Suresh C. Swain

Systems toxicology approaches for understanding the joint effects of environmental chemical mixtures

Environmental mixtures of chemicals constitute a prevalent issue in ecotoxicology and the development of new methods to reduce the uncertainties associated with their ecological risk assessment is a critical research need. Historically, a number of models have been explored to predict the potential combined effects of chemicals on species. These models, especially concentration addition and the independent action, have been applied to a number of mixtures. While often providing a good prediction of joint effect, there are cases where these models can have limitations: notably in cases where there are interactions for which they fail to adequately predict joint effects. To support the better mechanistic understanding of interactions in mixture toxicology a framework to support experimental studies to investigate the basis of observed interactions is proposed. The conceptual framework is derived from the extension of a three stage scheme which has previously been applied to understand chemical bioavailability. The framework considers that interactions in mixtures result from processes related to 1) the speciation, binding and transport of chemicals in the exposure medium (external exposure); 2) the adsorption, distribution, metabolism and excretion of chemicals within the organisms (toxicokinetics); 3) associations governing the binding and toxicity of the chemical(s) at the target site (toxicodynamics). The current state of the art in (eco)toxicology in relation to investigation of the mechanisms of interactions between chemicals is discussed with particular emphasis towards the multi-disciplinary tools and techniques within environmental chemistry; toxicology; biochemistry and systems biology that can be used to address such effects.

Gene expression profiling to characterize sediment toxicity – a pilot study using Caenorhabditis elegans whole genome microarrays

BackgroundTraditionally, toxicity of river sediments is assessed using whole sediment tests with benthic organisms. The challenge, however, is the differentiation between multiple effects caused by complex contaminant mixtures and the unspecific toxicity endpoints such as survival, growth or reproduction. The use of gene expression profiling facilitates the identification of transcriptional changes at the molecular level that are specific to the bio-available fraction of pollutants.ResultsIn this pilot study, we exposed the nematode Caenorhabditis elegans to three sediments of German rivers with varying (low, medium and high) levels of heavy metal and organic contamination. Beside chemical analysis, three standard bioassays were performed: reproduction of C. elegans, genotoxicity (Comet assay) and endocrine disruption (YES test). Gene expression was profiled using a whole genome DNA-microarray approach to identify overrepresented functional gene categories and derived cellular processes. Disaccharide and glycogen metabolism were found to be affected, whereas further functional pathways, such as oxidative phosphorylation, ribosome biogenesis, metabolism of xenobiotics, aging and several developmental processes were found to be differentially regulated only in response to the most contaminated sediment.ConclusionThis study demonstrates how ecotoxicogenomics can identify transcriptional responses in complex mixture scenarios to distinguish different samples of river sediments.

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

BackgroundPhysiologically 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).ResultsFor 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.ConclusionsOur 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.

Potential New Method of Mixture Effects Testing Using Metabolomics and Caenorhabditis elegans

The development of superior tools for molecular and computational biology in recent years has provided an opportunity for the creation of faster toxicological screens that are relevant for, but do not rely on, mammalian systems. In this study, NMR spectroscopy and GC-MS based metabolomics have been used in conjunction with multivariate statistics to examine the metabolic changes in the nematode Caenorhabditis elegans following exposure to different concentrations of the heavy metal nickel, the pesticide chlorpyrifos, and their mixture. Novel metabolic profiles were associated with both exposure and dose level. The biochemical responses were more closely matched when exposure was at the same effect level, even for different chemicals, than when exposure was for different levels of the same chemical (e.g., low versus high dose). Responses to the mixture reflected the contribution of the chemicals to the overall exposure. In common with the metabolic responses of several other species exposed to the same chemicals, we observed changes in branch chain amino acids and tricarboxylic acid cycle intermediates. These results form the basis for a rapid and economically viable toxicity test that defines the molecular effects of pollution/toxicant exposure in a manner that is relevant to higher vertebrates.

Hydrogen Sulfide Is an Endogenous Regulator of Aging in Caenorhabditis elegans

AIMS To investigate the role of endogenous hydrogen sulfide (H2S) in the control of aging and healthspan of Caenorhabditis elegans. RESULTS We show that the model organism, C. elegans, synthesizes H2S. Three H2S-synthesizing enzymes are present in C. elegans, namely cystathionine γ lyase (CSE), cystathionine β synthetase, and 3-mercaptopyruvate transferase (MPST or 3-MST). Genetic deficiency of mpst-1 (3-MST orthologue 1), but not cth-2 (CSE orthologue), reduced the lifespan of C. elegans. This effect was reversed by a pharmacological H2S donor (GYY4137). GYY4137 also reduced detrimental age-dependent changes in a range of physiological indices, including pharyngeal contraction and defecation. Treatment of C. elegans with GYY4137 increased the expression of several age-related, stress response, and antioxidant genes, whereas MitoSOX Red fluorescence, indicative of reactive oxygen species generation, was increased in mpst-1 knockouts and decreased by GYY4137 treatment. GYY4137 additionally increased the lifespan in short-lived mev-1 mutants with elevated oxidative stress and protected wild-type C. elegans against paraquat poisoning. The lifespan-prolonging and health-promoting effects of H2S in C. elegans are likely due to the antioxidant action of this highly cell-permeable gas. INNOVATION The possibility that novel pharmacological agents based on the principle of H2S donation may be able to retard the onset of age-related disease by slowing the aging process warrants further study. CONCLUSION Our results show that H2S is an endogenous regulator of oxidative damage, metabolism, and aging in C. elegans and provide new insight into the mechanisms, which control aging in this model organism.

A metabolomics based test of independent action and concentration addition using the earthworm Lumbricus rubellus

A major challenge in ecotoxicology is to understand the effects of multiple toxicants on organisms. Here we assess the effects on survival, weight change, cocoon production and metabolism caused by exposure to two similarly acting (imidacloprid/thiacloprid) and two dissimilarly acting (chlorpyrifos/Nickel) chemicals on the earthworm Lumbricus rubellus. We assessed the standard models of concentration addition (CA) and independent action (IA), in conjunction with a metabolomics based approach to elucidate mechanisms of effect. For imidacloprid and thiacloprid the reproductive effects indicated probable additivity. Although this suggests joint effects through a similar mechanism, metabolite changes for each pesticide actually indicated distinct effects. Further, earthworms exposed to a 0.5 toxic unit equitoxic mixture demonstrated metabolic effects intermediate between those for each pesticide, indicating a non-interactive, independent joint effect. For higher effect level mixtures (1 and 1.5 toxic units), metabolite changes associated with thiacloprid exposure began to dominate. The metabolomic effects of the two dissimilarly acting chemicals were distinct, confirming separate modes of action and both proved more toxic than anticipated from previous studies. In the mixtures, phenotypic effects were in accordance with IA estimates, while metabolite changes were dominated by Ni effects, even though chlorpyrifos contributed most to reproductive toxicity. This could be attributed to the greater systematic effect of Ni when compared to the more specifically acting chlorpyrifos.

Knock down of caenorhabditis elegans cutc-1 exacerbates the sensitivity toward high levels of copper

Copper, though toxic in excess, is an essential trace element that serves as a cofactor in many critical biological processes such as respiration, iron transport, and oxidative stress protection. To maintain this balance between requirement and toxicity, biological systems have developed intricate systems allowing the preservation of homeostasis while ensuring delivery of copper to the appropriate cellular component. The nematode Caenorhabditis elegans was exploited to assess the effects of copper toxicity at the population level to identify key changes in life cycle traits including, lethality, brood size, generation time, growth, and life span. To enhance our understanding of the complexities of copper homeostasis at the genetic level, the expression profile and functional significance of a putative copper cytoplasmic metallochaperone cutc-1 were analyzed. Using quantitative PCR technology, cutc-1 was found to be downregulated with increasing CuSO(4) concentrations. However, although total (whole body) copper levels increased in nematodes exposed to elevated levels of copper, wild-type and knock down of cutc-1 by RNA-mediated interference (RNAi) were statistically indistinguishable. Nevertheless, RNAi of cutc-1 affected brood size, growth and induced a marked increase in protruding vulva and bagging phenotypes at higher copper exposures. This indicates that cutc-1 plays a crucial role in the protection from excess copper.

Application of physiologically based modelling and transcriptomics to probe the systems toxicology of aldicarb for Caenorhabditis elegans (Maupas 1900)

The toxicity of aldicarb on movement, life cycle, population growth rate and resource allocation, and the gene expression changes underpinning these effects, were investigated for Caenorhabditis elegans. A clear effect of aldicarb on nematode movement was found suggesting that this pesticide acts as a neurotoxicant. Aldicarb also had an effect on life cycle traits including low concentration life-span extension; high concentration brood size reduction and a high concentration extension of time to first egg. All life-cycle and growth data were integrated into a biology-based model (DEBtox) to characterise aldicarb effects on life-history traits, resource allocation and population growth rate within a single modelling framework. The DEBtox fits described concentration dependent effects on individual traits and population growth rate and indicated that the most probable mechanism of action of the pesticide was an increase in energy demands for somatic and reproductive tissue maintenance. Transcriptomic profiling indicated that aldicarb was associated with changes in amino acid metabolism, DNA structure, fatty acid metabolism and cytochrome P450 mediated xenobiotic metabolism. The changes in the amino acid and fatty acid pathways suggest an effect of aldicarb on protein integrity; while effects on DNA suggests that aldicarb influence DNA morphology or replication. Both these effects have the potential to incur increased costs for structural maintenance of macromolecules. These effects, coupled to the effect on biotransformation enzymes also seen, represent the materialisation of the maintenance costs indicated by DEBtox modelling.

Meta-analysis of global transcriptomics suggests that conserved genetic pathways are responsible for Quercetin and Tannic acid mediated longevity in C. elegans

Recent research has highlighted that the polyphenols Quercetin and Tannic acid are capable of extending the lifespan of Caenorhabditis elegans. To gain a deep understanding of the underlying molecular genetics, we analyzed the global transcriptional patterns of nematodes exposed to three concentrations of Quercetin or Tannic acid, respectively. By means of an intricate meta-analysis it was possible to compare the transcriptomes of polyphenol exposure to recently published datasets derived from (i) longevity mutants or (ii) infection. This detailed comparative in silico analysis facilitated the identification of compound specific and overlapping transcriptional profiles and allowed the prediction of putative mechanistic models of Quercetin and Tannic acid mediated longevity. Lifespan extension due to Quercetin was predominantly driven by the metabolome, TGF-beta signaling, Insulin-like signaling, and the p38 MAPK pathway and Tannic acid’s impact involved, in part, the amino acid metabolism and was modulated by the TGF-beta and the p38 MAPK pathways. DAF-12, which integrates TGF-beta and Insulin-like downstream signaling, and genetic players of the p38 MAPK pathway therefore seem to be crucial regulators for both polyphenols. Taken together, this study underlines how meta-analyses can provide an insight of molecular events that go beyond the traditional categorization into gene ontology-terms and Kyoto encyclopedia of genes and genomes-pathways. It also supports the call to expand the generation of comparative and integrative databases, an effort that is currently still in its infancy.

Bio-electrospraying the nematode Caenorhabditis elegans: studying whole-genome transcriptional responses and key life cycle parameters

Bio-electrospray, the direct jet-based cell handling approach, is able to handle a wide range of cells (spanning immortalized, primary to stem cells). Studies at the genomic, genetic and the physiological levels have shown that, post-treatment, cellular integrity is unperturbed and a high percentage (more than 70%, compared with control) of cells remain viable. Although, these results are impressive, it may be argued that cell-based systems are oversimplistic. Therefore, it is important to evaluate the bio-electrospray technology using sensitive and dynamically developing multi-cellular organisms that share, at least some, similarities with multi-cell microenviorments encountered with tissues and organs. This study addressed this issue by using a well-characterized model organism, the non-parasitic nematode Caenorhabditis elegans. Nematode cultures were subjected to bio-electrospraying and compared with positive (heat shock) and negative controls (appropriate laboratory culture controls). Overall, bio-electrospraying did not modulate the reproductive output or induce significant changes in in vivo stress-responsive biomarkers (heat shock proteins). Likewise, whole-genome transcriptomics could not identify any biological processes, cellular components or molecular functions (gene ontology terms) that were significantly enriched in response to bio-electrospraying. This demonstrates that bio-electrosprays can be safely applied directly to nematodes and underlines its potential future use in the creation of multi-cellular environments within clinical applications.