Natural Protein Toxins Affecting Cutaneous Microvascular Permeability

Abstract

Natural toxins are found widespread from animal, plant and micro-organism sources. Presented here in review are recently discovered natural protein toxins that have in some way been shown to affect the permeability of the cutaneous micro-vasculature or skin capillaries.

Capillaries play a major role in the body by controlling the necessary normal balance of metabolites in the body's tissues and that of the blood. The trans-capillary exchanges of water and metabolites are regulated by the basal lamina, the internal activities of the endothelial cells and the driving forces on each side of the capillary wall. Edema, inflammation, urticaria, increased capillary permeability and cutaneous edema are conditions that are related. The main methods for detecting the increase in skin capillary permeability have been by using the rat paw edema assay, labelled albumin, or other detectable indicators.

Natural venoms and toxins sources causing edema include mammals, reptiles, amphibians, fishes, invertebrates, plants, and micro-organisms. Not all the substances causing increased capillary permeability are proteins such as certain alkaloids (Plants, fungi, fire ants and others) and normal endogenous substances e.g. histamine, serotonin, acetylcholine, (plants, snakes, and others) and are not considered in this review.

Recently, a large number of toxins (predominantly bacterial toxins) have been reported that produce increased cutaneous capillary permeability, some of which are known in some detail and selected toxins are discussed.

These studies have helped to understand the toxins, provided for more effective treatments, and helped to improve our knowledge of the capillaries and endothelial cells.     

Cellular Models in Natural Toxins Investigations: Intestinal and Kidney Cell Lines

Abstract

Cell cultures are increasingly used in toxicology. Cell lines, also of human origin are available, which have been widely studied for their capability of expressing in vitro the specialized functions of the tissues of origin.

When intestinal and renal cell lines are cultured on inserts, it is possible to study the effects of toxicants on epithelial barriers with regard to cell injury, transport and apical or basolateral cellular exposure.

This review describes natural toxins studies performed with the human intestinal adenocarcinoma cell lines Caco-2, HT-29, T84 and with the renal cell lines MDCK (canine) and LLC-PK1 (porcine).

These studies represent an interesting and fairly new approach in the field of natural toxins dealing with in vitro target-organ experimental models.     

Overview of Recent Liquid Chromatography Mass Spectrometry-Based Methods for Natural Toxins Detection in Food Products

Natural toxins include a wide range of toxic metabolites also occurring in food and products, thus representing a risk for consumer health. In the last few decades, several robust and sensitive analytical methods able to determine their occurrence in food have been developed. Liquid chromatography mass spectrometry is the most powerful tool for the simultaneous detection of these toxins due to its advantages in terms of sensitivity and selectivity. A comprehensive review on the most relevant papers on methods based on liquid chromatography mass spectrometry for the analysis of mycotoxins, alkaloids, marine toxins, glycoalkaloids, cyanogenic glycosides and furocoumarins in food is reported herein. Specifically, a literature search from 2011 to 2021 was carried out, selecting a total of 96 papers. Different approaches to sample preparation, chromatographic separation and detection mode are discussed. Particular attention is given to the analytical performance characteristics obtained in the validation process and the relevant application to real samples.     

Marine Toxins Implicated in Food Poisoning

Abstract

Various kinds of marine products make important contributions to our health, but a few of them cause serious health problems by being poisonous in limited or worldwide regions, and/or in a specific or all seasons. The food toxicity has a great impact on public health as well as the national economy. The role of marine toxins in food poisonings are described in the following from the viewpoints of public health and food hygiene.     

Toxins in anti-nociception and anti-inflammation.

The use of toxins as novel molecular probes to study the structure-function relationship of ion-channels and receptors as well as potential therapeutics in the treatment of wide variety of diseases is well documented. The high specificity and selectivity of these toxins have attracted a great deal of interest as candidates for drug development. This review highlights the involvement of the proteins and peptide toxins as well as non-proteinaceous compounds derived from both venomous and non-venomous animals, in anti-nociception and anti-inflammation. The possible mechanisms of these potential therapeutic agents and possible clinical applications in the treatment of pain and inflammation are also summarized.     

Research on Marine Toxins in Taiwan

Some health issues of marine toxins in Taiwan are introduced here. From 1986 to 2002, the causative agents of seafood poisoning incidents have been reported to be tetrodotoxin (TTX, 30 outbreaks), paralytic shellfish poisons (PSP, 3 outbreaks), grass carp bile toxins (sporadically), ciguateric toxins (about 1 outbreak per year), excess dose of vitamin A (2 outbreaks), histamine (more than 5 outbreaks), pyropheophorbide a (1 outbreak), fish roe of Ascrossochelius paradoxus (1 outbreak), and allergens (sporadically). Among them, health issue of TTX in Taiwan is the most concerned problem. Therefore, TTX‐containing animals have been found to include puffer, goby, xanthid crab, gastropod, starfish, and flatworm. The dried dressed fish fillets produced from the non‐toxic puffers have been found to be adulterated by toxic puffers. The SDS‐PAGE and PCR methods for identifying species of puffers and their products have been developed. PSP was distributed in the purple clam, xanthid crab, and gastropod in Taiwan. The toxic alga Alexandrium minutum appeared in December–March. The toxin production of alga was affected by a variety of nutritional, environmental, and physiological factors. Most shellfish possessed high resistance to PSP, but the susceptibility of shellfish to the toxic alga was quite different depending on species. The food safety of animal bile juice is another important issue in Taiwan. The toxicity of bile juice was in the order of grass carp ? chicken ? snake. The major component of grass carp bile powder was 5α‐cyprinol sulfate (more than 90%) and those of other animals are bile salts. The toxic potencies of 5α‐cyprinol sulfate and 5α‐cyprinol induced acute kidney failure, but bile salts induced chronic liver dysfunction. Other marine toxins including diarrheic shellfish poisons (DSP), neurotoxic shellfish poisons (NSP), and amnesic shellfish poisons (ASP) are also very important from the health viewpoint in the world, though no poisonings due to those toxins have so far occurred in Taiwan.     

Suspect and Target Screening of Natural Toxins in the Ter River Catchment Area in NE Spain and Prioritisation by Their Toxicity

This study presents the application of a suspect screening approach to screen a wide range of natural toxins, including mycotoxins, bacterial toxins, and plant toxins, in surface waters. The method is based on a generic solid-phase extraction procedure, using three sorbent phases in two cartridges that are connected in series, hence covering a wide range of polarities, followed by liquid chromatography coupled to high-resolution mass spectrometry. The acquisition was performed in the full-scan and data-dependent modes while working under positive and negative ionisation conditions. This method was applied in order to assess the natural toxins in the Ter River water reservoirs, which are used to produce drinking water for Barcelona city (Spain). The study was carried out during a period of seven months, covering the expected prior, during, and post-peak blooming periods of the natural toxins. Fifty-three (53) compounds were tentatively identified, and nine of these were confirmed and quantified. Phytotoxins were identified as the most frequent group of natural toxins in the water, particularly the alkaloids group. Finally, the toxins identified to levels 2 and 1 were prioritised according to their bioaccumulation factor, biodegradability, frequency of detection, and toxicity. This screening and prioritisation approach resulted in different natural toxins that should be further assessed for their ecotoxicological effects and considered in future studies.     

Naturally Occurring Seafood Toxins

Abstract

Incidents of illness reported to the Centers for Disease Control (CDC) in the period 1978–1987 were limited to ciguatera, scombroid fish poisoning (SFP), and paralytic shellfish poisoning (PSP). Natural intoxications are both highly regional and species associated, and toxins are present in seafood at the time of capture.

Ciguatera is a sometimes severe disease caused by consuming certain species of fish, usually island or reef, from tropical, subtropical, and other (temperate) waters, and was responsible for about half of all reported outbreaks to the CDC. Treatments are largely supportive, and mortality is usually low. Controls based on regulation of fishing or marketling of dangerous species, supported by testing suspect fish and educating consumers, sports fishers and health professionals, are recommended.

SFP caused nearly the same number of outbreaks as ciguatera, but was much more widespread. Tuna, mahimahi and bluefish are implicated as the major cause of SFP. The disease is generally mild and self-resolving, and symptoms can often be ameliorated by certain antihistamine drugs. Because the histamine poisoning is produced due to improper temperature control after the fish is caught, the disease can be prevented by rapid cooling and holding fish at low temperature(s) before cooking, as well as by not leaving the fish out too long when serving it under inadequate terperature control. A Hazard Analysis Critical Control Point-based system, and the education of subsistence and recreational/commercial fishers and cooks/servers may control this poisoning.

Paralytic shellfish poisoning was reported to the CDC as a minor cause of illness, with only two deaths. Although “other” natural seafood intoxications (e.g., puffer fish poisoning, and neurotoxic, diarrhetic and amnesic shellfish poisoning) have not been widely reported in U.S. consumers, the potential for their occurrence either from domestic or imported seafoods from imports is real. Increased vigilance concerning imported product, testing for these “other” toxins, and procedures to deal with outbreaks are needed.     

Natural Occurrence of Alternaria Toxins in Agricultural Products and Processed Foods Marketed in South Korea by LC–MS/MS

Alternaria mycotoxins including alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxin-I (ATX-I), tentoxin (TEN), and tenuazonic acid (TeA), are ubiquitous contaminants in agricultural products. A method for the simultaneous determination of these six toxins by ultrahigh performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) with solid phase extraction (SPE) was validated in rice, sesame, tomato, and apple juice matrices. The performance of the method was evaluated in terms of linearity (R² > 0.999), the limit of detection (0.04–1.67 μg/kg), the limit of quantification (0.12–5.06 μg/kg), recovery (80.0–114.7%), and precision (<17.7%). The validated method was applied to monitor 152 marketed food samples in South Korea, as well as to investigate the co-occurrence and correlation between Alternaria toxins. The mean occurrence levels were 2.77 μg/kg for AOH, 4.36 μg/kg for AME, 0.14 μg/kg for ALT, 0.11 μg/kg for ATX-I, 0.43 μg/kg for TEN, and 104.56 μg/kg for TeA. Mean and extreme (95th percentile) daily dietary exposures of South Koreans to Alternaria toxins were estimated to be 22.93 ng/kg b.w./day and 86.07 ng/kg b.w./day, respectively.     

Abstracts from Other Presentations at the International Symposium on Toxins and Natural Products in Honor of Professor Anthony T. Tu

Investigation of the Structure, Dynamics, and Folding of Snake Venom Proteins

Chin Yu* and T. K. C. Kumar

Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan

Cardiotoxins isolated from Taiwan cobra venom (Naja naja atra) are small molecular weight proteins (～ 7 kDa), containing four disulfide bonds. To‐date, different cardiotoxin isoforms (CTXI, CTXII, CTX III, CTXIV, and CTXV) have been isolated from the venom of Naja naja atra. The three‐dimensional structures of all the five cardiotoxin isoforms have been solved by multidimensional NMR techniques. Critical comparison of the structures of cardiotoxins reveal a common structural feature responsible for the lethal activity of cardiotoxins. Although the cardiotoxins show very high structural homology they exhibit significant differences in their lethal potencies. The observed differences in the lethal potencies are found to depend on the degree of exposure of the positive charge of an invariant lysine. Backbone dynamics of CTXIII has been studied by carbon‐13 relaxation measurements at natural abundance. The overall rotational correlation co‐efficient of the protein has been estimated to be 4.8 ns. Most of the residues in CTXIII have been observed to exhibit fast (τ_e < 30 ps) restricted motions (S² = 0.79–0.89). The functional important residues located at the tips of three lops are relatively flexible. The structural stability of CTXIII had been probed by hydrogen–deuterium exchange monitored by NMR spectroscopy. Among the five beta strands in the toxin, beta strand III is found to constitute the stability core. The stability of the triple stranded beta‐sheet domain is markedly higher than that of the double stranded beta‐sheet domain. The refolding of CTXIII monitored by a variety of biophysical techniques reveals that the toxin refolds completely within a time span of 200 milliseconds. The chronology of the folding events in CTXIII monitored by quenched‐flow H/D exchange shows that the triple‐stranded beta‐sheet domain folds faster than the double stranded beta‐sheet domain. These results will be extensively discussed.