Linking toxicant physiological mode of action with induced gene expression changes in Caenorhabditis elegans

Background Physiologically based modelling using DEBtox (dynamic energy budget in toxicology) and transcriptional profiling were used in Caenorhabditis elegans to identify how physiological modes of action, as indicated by effects on system level resource allocation were associated with changes in gene expression following exposure to three toxic chemicals: cadmium, fluoranthene (FA) and atrazine (AZ). Results For Cd, the physiological mode of action as indicated by DEBtox model fitting was an effect on energy assimilation from food, suggesting that the transcriptional response to exposure should be dominated by changes in the expression of transcripts associated with energy metabolism and the mitochondria. While evidence for effect on genes associated with energy production were seen, an ontological analysis also indicated an effect of Cd exposure on DNA integrity and transcriptional activity. DEBtox modelling showed an effect of FA on costs for growth and reproduction (i.e. for production of new and differentiated biomass). The microarray analysis supported this effect, showing an effect of FA on protein integrity and turnover that would be expected to have consequences for rates of somatic growth. For AZ, the physiological mode of action predicted by DEBtox was increased cost for maintenance. The transcriptional analysis demonstrated that this increase resulted from effects on DNA integrity as indicated by changes in the expression of genes chromosomal repair. Conclusions Our results have established that outputs from process based models and transcriptomics analyses can help to link mechanisms of action of toxic chemicals with resulting demographic effects. Such complimentary analyses can assist in the categorisation of chemicals for risk assessment purposes

Gene Expression Responses Linked to Reproduction Effect Concentrations (EC10,20,50,90) of Dimethoate, Atrazine and Carbendazim, in Enchytraeus albidus

BACKGROUND: Molecular mechanisms of response to pesticides are scarce and information on such responses from soil invertebrates is almost inexistent. Enchytraeus albidus (Oligochaeta) is a standard soil ecotoxicology model species for which effects of many pesticides are known on survival, reproduction and avoidance behaviour. With the recent microarray development additional information can be retrieved on the molecular effects. METHODOLOGY/PRINCIPAL FINDINGS: Experiments were performed to investigate the transcription responses of E. albidus when exposed to three pesticides - dimethoate (insecticide), atrazine (herbicide) and carbendazim (fungicide) - in a range of concentrations that inhibited reproduction by 10%, 20%, 50% and 90% (EC(10), EC(20), EC(50) and EC(90), respectively). The goal of this study was to further identify key biological processes affected by each compound and if dose-related. All three pesticides significantly affected biological processes like translation, regulation of the cell cycle or general response to stress. Intracellular signalling and microtubule-based movement were affected by dimethoate and carbendazim whereas atrazine affected lipid and steroid metabolism (also by dimethoate) or carbohydrate metabolism (also by carbendazim). Response to DNA damage/DNA repair was exclusively affected by carbendazim. CONCLUSIONS: Changes in gene expression were significantly altered after 2 days of exposure in a dose-related manner. The mechanisms of response were comparable with the ones for mammals, suggesting across species conserved modes of action. The present results indicate the potential of using gene expression in risk assessment and the advantage as early markers

Phenotypic responses in caenorhabditis elegans following Chronic low-level exposures to inorganic and organic Compounds

Responses of organisms to sub-lethal exposure of environmental stressors can be difficult to detect. We investigated phenotypic changes in the tissue of Caenorhabditis elegans via Raman spectroscopy, as well as survival and reproductive output when exposed to chronic low dose of exposure metals (copper, zinc or silver), an herbicide (diuron) and a pesticide (imidacloprid). Raman spectroscopy measures changes in phenotype by providing information about the molecular composition and relative abundance of biomolecules. Multivariate analysis was used to evaluate the significance of treatment phenotype segregation plots compared to controls. Dose-dependent responses were observed for copper, zinc, silver and diuron while imidacloprid exposure resulted in a small response over the tested concentrations. Concentrationdependent shifts in nematode biomolecular phenotype were observed for copper. Despite having a dose–dependent reproductive response, silver, diuron and imidaclorprid had inconsistent biological phenotype patterns. In contrast, there was a clear stepwise change between low concentrations (0.00625 - 0.5 mg/L) and higher concentration (1 – 2mg/L) of ionic zinc. The findings demonstrate that measuring phenotypic responses via Raman spectroscopy can provide an insight into the biomolecular mechanisms of toxicity. Despite the lack of consistency between survival and Raman-measured phenotypic changes, the results supports the effectiveness of Raman spectroscopy and multivariate analysis to detect sub-lethal responses of chemicals on whole organisms and to identify toxic effect thresholds

Gene-environment and protein degradation signatures characterize genomic and phenotypic diversity in wild Caenorhabditis elegans populations

Background: Analyzing and understanding the relationship between genotypes and phenotypes is at the heart of genetics. Research on the nematode Caenorhabditis elegans has been instrumental for unraveling genotype-phenotype relations, and has important implications for understanding the biology of mammals, but almost all studies, including forward and reverse genetic screens, are limited by investigations in only one canonical genotype. This hampers the detection and functional analysis of allelic variants, which play a key role in controlling many complex traits. It is therefore essential to explore the full potential of the natural genetic variation and evolutionary context of the genotype-phenotype map in wild C. elegans populations. Results: We used multiple wild C. elegans populations freshly isolated from local sites to investigate gene sequence polymorphisms and a multitude of phenotypes including the transcriptome, fitness, and behavioral traits. The genotype, transcriptome, and a number of fitness traits showed a direct link with the original site of the strains. The separation between the isolation sites was prevalent on all chromosomes, but chromosome V was the largest contributor to this variation. These results were supported by a differential food preference of the wild isolates for naturally co-existing bacterial species. Comparing polymorphic genes between the populations with a set of genes extracted from 19 different studies on gene expression in C. elegans exposed to biotic and abiotic factors, such as bacteria, osmotic pressure, and temperature, revealed a significant enrichment for genes involved in gene-environment interactions and protein degradation. Conclusions: We found that wild C. elegans populations are characterized by gene-environment signatures, and we have unlocked a wealth of genotype-phenotype relations for the first time. Studying natural isolates provides a treasure trove of evidence compared with that unearthed by the current research in C. elegans, which covers only a diminutive part of the myriad of genotype-phenotype relations that are present in the wild

Dos genes à população: efeitos de substâncias tóxicas em Enchytraeus albidus

Doutoramento em BiologiaNowadays, a systems biology approach is both a challenge as well as believed to be the ideal form of understanding the organisms’ mechanisms of response. Responses at different levels of biological organization should be integrated to better understand the mechanisms, and hence predict the effects of stress agents, usable in broader contexts. The main aim of this thesis was to evaluate the underlying mechanisms of Enchytraeus albidus responses to chemical stressors. Therefore, there was a large investment on the gene library enrichment for this species, as explained ahead. Overall, effects of chemicals from two different groups (metals and pesticides) were assessed at different levels of biological organization: from genes and biochemical biomarkers to population endpoints. Selected chemicals were: 1) the metals cadmium and zinc; 2) the insecticide dimethoate, the herbicide atrazine and the fungicide carbendazim. At the gene and sub-cellular level, the effects of time and dosage were also adressed. Traditional ecotoxicological tests - survival, reproduction and avoidance behavior - indicated that pesticides were more toxic than metals. Avoidance behaviour is extremely important from an ecological point of view, but not recommended to use for risk assessment purposes. The oxidative stress related experiment showed that metals induced significant effects on several antioxidant enzyme activities and substrate levels, as well as oxidative damage on the membrane cells. To increase the potential of our molecular tool to assess transcriptional responses, the existing cDNA library was enriched with metal and pesticide responding genes, using Suppression Subtractive Hybridization (SSH). With the sequencing information obtained, an improved Agilent custom oligonucleotide microarray was developed and an EST database, including all existing molecular data on E. albidus, was made publicly available as an interactive tool to access information. With this microarray tool, most interesting and novel information on the mechanisms of chemical toxicity was obtained, with the identification of common and specific key pathways affected by each compound. The obtained results allowed the identification of mechanisms of action for the tested compounds in E. albidus, some of which are in line with the ones known for mammals, suggesting across species conserved modes of action and underlining the usefulness of this soil invertebrate as a model species. In general, biochemical and molecular responses were influenced by time of exposure and chemical dosage and these allowed to see the evolution of events. Cellular energy allocation results confirmed the gene expression evidences of an increased energetic expenditure, which can partially explain the decrease on the reproductive output, verified at a later stage. Correlations found throughout this thesis between effects at the different levels of biological organization have further improved our knowledge on the toxicity of metals and pesticides in this species.Actualmente, em ecotoxicologia, uma abordagem ao nível da biologia de sistemas representa não só um desafio, como se acredita ser a forma ideal para a compreensão dos mecanismos de resposta dos organismos. A integração de respostas de diferentes níveis de organização biológica, permite a melhor percepção dos mecanismos envolvidos, e assim possibilita a previsão de efeitos de agentes tóxicos num contexto alargado. O objectivo principal desta tese foi a avaliação dos mecanismos de resposta de Enchytraeus albidus a stressores químicos. Desta forma, um grande investimento foi feito de forma a enriquecer a biblioteca genómica desta espécie, como explicado mais adiante. Em suma, os efeitos de compostos químicos pertencentes a duas classes diferentes (metais e pesticidas) foram avaliados a níveis de organização biológica distintos: desde parâmetros populacionais até bioquímicos e moleculares com a alteração de expressão genética. Os compostos seleccionados foram: 1) os metais cádmio e zinco; 2) o insecticida dimetoato, herbicida atrazina e fungicida carbendazim. Ao nível sub-cellular e genético, os efeitos do tempo de exposição e da dose aplicada também foram avaliados. Os testes de ecotoxicologia tradicionais – sobrevivência, reprodução e evitamento – mostraram uma maior toxicidade dos pesticidas em relação aos metais. O evitamento é um parâmetro de extrema importância do ponto de vista ecológico, apesar de não recomendado para avaliação de risco. O estudo dos indicadores de stress oxidativo revelou que o Zn e o Cd induziram efeitos significativos na actividade de várias enzimas antioxidantes e níveis de substratos, provocando igualmente danos oxidativos nas membranas celulares. De forma a aumentar as potencialidades da nossa ferramenta molecular disponível para avaliar respostas ao nível da transcrição, a biblioteca de cDNA existente foi enriquecida com genes de resposta a metais e a pesticidas, usando o método de SSH. A partir das sequências obtidas foi desenvolvido um microarray de oligonucleótidos (Agilent), assim como uma base de dados de utilização gratuita na internet que reúne todos os dados moleculares disponíveis para E. albidus e constitui uma ferramenta interactiva de acesso a informação. Com a aplicação do novo microarray, foram obtidos dados novos e relevantes acerca dos mecanismos de toxicidade, tendo sido possível a identificação de diferentes vias metabólicas afectadas por cada composto químico. Os resultados obtidos permitiram a identificação de mecanismos de acção destes compostos em E.albidus que, em grande parte, coincidem com mecanismos descritos para mamíferos, sugerindo que os modos de acção são conservados em várias espécies e sublinhando a utilidade deste invertebrado como espécie modelo. De um modo geral, as respostas bioquímicas e moleculares foram influenciadas pelo tempo de exposição e concentração do composto tóxico, permitindo seguir a evolução dos eventos. Os resultados da determinação da alocação energética confirmaram as evidências de um aumento de gastos energéticos, sugeridas pelo microarray, e que podem explicar parcialmente o decréscimo na reprodução verificado numa fase posterior. As correlações encontradas no decorrer desta tese entre parâmetros de vários níveis de organização biológica, contribuíram para uma melhor compreensão da toxicidade de metais e pesticidas nesta espécie

Identification and characterization of molecular modulators of methylmercury-induced toxicity and dopamine neuron degeneration in Caenorhabditis elegans

Indiana University-Purdue University Indianapolis (IUPUI)Methylmercury (MeHg) exposure from occupational, environmental and food sources is a significant threat to public health. MeHg poisonings in adults may result in severe psychological and neurological deficits, and in utero exposures can confer significant damage to the developing brain and impair neurobehavioral and intellectual development. Recent epidemiological and vertebrate studies suggest that MeHg exposure may contribute to dopamine (DA) neuron vulnerability and the propensity to develop Parkinson’s disease (PD). I have developed a novel Caenorhabditis elegans (C. elegans) model of MeHg toxicity and have shown that low, chronic exposure confers embryonic defects, developmental delays, reduction in brood size, decreased animal viability and DA neuron degeneration. Toxicant exposure results in an increase in reactive oxygen species (ROS) and the robust induction of several glutathione-S-transferases (GSTs) that are largely dependent on the PD-associated phase II antioxidant transcription factor SKN-1/Nrf2. I have also shown that SKN-1 is expressed in the DA neurons, and a reduction in SKN-1 gene expression increases MeHg-induced animal vulnerability and DA neuron degeneration. Furthermore, I incorporated a novel genome wide reverse genetic screen that identified 92 genes involved in inhibiting MeHg-induced animal death. The putative multidrug resistance protein MRP-7 was identified in the screen. I have shown that this transporter is likely expressed in DA neurons, and reduced gene expression increases cellular Hg accumulation and MeHg-associated DA neurodegeneration. My studies indicate that C. elegans is a useful genetic model to explore the molecular basis of MeHg-associated DA neurodegeneration, and may identify novel therapeutic targets to address this highly relevant health issue