether a mycotoxin adsorbing agent included into diets interacts with those antibiotics. Experiments were conducted with pigs utilizing diets that contained blank feed, mycotoxin-contaminated feed (T-2 toxin or deoxynivalenol), mycotoxin-contaminated feed supplemented with a glucomannan mycotoxin binder, or blank feed supplemented with mycotoxin binder. Diets with T-2 toxin and binder or deoxynivalenol and binder induced increased plasma concentrations of doxycycline administered as single bolus in pigs compared to diets containing blank feed. These results suggest that complex interactions may occur between mycotoxins, mycotoxin binders, and antibiotics which could alter antibiotic bioavailability. This could have consequences for animal toxicity, withdrawal time for oral antibiotics, or public health.     

Influence of different soluble dietary fibers on the bioaccessibility of the minor Fusarium mycotoxin beauvericin

Beauvericin (BEA) is a bioactive compound produced by the secondary metabolism of several Fusarium strains and is known to have various biological activities.     

A molecular mechanism for the toxic action of moniliformin,a mycotoxin produced by fusarium moniliforme

Molecular mechanisms of the toxic action of moniliformin, an extremely toxic fungal metabolite produced by Fusarium moniliforme, were investigated. Exceedingly low concentrations of moniliformin (<5 μM) selectively inhibited mitochondrial pyruvate and α-ketoglutarate oxidations by 50 per cent. It is suggested that these inhibitory effects could constitute the major molecular mechanism of toxic action and could largely, if not exclusively, account for the clinical symptoms of moniliformin poisoning.     

Temperature dependence of the effects of isoprenaline, aminophylline and calcium ionophores on the isometric contraction of the isolated hemidiaphragm of the rat.

Both parameters of isometric contraction, Td and dT/dt max, of the isolated hemidiaphragm of the rat, as measured during the action of isoprenaline in the course of direct electrical stimulation, were significantly affected by lowering the temperature of the bath to 18°C. The action of isoprenaline was only depressed, whereas the effect of aminophylline was completely blocked by this procedure. The effect of calcium chloride in antagonizing the blocking action of di-Na-EDTA was even stronger at 18°C than at 36°C. Calcium ionophores Ro-2-2985 and A 23187 produced an increase in Td and dT/dt max when used in lower concentrations, but higher concentrations regularly produced depression or block of the isometric contraction. The increasing effect of Ro-2-2985 on Td and dT/dt max was completely reversed by lowering the temperature to 18°C. It was shown that at 18°C neither calcium nor isoprenaline antagonized the blocking action of verapamil on Td and dT/dt max, thus indicating the alteration of calcium channels at low temperatures. A 23187 potentiated the action of isoprenaline on Td and dT/dt max, whereas Ro-2-2985 blocked it. Similarly, the inrcreasing effect of db-cAMP on Td and dT/dt max was reversed by Ro-2-2985. It is concluded that both lowering the temperature of the medium and the calcium ionophores produce changes in the response to isoprenaline and aminophylline, during direct electrical stimulation, in a way which is compatible with the view that the interaction between calcium and cAMP system is essential to the isometric contraction of skeletal muscle.     

Toxicity and differential protein analysis following destruxin A treatment of Spodoptera litura (Lepidoptera: Noctuidae) SL-1 cells

The cytotoxicity of a destruxin A (DA) treatment of Spodoptera litura SL-1 cells was investigated. An MTT assay showed that DA was highly toxic to SL-1 cells in a concentration- and time-dependent manner. The IC₅₀ values of DA, after 24 h and 48 h of treatment, were 17.86 μg/mL and 7.80 μg/mL, respectively. Under inverted phase contrast microscopy (IPCM), it was found that prolonged treatment with DA could induce cell rounding, cellular membrane shrinking, formation of apoptotic bodies, vacuole appearance and cytoplasm leak out. Apoptosis induced by DA was further confirmed by fluorescence microscopy (FM) and flow cytometry (FCM) studies. SL-1 cells entered early apoptosis following a treatment with 2.5 μg/mL DA and entered late apoptosis following a treatment with increasing concentrations of DA. Furthermore, two-dimensional gel electrophoresis (2-DE) analysis was used to identify 22 proteins which were differentially expressed (≥2-fold difference) between control cells and DA-treated cells, and the expression level of these proteins was significantly different between the treated and untreated cells. Our results suggest that these differentially expressed proteins may help explain the diverse biological effects caused by the destruxin A treatment of cells; additionally, some of the identified proteins may have roles in SL-1 cellular proliferation and apoptosis.     

A biochemical characterization of the major peptides from the Venom of the giant Neotropical hunting ant Dinoponera australis

Venom from the “false tocandira” Dinoponera australis, a giant Neotropical hunting ant, paralyzes small invertebrate prey and induces a myriad of systemic effects in large vertebrates. HPLC/DAD/MS analyses revealed that the venom has over 75 unique proteinaceous components with a large diversity of properties ranging in size, hydrophobicity, and overall abundance. The six most abundant peptides, demonstrative of this diversity and hereafter referred to as Dinoponeratoxins, were de novo sequenced by exact mass precursor ion selection and Edman degradation. The smallest peptide characterized, Da-1039, is hydrophilic and has similarities to vasoactive peptides like kinin and bombesin. The two largest and most abundant peptides, Da-3105 and Da-3177, have a 92.9% identity in a 28 residue overlap and share ∼50 of their sequence with ponericin G2 (an antimicrobial from another ponerine ant Pachycondyla goeldii). One peptide, Da-1585, is a hydrophilic cleavage product of an amphipathic peptide, Da-2501. The most hydrophobic peptide, Da-1837, is amidated (a PTM observed in one half of the major peptides) and shares homology with poneratoxin, a sodium channel modifier found in the bullet ant Paraponera clavata. This study is the first examination of potential pharmacophores from venom of the genus Dinoponera (Order: Hymenoptera).     

Effects of anticonvulsants on the in vivo and in vitro release of gaba

The actions of various anticonvulsant compounds on GABA release in vivo and in vitro were studied. An in vivo, superfusion of sensorimotor cerebral cortex was employed and drugs were administered either by intraperitoneal injection, or in superfusion fluid and release of endogenous amino acids was measured. The in vitro method involved superfusion of synaptosomes, with drugs dissolved in superfusate, with monitoring of the release of pre-loaded [U¹⁴C]-GABA. Two alkyl-GABA analogues, γ-acetylenic GABA and γ-vinyl GABA caused enhanced release of GABA to superfusate both in vivo and in vitro. However, phenobarbitone, diphenyl hydantoin, sodium n-dipropyl acetate and carbamazepine were without effect on GABA release in either test system. Taurine caused no detectable GABA release in vivo, or from purified synaptosomes in vitro, but did stimulate release in vitro, from crude synaptosome preparations containing mitochondria in large quantities, though histidine and leucine were equally effective.     

Protonectin (1-6): A novel chemotactic peptide from the venom of the social wasp Agelaia pallipes pallipes

Peptides constitute the largest group of Hymenoptera venom toxins; some of them interact with GPCR, being involved with the activation of different types of leukocytes, smooth muscle contraction and neurotoxicity. Most of these toxins vary from dodecapeptides to tetradecapeptides, amidated at their C-teminal amino acid residue. The venoms of social wasps can also contains some tetra-, penta-, hexa- and hepta-peptides, but just a few of them have been structurally and functionally characterized up to now. Protonectin (ILGTILGLLKGL-NH₂) is a polyfunctional peptide, presenting mast cell degranulation, release of lactate dehydrogenase (LDH) from mast cells, antibiosis against Gram-positive and Gram-negative bacteria and chemotaxis for polymorphonucleated leukocytes (PMNL), while Protonectin (1-6) (ILGTIL-NH₂) only presents chemotaxis for PMNL. However, the mixture of Protonectin (1-6) with Protonectin in the molar ratio of 1:1 seems to potentiate the biological activities dependent of the membrane perturbation caused by Protonectin, as observed in the increasing of the activities of mast cell degranulation, LDH releasing from mast cells, and antibiosis. Despite both peptides are able to induce PMNL chemotaxis, the mixture of them presents a reduced activity in comparison to the individual peptides. Apparently, when mixed both peptides seems to form a supra-molecular structure, which interact with the receptors responsible for PMNL chemotaxis, disturbing their individual docking with these receptors. In addition to this, a comparison of the sequences of both peptides suggests that the sequence ILGTIL is conserved, suggesting that it must constitute a linear motif for the structural recognition by the specific receptor which induces leukocytes migration.     

Vejovine, a new antibiotic from the scorpion venom of Vaejovis mexicanus

Multidrug resistant bacterial infections are one of the most important health problems in recent years. Resistance to conventional antibiotics limits the therapeutic options causing increase rate in morbid-mortality in hospitals. Therefore, new antibacterial agents with new bacterial targets have been searched and found in many different sources, including scorpion venom and scorpion hemolymph. Here, we report a new anti-microbial peptide named Vejovine. This peptide was isolated from the venom of the Mexican scorpion Vaejovis mexicanus by two steps of reversed phase high performance liquid chromatography (RP-HPLC). It is composed of 47 amino acid residues with no cysteine residues in its sequence, with a molecular weight of 4873 Da. The chemical synthesis of Vejovine was performed by the solid phase method of Merrifield, using fluoren-9-ylmethoxycarbonyl (Fmoc)-amino acids. Both the native and synthetic peptides were shown to have essentially the same activity. Vejovine inhibits growth of clinical isolates of Gram-negative multidrug resistant (Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Acinetobacter baumanii) causing nosocomial infections with a minimum inhibitory concentration (MIC) of 4.4 μM up to 50 μM. This peptide has also hemolytic activity against human erythrocytes with a HC₅₀ value of 100 μM. A cDNA library of the venomous gland of this scorpion provided material for cloning the gene encoding Vejovine. This peptide is a new type of antibiotic, showing less than 50% similarity to other known scorpion peptides. Vejovine is a candidate to be used as a leading compound for future development of an effective peptide against multidrug resistant bacteria.     

Effects of the mycotoxin ochratoxin A and some of its metabolites on human kidney cell lines.

The modulations of complement-regulating surface proteins on a human embryonic and a renal carcinoma cell line are described regarding the effects of ochratoxin A and some of its metabolites on the surface markers CD46, CD55 and CD59. Membrane integrity, cell proliferation and metabolic activity were reduced to different extents, depending on the kind of mycotoxin and the dosage, which was ranging from 10 to 1000 ng/ml. The number of cells carrying surface markers was suppressed significantly at 1000 ng/ml, in some cases even at 100 ng/ml, whereas the intensity of receptor expression on the positive cells was found to be stimulated. The fraction RE2 (OTC) isolated from an OTA-containing crude toxin surpassed the effects of all other ochratoxin metabolites. Apart from well-known cytotoxic and genotoxic effects modulation of cell surface marker expression by low concentrations of OTA and OTC deserves more attention with regard to its immuno-pathogenic importance. Furthermore, occurrence and impact of the mycotoxin OTC should be studied more into detail.     

A Review on Mycotoxins and Microfungi in Spices in the Light of the Last Five Years

Spices are imported worldwide mainly from developing countries with tropical and/or subtropical climate. Local conditions, such as high temperature, heavy rainfall, and humidity, promote fungal growth leading to increased occurrence of mycotoxins in spices. Moreover, the lack of good agricultural practice (GAP), good manufacturing practice (GMP), and good hygienic practice (GHP) in developing countries are of great concern. This review summarizes recent data from a total of 56 original papers dealing with mycotoxins and microfungi in various spices in the last five years. A total of 38 kinds of spices, 17 mycotoxins, and 14 microfungi are discussed in the review. Worldwide, spices are rather overlooked in terms of mycotoxin regulations, which usually only cover aflatoxins (AFs) and ochratoxin A (OTA). In this paper, an extensive attention is devoted to the limits on mycotoxins in spices in the context of the European Union (EU) as well as other countries. As proven in this review, the incidence of AFs and OTA, as well as other mycotoxins, is relatively high in many spices; thus, the preparation of new regulation limits is advisable.     

Effects of the mycotoxin ochratoxin A and some of its metabolites on the human cell line THP-1

The immunomodulatory effects of ochratoxin A (OTA) and some of its metabolites on the human monocyte/macrophage line THP-1 are described. Metabolic activity, cell proliferation, cell membrane integrity, cell differentiation, phagocytic behaviour, nitrogen oxide synthesis and cell surface markers were largely suppressed by these mycotoxins at concentrations between 10 and 1000 ng/ml, in individual cases already at 1 ng/ml. After analysis of a crude toxin, a substance designated RE2 could be isolated besides OTA, which was identified as ochratoxin C (OTC). The latter showed a stronger suppressive effect on most functions studied than all other metabolites of OTA. Because of the immunomodulatory effects of OTA and OTC, more attention should be paid to their immunopathogenic importance in addition to their known cytotoxic and genotoxic effects. The occurrence and importance of the mycotoxin OTC should be more closely examined in this context.     

Central effects of the neurotropic mycotoxin fumitremorgin A in the rabbit (I). Effects on the spinal cord

Effects of a potent neurotropic mycotoxin, fumitremorgin A (FTA), on the spinal cord were studied, using rabbits lightly anesthetized with urethane and chloralose. Spontaneous discharges of L7 spinal ventral roots and common peroneal nerves were increased after intravenous injection of 100-200 micrograms/kg of FTA. Their abnormal discharge pattern represented the convulsive effect of FTA. Spinal monosynaptic reflexes became irregular in amplitude, with a slight predominance of smaller reflexes. Polysynaptic reflexes were inhibited in many cases. These FTA-induced changes in ventral root discharges and spinal reflexes were abolished by spinal transection at a segment of the upper level. These results suggest that FTA may have no direct facilitatory action on spinal motoneurons and that its remarkable motor effect has its origin in the supraspinal central nervous system.     

Calcium/calmodulin signaling elicits release of cytochrome c during 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced apoptosis in the human lymphoblastic T-cell line,L-MAT

We have reported previously that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces apoptosis in the human lymphoblastic T-cell line L-MAT, although these cells do not express the aromatic hydrocarbon receptor (AhR). The AhR-dependent pathway for the induction of immunotoxicity by TCDD has been studied extensively, but the AhR-independent pathway is not understood. Several studies have reported that TCDD elevates the concentration of free intracellular calcium ([Ca²⁺]_i) in various types of cells. However, the precise mechanism of the increase in [Ca²⁺]_i that occurs during apoptosis induced by TCDD has not been elucidated.     

Assessment of toxic potential of mycotoxin contaminated bread during in vitro human digestion on human B lymphoid cell line

Ingestion of food is considered a major route of exposure to many contaminants including mycotoxins. The amount of mycotoxin resisting to the digestion process and potentially absorbable by the systemic circulation is only a smaller part of that ingested. In vitro digestion models turn useful for evaluating mycotoxins bioaccessibility during the intestinal transit and can be intended as a valuable tool for the assessment of mycotoxin bioavailability in food. In this paper we describe a study aimed at investigating toxicity of in vitro gastro-duodenal digests of mycotoxin contaminated bread collected along the digestion time-course. Toxicity tests were carried out on a sensitive RPMI lymphoid B cell line chosen as the most suitable lineage to assess toxicity retained by gastro-duodenal digests. In parallel, a chemical quantification of T-2 and HT-2 toxins contaminating the bread digests was accomplished during the gastric and duodenal transit. The digestive fluids undergoing chemical and toxicological analysis were collected at the beginning and end of gastric phase, and after completion of the duodenal phase. Results proved that a correlation between HT-2 content and toxicity did exist although a more persistent toxic activity was displayed in the later stage of the duodenal phase. This persistent toxicity might be explained by the co-occurrence of unknown HT-2-related conjugates or metabolites formed during digestion.