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14.4A: Toxins - Biology

14.4A: Toxins - Biology



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Learning Objectives

  • Describe the major toxin types (bacterial toxins and mycotoxins) and their mechanisms of action

Toxins are poisonous substances produced within living cells or organisms and can include various classes of small molecules or proteins that cause disease on contact. The severity and type of diseases caused by toxins can range from minor effects to deadly effects. The organisms which are capable of producing toxins include bacteria, fungi, algae, and plants. Some of the major types of toxins include, but are not limited to, environmental, marine, and microbial toxins. Microbial toxins may include those produced by the microorganisms bacteria (i.e. bacterial toxins) and fungi (i.e. mycotoxins).

Bacterial Toxins

Bacterial toxins are typically classified under two major categories: exotoxins or endotoxins. Exotoxins are immediately released into the surrounding environment whereas endotoxins are not released until the bacteria is killed by the immune system. The release of toxins into the surrounding environment, regardless of when released, results in the disruption of metabolic pathways in the host eukaryote. These metabolic pathways include damaging cell membranes, disrupting protein synthesis, inhibiting neurotransmitter release, or activating the host immune system. The mechanisms of action by which toxins disrupt eukaryotic cell processes are dependent on the target. For example, the bacteria Listeria monocytogenes, associated with food-borne illnesses, specifically targets cholesterol by producing a pore-forming toxin protein, listeriolysin O. This exotoxin affects intracellular processes and creates unregulated pores within the cell membranes of the host. Another example of an exotoxin includes an enterotoxin produced by the bacteria Staphlycoccal aureus. S. aureus can producestaphylococcal enterotoxin B (SEB), associated with intestinal illness, which promotes activation of the immune system. Upon activation of the immune system, the release of large amounts of cytokines, inflammatory related molecules, causes significant inflammation. Lastly, an example of an endotoxin, includes the protein lipopolysaccharide (LPS) produced by gram-negative bacteria. The LPS is a component of the bacteria’s outer membrane and promotes structural integrity. Upon destruction of the membrane by an immune response, the LPS is released and functions as a toxin.

However, bacterial toxins are also currently serving as new sources for potential drug development. Toxins have been shown to exhibit anticancer characteristics and fight again microbial virulence. The investigation of toxins as potential medicinal compounds is currently underway.

Mycotoxins

Mycotoxins are the classes of toxins produced by fungi. Mycotoxins are numerous and production of a specific mycotoxin is not restricted to one specific species. Mycotoxins are secondary metabolites that are toxic to humans and produced by fungi. There are various types of mycotoxins including, but not limited to, aflatoxins, ochratoxins, citrinin, and ergot alkaloids.

Aflatoxins

Aflatoxins are a type of mycotoxin that are produced by certain strains of Aspergillus fungi. The aflatoxins are further broken down into types: AFB1, AFB2, AFG1, and AFG2. These strains are present in a wide range of agricultural commodities associated with tropic and subtropic zones. These commodities include species of peanuts and corn. The most potent toxin is AFB1 and it is associated with carcinogenic effects.

Ochratoxin

Ochratoxin is a type of toxin produced by both Penicillium and Aspergillus species. Ochratoxins are further classified in types A, B and C and differ in structure. Ochratoxins have demonstrated carcinogenic properties and are often found in beverages such as beer and wine, as the fungal species which produce ochratoxins are often found on the plants used to produce these products.

Citrinin

Citrinin is a mycotoxin that has been isolated in numerous species of both Penicillium and Aspergillus. Many of these fungal species are utilized in food processing and are often found in foods including cheese, wheat, rice, corn, and soy sauce. Citrinin is known to function as a nephrotoxin, indicating it has toxic effects on kidney function.

Ergot Alkaloids

Ergot Alkaloids are specific compounds that are produced as toxic alkaloids in Claviceps, a group of fungi associated with grasses, rye, and related plants. The disease caused by ingestion of this fungi is called ergotism. Ergotism is characterized by detrimental effects on the vascular system in particular, including vasoconstriction of blood vessels resulting in gangrene, and eventually, limb loss if left untreated. Additionally, ergotism can present as hallucinations and convulsions as ergot alkaloids target the central nervous system. Due to the vascular system effects of ergot alkaloids, they have been used for medicinal purposes.

Key Points

  • Microbial toxins may include those produced by the microorganisms bacteria (i.e. mycotoxins ).
  • Bacterial toxins can include both endotoxins and exotoxins, which vary in mechanism of action and are species -specific.
  • Exotoxins are immediately released into the surrounding environment whereas endotoxins are not released until the bacteria is killed by the immune system.
  • Mycotoxins can be classified into numerous categories and are not species-specific because the same mycotoxin can be produced by different fungal species.

Key Terms

  • endotoxin: Any toxin secreted by a microorganism and released into the surrounding environment only when it dies.
  • exotoxin: Any toxin secreted by a microorganism into the surrounding environment.
  • cytokines: Regulatory proteins that function in the regulation of the cells involved in immune system function

Tetrodotoxin

Tetrodotoxin (TTX) is a potent neurotoxin. Its name derives from Tetraodontiformes, an order that includes pufferfish, porcupinefish, ocean sunfish, and triggerfish several of these species carry the toxin. Although tetrodotoxin was discovered in these fish and found in several other animals (e.g., in blue-ringed octopus, rough-skinned newts, and moon snails), it is actually produced by certain infecting or symbiotic bacteria like Pseudoalteromonas, Pseudomonas, and Vibrio as well as other species found in animals. [1] [2]

  • 4368-28-9 Y
  • CHEBI:9506 N
  • ChEMBL507974 N
  • 9349691 N
  • C11692 N
  • 3KUM2721U9 Y
InChI=1S/C11H17N3O8/c12-8-13-6(17)2-4-9(19,1-15)5-3(16)10(2,14-8)7(18)11(20,21-4)22-5/h2-7,15-20H,1H2,(H3,12,13,14)/t2-,3-,4-,5+,6-,7+,9+,10-,11+/m1/s1 N Key: CFMYXEVWODSLAX-QOZOJKKESA-N N

Tetrodotoxin is a sodium channel blocker. It inhibits the firing of action potentials in neurons by binding to the voltage-gated sodium channels in nerve cell membranes and blocking the passage of sodium ions (responsible for the rising phase of an action potential) into the neuron. This prevents the nervous system from carrying messages and thus muscles from contracting in response to nervous stimulation. [3]

Its mechanism of action, selective blocking of the sodium channel, was shown definitively in 1964 by Toshio Narahashi and John W. Moore at Duke University, using the sucrose gap voltage clamp technique. [4]


Clostridium difficile Toxin Biology

Clostridium difficile is the cause of antibiotics-associated diarrhea and pseudomembranous colitis. The pathogen produces three protein toxins: C. difficile toxins A (TcdA) and B (TcdB), and C. difficile transferase toxin (CDT). The single-chain toxins TcdA and TcdB are the main virulence factors. They bind to cell membrane receptors and are internalized. The N-terminal glucosyltransferase and autoprotease domains of the toxins translocate from low-pH endosomes into the cytosol. After activation by inositol hexakisphosphate (InsP6), the autoprotease cleaves and releases the glucosyltransferase domain into the cytosol, where GTP-binding proteins of the Rho/Ras family are mono-O-glucosylated and, thereby, inactivated. Inactivation of Rho proteins disturbs the organization of the cytoskeleton and affects multiple Rho-dependent cellular processes, including loss of epithelial barrier functions, induction of apoptosis, and inflammation. CDT, the third C. difficile toxin, is a binary actin-ADP-ribosylating toxin that causes depolymerization of actin, thereby inducing formation of the microtubule-based protrusions. Recent progress in understanding of the toxins' actions include insights into the toxin structures, their interaction with host cells, and functional consequences of their actions.

Keywords: ADP ribosylation CDT Clostridium difficile infection Clostridium difficile toxins Clostridium difficile transferase toxin Rho proteins actin glucosylation microtubules toxin receptors toxin uptake.


Multifunctional-autoprocessing repeats-in-toxin (MARTX) Toxins of Vibrios

Multifunctional-autoprocessing repeats-in-toxin (MARTX) toxins are a heterogeneous group of toxins found in a number of Vibrio species and other Gram-negative bacteria. The toxins are composed of conserved repeat regions and an autoprocessing protease domain that together function as a delivery platform for transfer of cytotoxic and cytopathic domains into target eukaryotic cell cytosol. Within the cells, the effectors can alter biological processes such as signaling or cytoskeletal structure, presumably to the benefit of the bacterium. Ten effector domains are found in the various Vibrio MARTX toxins, although any one toxin carries only two to five effector domains. The specific toxin variant expressed by a species can be modified by homologous recombination to acquire or lose effector domains, such that different strains within the same species can express distinct variants of the toxins. This review examines the conserved structural elements of the MARTX toxins and details the different toxin arrangements carried by Vibrio species and strains. The catalytic function of domains and how the toxins are linked to pathogenesis of human and animals is described.

Figures

The multifunctional-autoprocessing repeats-in-toxin (MARTX) toxins…

The multifunctional-autoprocessing repeats-in-toxin (MARTX) toxins are a form of effector delivery similar in…

(A) The general structure of…

(A) The general structure of a multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin showing the number…

Schematic representation of all multifunctional-autoprocessing…

Schematic representation of all multifunctional-autoprocessing repeats-in-toxin (MARTX) toxins described in text. Toxins are…


Discussion

Different pH values 4, 4.5, 5.5, 6, and 6.5 were used in the culture media showed that the concentration of pH 4.5 had the highest growth rate. While, the growth was decreased with the increasing of pH values, this result is concordant with Banjara et al. (2016), who tested the killer activity of 23 strains against C. albicans and C. tropicalis, and noticed that there was no killer activity for any strain at pH ≥ 6.5 or at ≥ 35 °C.

The temperature was an another factor to impact the production of enzymes, since the growth at 22, 25, 30, and 37 °C gave varied amount of production enzymes for the killer toxins with a maximum level of growth rate observed at 30 °C. This result is similar to the finding reported by Al-Qaysi et al. (2017b), who studied the effects of temperature and the production of killer toxins against several pathogenic microorganisms, such as C. albicans, C. neoformans Staphylococcus aureus, and others. Their results confirmed that the ideal inhibitory influence of killer toxin was at ≥ 25 °C.

The incubation time in fermentation media was monitored at 72, 96, and 168 h post inoculation. The result is in concord with the finding of Thammasittirong et al. (2013), who reported that the time was accelerated the vegetative phase of yeasts and encouraged them to reach the production phase at 72 h. To guarantee the efficient production and supply of oxygen to yeast, the rotation rate was tested at 120, 150, 180, and 200 rpm. The production rate was stimulated at highest level with 150 rpm. Culture media was also supplemented with sodium dodecyl sulphate (SDS) at concentration of 2%, which enhanced the growth rate. This may be attributed to the efficiency of this medium that contains nitrogen and carbon sources, which are used by the yeasts, similar explanation was giving by Çorbacı and Uçar (2017b). Besides, the addition of SDS and the detergent that destroys yeast wall and release killer toxin intracellular production. According to Grzegorczyk et al. (2017), crude killer toxins have the ability to counteract the growth of bacteria and yeasts through pore formation by hydrolytic enzymes production. The toxin binds to its specific receptors on the surfaces of the bacterial and yeast cell walls, then forms pores to release the contents inside the target cells leading to death. This can be attributed to the different mechanism actions reported by Becker and Schmitt (2017). In terms of K28, it has a specific primary receptor in the yeast cell wall, which allows rapid K28 absorption via an energy-independent binding mechanism. Afterward, α-1, 3- or α-1,2- mannotriose side-chains of a 185 kDa cell wall mannoprotein were identified as primary K28 receptor and cell wall binding sites. In contrast, Vadasz et al. (2000) stated that the ionophoric killer toxin (K2) has the ability of killing cells by causing shrinkage as a characteristic of cytosol efflux, which can be seen clearly by electron microscope. The cytosolic efflux and cell death are the results of initial interaction between yeast toxin and specific cell wall receptors, followed by the formation or activation of endogenous ion channels in the plasma membrane of target cells (Ahmed et al. 1999).


Endotoxins Vs Exotoxins

Micro-organisms such as bacteria, fungi produce toxic substances that boost up the infection and diseases through damaging the host tissues and by troubling the immune system. The most potent and known natural bacterial toxin is Botulinum neurotoxin which has essential uses in research and medical science. At present, toxins are used as tools in cellular biology and neurobiology to develop anticancer drugs and other medicines. Toxins from bacteria act as virulence factors that influence the functions of the host cell to help microbial infections. Toxins produced from bacteria can be either endotoxins or exotoxins. These toxins play a pivotal role in leading various diseases and infections.

Endotoxins

Endotoxins are lipopolysaccharides toxic substances which are part of the outer membrane of the gram-negative bacteria. Generally, it is released when the bacterium is killed, or cell lyses by the immune system due to action of phagocytic digestion or specific antibiotic actions. It is found in the body of the following bacteria species:


Results

Preparation of phallotoxin derivatives

Attachment sites in phalloidin ( Figure 1 ) for conjugation with uptake-mediating moieties were chosen based on our knowledge of structure�tivity relationships in phalloidin [1]: As the side chains in the small heterodet peptide ring are known to be involved in actin binding they were not used for derivatization. In the larger peptide ring, on the other hand, the dihydroxylated leucine moiety is juxtaposed to the actin binding site and thus appeared as most promising for derivatization. Accordingly, all residues investigated in this study were attached to the Cδ-atom of the dihydroxylated leucine moiety, either as esters or amides ( Table 1 ). Large residues, which might disturb the interaction of a phalloidin derivative with actin through steric hindrance, were coupled via disulfide-containing linkers, which would be reduced inside the cell so as to release a defined thiol derivative of phalloidin ( Table 1 ).

Chemical structure of phalloidin with the attachment site (R) used for conjugation to uptake-mediating moieties.

Table 1

Structures of phalloidin derivatives, their relative affinity values for α-actin as compared to phalloidin and their IC50 values of cell growth inhibition after incubation for 72 h as determined by MTT cell proliferation assay (n.a.: not assayed).

RName/designationRelative affinity to α-actin [%]IC50 values NIH 3T3 fibroblasts [µM]IC50 values Jurkat cells [µM]
1OHphalloidin100ca. 1,000ca. 1,000
1aOCOC6H5(1)-benzoate769294
1bOCOC6H4o-OH(1)-salicylate8136496
1cOCOC7H15(1)-octanoate124437
1dOCOC13H27(1)-myristate7119
1eOCO(CH2)7CHCH(CH2)7CH3(1)-oleate432
2NH2aminophalloidin2ca. 1,000ca. 1,000
2aNHCOC6H5N-benzoyl-(2)74693599
2bNHCO(CH2)7CHCH(CH2)7CH3N-oleoyl-(2)5883630
2cNHCO(CH2)2SS(Ac)CysGlyTyrGly- -Arg(Lys)2(Arg)2Glu(Arg)3OH(2)-Tat-peptide343
2dNHCO(CH2)2SS(Ac)CysGly(Arg)8OH(2)-octarginine2117
2eNHCO(CH2)6CONH(Lys)210(2)-poly-(L)-lysine28,0004054
(2)-poly-(D)-lysine28,000385437
2fNHCO(CH2)2SS(CH2)2CONH(Lys)210(2)-(SS) poly-(L)-lysine28,0003932
(2)-(SS) poly-(D)-lysine28,0003942
2gNH(CH2)2SS(CH2)2CONH(PEG)800(2)-(SS) PEG800816798
2hNH(CH2)2SS(CH2)2CONH(PEG)5.200(2)-(SS) PEG5,20047750
2iNH(CH2)2SS(CH2)2CONH(PEG)23.000(2)-(SS) PEG23,00011010
2f +DTTNH(CH2)2SHN-(2-mercaptoethyl)-(2)-SH37n.a.n.a.
3for structure see Figure 6a dithiolanoaminophalloidin TRITC labeled2511n.a

Actin binding

All phalloidin derivatives were tested for their affinity to muscle actin (α-actin), which is used as a model for β-actin present in nonmuscle cells. This parameter was important, since cytotoxicity depends not only on membrane permeability but also on actin affinity. Moreover, this parameter will hint on whether phalloidin was cleaved from its carrier inside the cell, in cases where low actin affinity of a phalloidin derivative was combined with high cytotoxicity. Relative affinity values of the phalloidin derivatives to muscle actin are shown in Table 1 .

Growth inhibition of mouse fibroblasts

By using the MTT cell proliferation assay, each phalloidin conjugate was examined for its capacity to inhibit the growth of mouse fibroblasts in vitro after 72 h incubation time ( Table 1 ). Phalloidin displayed no antiproliferative activity up to a concentration of 10 𢄣 M in the culture medium. In contrast, the most lipophilic ester derivative, phalloidin oleate (1e), showed an IC50 value of proliferation inhibition of 2.5 × 10 𢄦 M, and was thus ca. 1000 times more active than phalloidin. Other esters (1a1d) exhibited increasing cytotoxic activities with increasing hydrophobicity ( Figure 2 ). In order to examine a possible relationship between cytotoxicity and hydrophobicity we determined the octanol/water equilibrium distribution coefficient of the ester derivatives (log Pow values) and found a linear relationship between log Pow and IC50 values of cellular cytotoxicity ( Figure 3 ). The ester derivatives of phalloidin are probably hydrolyzed inside the cell, e.g., by esterases or proteases, as suggested by the fact that the ester 1e and the corresponding amide 2b possess comparable affinities to actin ( Table 1 ), but differ in their IC50 values by a factor of ca. 300. The effect is presumably a consequence of the faster hydrolysis of esters over amides by intracellular hydrolases.

Cytotoxicity of phalloidin derivatives. NIH 3T3 mouse fibroblasts were incubated with various concentrations of phalloidin and phalloidin derivatives. Cell viability was determined after 72 h incubation time by MTT assay.

n-Octanol/water distribution coefficients (log Pow) of the hydrophobic phalloidin derivatives of Figure 2 plotted versus cell toxicity (log (1/IC50) in NIH 3T3 mouse fibroblasts.

Polycationic derivatives of phalloidin, such as the polylysine conjugates, were highly toxic for mouse fibroblasts, and their antiproliferative activity was comparable to the most lipophilic derivative, phalloidin oleate ( Figure 4a ). Their toxicity was found to be dependent on the configuration of the polymer, since phalloidin bound to D-configurated polylysine was about 10 times less toxic than when bound to poly-(L)-lysine. This suggests that the release of a toxic phalloidin species inside the cell includes the enzymatic breakdown of the carrier. In agreement with this we found no difference between the L-configurated and the D-configurated carrier when the linker contained a disulfide bridge, arguing for the presence of a disulfide-reducing compartment inside the cells.

(a) and (b): NIH 3T3 mouse fibroblasts were incubated with various concentrations of phalloidin and phalloidin derivatives. Cell viability was determined after 72 h incubation time by MTT assay.

Likewise, high cytotoxicity was found for the octarginine conjugate ( Figure 4a ). From the fact that the cytotoxic activities of the polylysine derivative (ca. 150 residues) and the oligoarginine derivative (8 residues) are comparable, we argue that arginine residues are more effective in mediating internalization of phalloidin than are lysine residues.

For phalloidin bound to methoxy-polyethylene-glycol we found that cytotoxicity strictly depended on the molecular weight of the polymer chain ( Figure 4b ). Most active was the conjugate with the longest chain (Mr = 22,600). Cytotoxicity fell strikingly when the polymer chain was shortened ( Table 1 ).

Coupling to Tat-peptide was also very effective in enhancing the cytotoxicity of phalloidin ( Figure 4b ), while the phalloidin conjugate with the Kaposi protein fragment (likewise claimed to enhance membrane permeability) was much less effective (data not shown).

Beside mouse fibroblasts, we investigated the phalloidin derivatives in several human leukemia and lymphoma cell lines, in order to find possible specificities for one or the other kind of tumor cell. However, Jurkat cells ( Table 1 ) and all other cell lines tested (K562 cells, HL-60 cells, and Daudi cells) showed sensitivities comparable to mouse fibroblasts (see Supporting Information File 1).

Uptake kinetics

Since the toxic effects of phalloidin develop slowly, growth inhibition was measured only after 72 h. During this period several partial processes must occur such as binding to the plasma membrane, internalization, processing and toxin release, etc., which cannot be distinguished. However, replacement of the toxin medium by toxin-free medium after various times of incubation would provide information on the time required for each of the toxin derivatives to bind to the cell surface. We compared the three most effective phalloidin conjugates, phalloidin oleate (1e), phalloidin-Tat conjugate (2c), and phalloidin polylysine (2e) ( Figure 5 ) and found that the exposure times necessary to achieve, e.g., a 50% growth inhibition by a given toxin concentration after 72 h indeed varied considerably, from 2 h to 24 h. The results show that the polycationic derivative was bound much more rapidly than the lipophilic conjugate.

Time course of uptake in NIH 3T3 cells of three phalloidin derivatives as measured by the cytotoxicity after 72 h incubation during which the toxins were washed off after various exposure times.

A fluorescent residue that enhances uptake of phalloidin into cells

Tetramethylrhodaminyl-phalloidin ( Figure 6a ) has been used to visualize actin fibers in fixed cells for 30 years. Here we show that the rhodamine residue also strongly enhanced cellular uptake, making this phalloidin derivative a tool for cell biology. With an IC50 value of 11 µM its toxicity is comparable to those of the most toxic phalloidin derivatives, phalloidin oleate (1e) and phalloidin-poly-(L)-lysine (2e). More importantly, rhodaminyl-phalloidin seems not to be cleaved inside the cell and, through its fluorescence, can report on the structure of its target protein, the actin filaments, albeit under toxic conditions.

(a) Chemical structure of tetramethylrhodaminyl-phalloidin (3). (b) Growth inhibition of NIH 3T3 mouse fibroblasts by tetramethylrhodaminyl-phalloidin (3) versus phalloidin (1) after 72 h incubation time. (c) Binding and uptake of tetramethylrhodaminyl-phalloidin (3) in NIH 3T3 mouse fibroblasts after 1, 6 and 24 h, as documented by fluorescence microscopy.

Using rhodamine-labeled phalloidin, we could also study how membrane-permeable peptides are incorporated into the cell. Immediately after exposure the toxin was located on the plasma membrane of the cells as shown by fluorescence microscopy ( Figure 6c ), while after 6 h increasing amounts of the toxin were found in endocytotic vesicles. After 24 h most of the rhodamine-labeled toxin was still in endosomes, while some of it had found its target, as concluded from the decoration of filaments.

Phalloidin causes apoptosis of cells

Under the microscope, cells treated with membrane-permeable phalloidin derivatives appeared shrunken and developed blebs, as described for cells undergoing apoptosis. Treatment with annexin followed by flow cytometric analysis showed a fluorescence distribution typical for apoptosis and similar to that induced by camptothecin. Cells treated with native phalloidin were indistinguishable from controls [13].

Growth inhibition by amatoxin derivatives

Unlike the phallotoxins, the natural amatoxins are toxic in cell cultures, exhibiting IC50 values around 10 𢄦 M. As amatoxins are more hydrophilic than phallotoxins, it seems unlikely that their membrane permeation capacity is larger than that of phallotoxins. The more likely explanation is that for amatoxins the threshold concentration lethal for cells is much lower than for phallotoxins (see Discussion).

Two of the internalization-mediating residues investigated in the phallotoxin series, oleic acid and polylysine, were also tested for their uptake capacity in the amatoxin series. Structure�tivity studies had shown that the primary OH group of the dihydroxy-isoleucine moiety ( Figure 7 ) is not involved in RNA polymerase II binding and, hence, may be used for derivatization with oleic acid chloride ( Table 2 ). In order to avoid concurrent acylation of the 6’-OH of tryptophan, the phenolic OH was methylated before acetylation. For coupling to polylysine the natural carboxy group of aspartic acid as present in β-amanitin was used, which after activation as N-hydroxysuccinimide ester reacted with ε-amino groups in polylysine. Since the polymeric carrier may be broken down inside the cell by proteases, we investigated both poly-(L)-lysine and poly-(D)-lysine as carriers, in order to study whether the biological availability of the amatoxin depended on the configuration of lysine in the polymeric carrier.

Chemical structure of amanitin with attachment site for conjugation to internalization-mediating moieties (R 1 , R 3 ).

Table 2

Structures of amanitin derivatives and IC50 concentrations of growth inhibition in mouse fibroblasts after 72 h incubation time (MTT cell proliferation assay).

R 1 R 2 R 3 IC50 values NIH 3T3 fibroblasts [nM]
4OHOHNH2α-amanitin6,200
4aOCO(CH2)7CHCH(CH2)7CH3OCH3NH2α-amanitin oleate39
5OHOHOHβ-amanitin1,000
5aOHOHβ-amanitin poly-(L)-lysine10
5bOHβ-amanitin poly-(D)-lysine18,000

Conjugation of amanitin to oleic acid increased the cytotoxicity by a factor of 150 (see Table 2 ). In analogy with the phallotoxins, we conclude that the oleic acid ester is hydrolysed inside the cell. β-Amanitin, when conjugated to poly-(L)-lysine, became 100-fold more toxic for mouse fibroblasts than the native toxin, showing IC50 values in the nanomolar range ( Figure 8 ). As in the phallotoxin series, toxicity of β-amanitin conjugates depends on the cleavage of the toxin from the polymeric carrier, as shown by the fact that β-amanitin coupled to the (L)-polymer was 1800 times more toxic than when coupled to the (D)-polymer ( Table 2 ).

NIH 3T3 mouse fibroblasts were incubated with various concentrations of α-amanitin and amanitin derivatives. Cell viability was determined after 72 h incubation time by MTT assay.


Contents

Aflatoxin B1 is considered the most toxic and is produced by both Aspergillus flavus and Aspergillus parasiticus. Aflatoxin M1 is present in the fermentation broth of Aspergillus parasiticus, but it and aflatoxin M2 are also produced when an infected liver metabolizes aflatoxin B1 and B2.

  • Aflatoxin B1 and B2 (AFB), produced by Aspergillus flavus and A. parasiticus
  • Aflatoxin G1 and G2 (AFG), produced by some Group II A. flavus and Aspergillus parasiticus[16]
  • Aflatoxin M1 (AFM1), metabolite of aflatoxin B1 in humans and animals (exposure in ng levels may come from a mother's milk)
  • Aflatoxin M2, metabolite of aflatoxin B2 in milk of cattle fed on contaminated foods [17]
  • Aflatoxicol (AFL): metabolite produced by breaking down the lactone ring
  • Aflatoxin Q1 (AFQ1), major metabolite of AFB1 in in vitro liver preparations of other higher vertebrates [18]

AFM, AFQ, and AFL retain the possibility to become an epoxide. Nevertheless, they appear much less capable of causing mutagenesis than the unmetabolized toxin. [19]

Aflatoxins are produced by both Aspergillus flavus and Aspergillus parasiticus, which are common forms of 'weedy' molds widespread in nature. The presence of those molds does not always indicate that harmful levels of aflatoxin are present, but does indicate a significant risk. The molds can colonize and contaminate food before harvest or during storage, especially following prolonged exposure to a high-humidity environment, or to stressful conditions such as drought.

The native habitat of Aspergillus is in soil, decaying vegetation, hay, and grains undergoing microbiological deterioration, but it invades all types of organic substrates whenever conditions are favorable for its growth. Favorable conditions for production of aflatoxins include high moisture content (at least 7%) and temperatures from 55 °F (13°C) to 104 °F (40°C) (optimum 81--86 °F). [20] [21] Aflatoxins have been isolated from all major cereal crops, and from sources as diverse as peanut butter and cannabis. The staple commodities regularly contaminated with aflatoxins include cassava, chilies, corn, cotton seed, millet, peanuts, rice, sorghum, sunflower seeds, tree nuts, wheat, and a variety of spices intended for human or animal consumption. Aflatoxin transformation products are sometimes found in eggs, milk products, and meat when animals are fed contaminated grains. [1] [22]

A study conducted in Kenya and Mali found that the predominant practices for drying and storage of maize were inadequate in minimizing exposure to aflatoxins. [23]

Organic crops, which are not treated with fungicides, may be more susceptible to contamination with aflatoxins. [24]

There is very limited evidence to show that agricultural and nutritional education can reduce exposure to aflatoxin in low to middle income countries. [25]

No animal species is immune to the acute toxic effects of aflatoxins. Adult humans have a high tolerance for aflatoxin exposure and rarely succumb to acute aflatoxicosis, [26] but children are particularly affected, and their exposure can lead to stunted growth and delayed development, in addition to all the symptoms mentioned below. [4]

High-level aflatoxin exposure produces an acute hepatic necrosis (acute aflatoxicosis), resulting later in cirrhosis or carcinoma of the liver. Acute liver failure is made manifest by bleeding, edema, alteration in digestion, changes to the absorption and/or metabolism of nutrients, and mental changes and/or coma. [26]

Chronic, subclinical exposure does not lead to symptoms so dramatic as acute aflatoxicosis. Chronic exposure increases the risk of developing liver and gallbladder cancer, [27] as aflatoxin metabolites may intercalate into DNA and alkylate the bases through epoxide moiety. This is thought to cause mutations in the p53 gene, an important gene in preventing cell cycle progression when there are DNA mutations, or signaling apoptosis (programmed cell death). These mutations seem to affect some base pair locations more than others, for example, the third base of codon 249 of the p53 gene appears to be more susceptible to aflatoxin-mediated mutations than nearby bases. [28] As with other DNA-alkylating agents, Aflatoxin B1 can cause immune suppression, and exposure to it is associated with an increased viral load in HIV positive individuals. [29] [30]

The expression of aflatoxin-related diseases is influenced by factors such as species, age, nutrition, sex, and the possibility of concurrent exposure to other toxins. The main target organ in mammals is the liver, so aflatoxicosis primarily is a hepatic disease. Conditions increasing the likelihood of aflatoxicosis in humans include limited availability of food, environmental conditions that favour mould growth on foodstuffs, and lack of regulatory systems for aflatoxin monitoring and control. [31]

A regular diet including apiaceous vegetables, such as carrots, parsnips, celery, and parsley may reduce the carcinogenic effects of aflatoxin. [32]

There is no specific antidote for aflatoxicosis. Symptomatic and supportive care tailored to the severity of the liver disease may include intravenous fluids with dextrose, active vitamin K, B vitamins, and a restricted, but high-quality protein diet with adequate carbohydrate content.

In other animals Edit

In dogs, aflatoxin has potential to lead to liver disease. Low levels of aflatoxin exposure require continuous consumption for several weeks to months in order for signs of liver dysfunction to appear. [33] Some articles have suggested the toxic level in dog food is 100–300 ppb and requires continuous exposure or consumption for a few weeks to months to develop aflatoxicosis. [34] No information is available to suggest that recovered dogs will later succumb to an aflatoxin-induced disease.

Turkeys are extremely susceptible to aflatoxicosis. Recent studies have revealed that this is due to the efficient cytochrome P450 mediated metabolism of aflatoxin B1 in the liver of turkeys and deficient glutathione-S-transferase mediated detoxification. [35] [36]

Some studies on pregnant hamsters showed a significant relationship between exposure of aflatoxin B1 (4 mg/kg, single dose) and the appearance of developmental anomalies in their offspring. [37]

In 2005, Diamond Pet Foods discovered aflatoxin in a product manufactured at their facility in Gaston, South Carolina. [38] [39] In 23 states, Diamond voluntarily recalled 19 products formulated with corn and manufactured in the Gaston facility. Testing of more than 2,700 finished product samples conducted by laboratories confirmed that only two date codes of two adult dog formulas had the potential to be toxic. [40]

In December 2020 and January 2021, Midwestern Pet Foods recalled dog food that contained fatal levels of aflatoxin. [41] As many as 70 dogs had died from aflatoxin poisoning by January 12, 2021. [42]

There are two principal techniques that have been used most often to detect levels of aflatoxin in humans.

The first method is measuring the AFB1-guanine adduct in the urine of subjects. The presence of this breakdown product indicates exposure to aflatoxin B1 during the past 24 hours. This technique measures only recent exposure, however. Due to the half-life of this metabolite, the level of AFB1-guanine measured may vary from day to day, based on diet, it is not ideal for assessing long-term exposure.

Another technique that has been used is a measurement of the AFB1-albumin adduct level in the blood serum. This approach provides a more integrated measure of exposure over several weeks or months.


Contents

When an insect ingests these proteins, they are activated by proteolytic cleavage. The N-terminus is cleaved in all of the proteins and a C-terminal extension is cleaved in some members. Once activated, the endotoxin binds to the gut epithelium and causes cell lysis by the formation of cation-selective channels, which leads to death. [2] [1]

The activated region of the delta toxin is composed of three distinct structural domains: an N-terminal helical bundle domain (InterPro: IPR005639) involved in membrane insertion and pore formation a beta-sheet central domain involved in receptor binding and a C-terminal beta-sandwich domain (InterPro: IPR005638) that interacts with the N-terminal domain to form a channel. [1] [2]

B. thuringiensis encodes many proteins of the delta endotoxin family (InterPro: IPR038979), with some strains encoding multiple types simultaneously. [3] A gene mostly found on plasmids, [4] delta-entotoxins sometimes show up in genomes of other species, albeit at a lower proportion than those found in B. thuringiensis. [5] The gene names looks like Cry3Bb , which in this case indicates a Cry toxin of superfamily 3 family B subfamily b. [6]

Cry proteins that are interesting to cancer research are listed under a parasporin (PS) nomenclature in addition to the Cry nomenclature. They do not kill insects, but instead kill leukemia cells. [7] [8] [9] The Cyt toxins tend to form their own group distinct from Cry toxins. [10] Not all Cry -- crystal-form -- toxins directly share a common root. [11] Examples of non-three-domain toxins that nevertheless have a Cry name include Cry34/35Ab1 and related beta-sandwich binary (Bin-like) toxins, Cry6Aa, and many beta-sandwich parasporins. [12]

Specific delta-endotoxins that has been used for genetic engineering include Cry3Bb1 found in MON 863 and Cry1Ab found in MON 810, both of which are corn species. Cry3Bb1 is particularly useful because it kills the coleopteran insects such as the corn rootworm, an activity not seen in other Cry proteins. [1] Other common toxins include Cry2Ab and Cry1F in cotton and corn. [13] In addition, Cry1Ac is effective as a vaccine adjuvant in humans. [14]

Some insects populations have started to develop resistance towards delta endotoxin, with five resistant species found as of 2013. Plants with two kinds of delta endotoxins tend to make resistance happen slower, as the insects have to evolve to overcome both toxins at once. Planting non-Bt plants with the resistant plants will reduce the selection pressure for developing the toxin. Finally, two-toxin plants should not be planted with one-toxin plants, as one-toxin plants act as a stepping stone for adaption in this case. [13]


Why/How do Cyanobacteria Produce Toxins?

I've been doing some research on the versatility of nitrogen-fixing cyanobacteria, particularly of the genus Anabaena, and I often run into safety hazards and have to add extra steps to my procedures because of the toxins produced by the genus. After doing some reading, I've found that most cyanobacteria produce toxins (collectively dubbed "cyanotoxins"). The wide variety of different structures and mechanisms of the toxins across multiple genera is astounding, especially because some of them are carcinogens, which would logically affect the host unless a mechanism to prevent this had evolved. This tells me that a lot of evolutionary "effort" must have been put into the evolution of toxin production in cyanobacteria. Originally, I just thought that the toxins were a natural part of their metabolism that evolved normally because these compounds didn't affect them in the same way as they did us. Now that I see the Huge, variable trend throughout the class, I have begun to think otherwise. My questions are as follows:

  1. What is the evolutionary basis for the production of cyanotoxins by cyanobacteria
  2. Are the cyanotoxins a necessary part of the bacteria's metabolism, or can they be eliminated by way of genetic engineering without directly affecting the success of the organism?
  3. How do cyanobacteria cope with the constant exposure to native carcinogens?

These questions are only the major ones that I have, so any extra information is greatly appreciated