Low potency toxins reveal dense interaction networks in metabolism

Background The chemicals of metabolism are constructed of a small set of atoms and bonds. This may be because chemical structures outside the chemical space in which life operates are incompatible with biochemistry, or because mechanisms to make or utilize such excluded structures has not evolved. In this paper I address the extent to which biochemistry is restricted to a small fraction of the chemical space of possible chemicals, a restricted subset that I call Biochemical Space. I explore evidence that this restriction is at least in part due to selection again specific structures, and suggest a mechanism by which this occurs. Results Chemicals that contain structures that our outside Biochemical Space (UnBiological groups) are more likely to be toxic to a wide range of organisms, even though they have no specifically toxic groups and no obvious mechanism of toxicity. This correlation of UnBiological with toxicity is stronger for low potency (millimolar) toxins. I relate this to the observation that most chemicals interact with many biological structures at low millimolar toxicity. I hypothesise that life has to select its components not only to have a specific set of functions but also to avoid interactions with all the other components of life that might degrade their function. Conclusions The chemistry of life has to form a dense, self-consistent network of chemical structures, and cannot easily be arbitrarily extended. The toxicity of arbitrary chemicals is a reflection of the disruption to that network occasioned by trying to insert a chemical into it without also selecting all the other components to tolerate that chemical. This suggests new ways to test for the toxicity of chemicals, and that engineering organisms to make high concentrations of materials such as chemical precursors or fuels may require more substantial engineering than just of the synthetic pathways involved

In vitro and in vivo toxicity study of compounds of natural origin: marine biotoxins

This doctoral thesis provides data on different phases necessary for a complete risk assessment of the most common marine biotoxins in Galicia: okadaic acid (OA), their derivatives the dinophysistoxins (DTXs), the azaspiracids (AZAs), and also the emerging toxin: tetrodotoxin (TTX). For this, elimination of the toxic source from the natural environment, the passage of toxins through the gastrointestinal tract, toxic effects, and oral toxicity studies are evaluated. Natural clays with smaller particle size result more efficient in eliminating dinoflagellates producing toxins. Azaspiracid-1 (AZA1) crosses the intestinal barrier in a dose- dependent manner causing alterations in intestinal epithelial cells. In vivo studies reveal highest toxicity for DTX1 and lowest for DTX2 with respect to OA, while a relationship between the diarreal effect of OA and its neuronal action is suggested in vitro. Besides, this is the first time that oral lethal dose 50 and NOAEL (No-Observed-Adverse-Effect-Level) values for TTX are established

Genomic and Proteomic Analyses of the Fungus Arthrobotrys oligospora Provide Insights into Nematode-Trap Formation

Nematode-trapping fungi are “carnivorous” and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions

The biotechnological value of a novel potent marine biotoxin from the polychaete worm Eulalia viridis: chemical and toxicological evaluation

The ocean’s vastness holds still many unexplored organisms with unique adaptive features that enable them to thrive in their environment. Eulalia viridis is one of them. This uncanny bright green worm is a predatory marine Polychaeta of the rocky intertidal that lacks jaws, but it is equipped with a toxin-containing mucus that enables predating larger invertebrates from which soft tissue is extracted through suction. Motivated by the high-value of toxins as bioreactives and by the European Union’s ambitions of leading the Blue Growth Strategy, for which Portugal’s vast EEZ offers high perspectives; a case-study to explore the biotechnological potential of this species was undertaken. The worm’s microanatomy revealed key adaptations for feeding such as special sensorial papillae and cells responsible for the secretion of mucus and toxins. Whole-transcriptome sequencing and toxicity assays yielded a complex pattern of proteinaceous toxins and enzymes with several functions: tissue permeabilization, coagulation impairment and blocking of neuromuscular activity. The main neurotoxins, “phyllotoxins”, found to be cysteine-rich proteins, act as immobilising agents against mussels and other invertebrate prey and are delivered by contact with mucus. In turn, higher toxicity towards ovarian cancer cell line (A2780) than normal cells, involved cytotoxic and cytostatic properties through cell cycle arrest and extrinsic programmed cell death. Several proteins involved in these effects were identified by combining transcriptomics and proteomics. The worm’s mucosecretions also hold internalisable proteinaceous complexes that display fluorescent properties and whose emission is reversibly switched by redox status. Altogether, the features found in the secretions of a single species disclose the immense biotechnological potential of marine annelids, and invertebrates in general, as source of valuable bio-reactives, even in temperate waters. The methodological pipeline, combining ecology, toxicology and molecular biology, circumvented many difficulties of marine bioprospecting and is an important contribution for the sustainable exploitation of novel marine bioresources.O oceano contém muitos organismos por explorar com características adaptativas que os permitem prosperar no seu meio ambiente. Eulalia viridis é um desses organismos. Esta poliqueta verde clara é predador do intertidal rochoso sem mandibulas, equipado dum muco tóxico, permitindo assim predar invertebrados maiores extraindo pedaços de tecido através da sucção. Motivado pelo valor das toxinas como substâncias bioreactivas e pela ambição da UE de conduzir a Estratégia para o Crescimento Azul, à qual a vasta ZEE portuguesa oferece grandes perspetivas, o potencial biotecnológico desta espécie foi analisado. A análise microanatómica desta minhoca revelou adaptações cruciais na alimentação: existência de papilas sensoriais e células responsáveis pela secreção de muco e toxinas. A sequenciação completa do transcritoma e ensaios toxicológicos revelaram um complexo padrão de toxinas proteicas e enzimas com diversas funções: permeabilização de tecidos, anti-coagulação e bloqueio da atividade neuromuscular. As principais neurotoxinas da Eulalia, “filotoxinas”, são proteínas ricas em cisteínas, que atuam como agentes imobilizadores em mexilhões e outros invertebrados, que são administradas por contacto através do muco. Por sua vez, uma maior toxicidade para com a linha celular do cancro do ovário (A2780) do que células normais, demonstraram propriedades citotóxicas e citoestáticas através da paragem do ciclo celular e indução de morte celular programada pela via extrínseca. As proteínas responsáveis por estes efeitos foram identificadas, através de transcritómica e proteómica. As secreções mucosas da minhoca contêm também complexos proteicos internalizáveis que apresentam fluorescência, cuja emissão é reversivelmente regulada pelo estado redox. Resumindo, as características encontradas nas secreções de uma espécie demonstram o vasto potencial biotecnológico de anelídeos marinhos, como uma fonte valiosa de bioreactivos, mesmo em águas temperadas. O percurso metodológico utilizado, a combinação da ecologia, toxicologia e biologia molecular, permitiu contornar dificuldades da bioprospecção marinha, revelando ser um importante contributo para exploração sustentável de novos biorecursos marinhos

The Contribution of Acyl Amides to the Toxicity of the Harmful Alga Prymnesium parvum

The golden alga Prymnesium parvum has been implicated in fish and aquatic animal kills globally for over a century. In addition to widespread ecological impacts through the loss of entire fish populations within lakes, an economic burden is also felt by state and local agencies due to losses of fish raised for stocking lakes and the loss of fishing and recreational use of the affected water body. Multiple compounds have been implicated in P. parvum toxicity, but the unequivocal identification and characterization of all P. parvum toxins remains to be accomplished. In this work we isolated and structurally characterized toxic metabolites from P. parvum, examining uni-algal laboratory cultures and field collections of algal biomass from fish kill sites. An aggregate of saturated and unsaturated acyl amides are a previously undescribed class of P. parvum toxins. The amount of acyl amides present at multiple P. parvum bloom sites and reference sites was quantified. Further analysis of these compounds through selected bioassays demonstrated that acyl amides were cytotoxic, hemolytic, and ichthyotoxic, with the gill as the site of action. In addition, we demonstrated that the presence of divalent cations and increased pH significantly increased the toxicity of unsaturated acyl amides. Moreover, increased pH decreased the toxicity of fatty acids, a class of compounds previously implicated in P. parvum toxicity. Multiple abiotic and biotic factors are associated with P. parvum blooms and the toxicity of these blooms. We analyzed the effect of multiple physicochemical parameters on the toxicity of acyl amides using an artificial neural network and linear statistical tests. Our results demonstrated that increasing pH is significantly associated with an increase in the toxicity of acyl amides. Mixtures of environmentally relevant concentrations of unsaturated acyl amides at ecologically relevant pH levels resulted in a significant loss of viability of rainbow trout gill cells. Our results have identified a new toxin class from P. parvum. This is the first report to demonstrate that a toxin class implicated in P. parvum toxicity accumulates to lethal levels in the environment. We have developed a new method for analyzing complex mixtures that can be utilized to assess the effect of multiple chemical and physical factors on toxicity. These results highlight the potential role of physicochemical factors and their effect on algal toxins after they are released into the environment, illustrating the necessity to examine toxin chemistry in an environmentally relevant context

Abundant toxin-related genes in the genomes of beneficial symbionts from deep-sea hydrothermal vent mussels

Bathymodiolus mussels live in symbiosis with intracellular sulfur-oxidizing (SOX) bacteria that provide them with nutrition. We sequenced the SOX symbiont genomes from two Bathymodiolus species. Comparison of these symbiont genomes with those of their closest relatives revealed that the symbionts have undergone genome rearrangements, and up to 35% of their genes may have been acquired by horizontal gene transfer. Many of the genes specific to the symbionts were homologs of virulence genes. We discovered an abundant and diverse array of genes similar to insecticidal toxins of nematode and aphid symbionts, and toxins of pathogens such as Yersinia and Vibrio. Transcriptomics and proteomics revealed that the SOX symbionts express the toxin-related genes (TRGs) in their hosts. We hypothesize that the symbionts use these TRGs in beneficial interactions with their host, including protection against parasites. This would explain why a mutualistic symbiont would contain such a remarkable 'arsenal' of TRG

UNDERSTANDING CONDITIONAL MODES OF ACTIONS IN CHEMICAL-INDUCED TOXICITY USING RULE MODELS

It is estimated that 115 million animals are used in experimental testing each year. Hence, shifting efforts toward alternative methods for toxicity assessment is essential. However, slow regulatory acceptance of new approaches is governed by knowledge gaps in toxicity modes of action. In this thesis, I describe these challenges and the use of in vitro screening as an alternative of animal testing. I also discuss common data-based methods to derive hypotheses about toxicity modes of actions, and the associated limitations in capturing multiple biological perturbations. I applied novel data-based workflows, using rule models, to prioritize in vitro assays predictive of toxicity as well as to detect significant polypharmacology profiles. I explain how constraints were applied to rule-based models to inform meaningful mechanistic interpretation for two toxicity endpoints: rat hepatotoxicity and acute toxicity. I compared assays selected, by rules, for predicting hepatotoxicity with endpoints used in in vitro models from commercial sources. An overlap was observed including cytochrome activity, mitochondrial toxicity and immunological responses. However, nuclear receptor activity, identified in rules, is not currently covered in commercial setups. I also demonstrate that endocrine disruption endpoints extrapolate better into in vivo toxicity when a set of specific conditions are met, such as physicochemical properties associated with good bioavailability. Next, I examined synergistic interactions between conditions in rules describing acute toxicity. I gained novel insights into how specific stressors potentiate the perturbation by known key events, such as acetylcholinesterase inhibition and neuro-signalling disruption. I show that examining polypharmacology profiles is particularly important at low bioactive potencies. Further, the overall predictive performance of rules describing acute toxicity was tested against a benchmark Random Forest model in a conformal prediction framework. Irrespective to the data type used in the training, the models were prone to bias over compounds promiscuity, by which high promiscuous compounds were more likely to be predicted as toxic. Overall, the studies conducted in this thesis provide novel insights into molecular mechanisms of toxicity, namely hepatotoxicity and acute toxicity, and with regards to chemical properties and polypharmacology. This knowledge can be used to improve the utility and design of alternative methods for toxicity, and hence, accelerate the regulatory acceptance.Islamic Development Bank Cambridge Trust Fun