[Show abstract][Hide abstract] ABSTRACT: Humans are routinely exposed to mixtures of flame retardants (FRs) from multiple sources including indoor dust. As a model to explore the potential effects of FR exposure from indoor dust on human health, the molecular responses of human hepatoma cells (HepG2/C3A cells) to a defined mixture of FRs and to a dust extract were investigated using multiple non-targeted omics approaches. A solvent extract of an indoor dust standard reference material SRM2585 was used as the surrogate dust sample, while a mixture of four FRs (TCEP, TCIPP, TDCIPP and HBCD) was used to mimic the FR mixture in the indoor dust. Cytotoxicity tests indicated there were no significant changes to cell viability or cell integrity after a 24- or 72-h exposure of HepG2/C3A cells to the FR mixture or to the dust extract. However, transcriptomics revealed changes in gene expression associated with the metabolism of xenobiotics (e.g. CYP1A1, CYP1A2, CYP2B6) in the dust extract group but not in the FR mixture group after a 72-h exposure. Few metabolic or lipidomic changes were detected in response to either the FR mixture or to the dust extract group. Given that the dust extract contained components that elicited a biological response, in contrast to the lack of response induced by the FR mixture, our findings suggest that the most likely causes of the molecular responses to indoor dust exposure lie in components other than the four FRs investigated, e.g. caused by PAHs or PCBs.
[Show abstract][Hide abstract] ABSTRACT: Altered expression of tumor suppressor genes and oncogenes, which is regulated in part at the level of DNA methylation, is an important event involved in non-genotoxic carcinogenesis. This may serve as a marker for early detection of non-genotoxic carcinogens. Therefore, we evaluated the effects of non-genotoxic hepatocarcinogens, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), hexachlorobenzene (HCB), methapyrilene (MPY) and male rat kidney carcinogens, d-limonene, p-dichlorobenzene (DCB), chloroform and ochratoxin A (OTA) on global and CpG island promoter methylation in their respective target tissues in rats. No significant dose-related effects on global DNA hypomethylation were observed in tissues of rats compared to vehicle controls using LC-MS/MS in response to short-term non-genotoxic carcinogen exposure. Initial experiments investigating gene-specific methylation using methylation-specific PCR and bisulfite sequencing, revealed partial methylation of p16 in the liver of rats treated with HCB and TCDD. However, no treatment related effects on the methylation status of Cx32, e-cadherin, VHL, c-myc, Igfbp2, and p15 were observed. We therefore applied genome-wide DNA methylation analysis using methylated DNA immunoprecipitation combined with microarrays to identify alterations in gene-specific methylation. Under the conditions of our study, some genes were differentially methylated in response to MPY and TCDD, whereas d-limonene, DCB and chloroform did not induce any methylation changes. 90-day OTA treatment revealed enrichment of several categories of genes important in protein kinase activity and mTOR cell signaling process which are related to OTA nephrocarcinogenicity.
Full-text · Article · Oct 2015 · Toxicology and Applied Pharmacology
[Show abstract][Hide abstract] ABSTRACT: DNA methyltransferases (DNMTs) are an enzyme family that catalyzes the transfer of a methyl group to DNA for a wide variety of biological functions. To determine whether the number and composition of the Dnmt gene family affects the 5-methylcytosine (5mc) ratio at the genome level, genome-wide 5mc ratios from three marine animals, the mangrove killifish (Kryptolebias marmoratus), an intertidal copepod (Tigriopus japonicus), and a monogonont rotifer (Brachionus koreanus), were analyzed in each organism after the cloning of Dnmt genes. Lineage- and teleost-specific gene evolution was observed in the vertebrate Dnmt3 gene family, while unique gene expansion was found in the T. japonicus
Dnmt1 gene family. However, the rotifer did not have any apparent homologue of Dnmt1 or Dnmt3 in its genome. This gene information was highly supportive of genome-wide 5mc levels in the three marine animals. Therefore, the absence or presence of the Dnmt gene family could be an important evolutionary parameter of how this could affect genome-wide epigenetic metabolism.
Full-text · Article · Aug 2015 · Genes and Genomics
[Show abstract][Hide abstract] ABSTRACT: Abstract Ceria nanoparticles (NPs) are widely used as fuel catalysts and consequently are likely to enter the environment. Their potential impacts on. biota at environmentally relevant concentrations, including uptake and toxicity, remain to be elucidated and quantitative data on which to assess risk are sparse. Therefore, a definitive assessment of the molecular and phenotypic effects of ceria NPs was undertaken, using well-characterised mono-dispersed NPs as their toxicity is likely to be higher, enabling a conservative hazard assessment. Unbiased transcriptomics and metabolomics approaches were used to investigate the potential toxicity of tightly constrained 4-5 nm ceria NPs to the unicellular green alga, Chlamydomonas reinhardtii, a sentinel freshwater species. A wide range of exposure concentrations were investigated from predicted environmental levels, to support hazard assessment, to supra-environmental levels to provide insight into molecular toxicity pathways. Ceria NPs were internalised into intracellular vesicles within C. reinhardtii, yet caused no significant effect on algal growth at any exposure concentration. Molecular perturbations were only detected at supra-environmental ceria NP-concentrations, primarily down-regulation of photosynthesis and carbon fixation with associated effects on energy metabolism. For acute exposures to small mono-dispersed particles, it can be concluded there should be little concern regarding their dispersal into the environment for this trophic level.
[Show abstract][Hide abstract] ABSTRACT: Prediction of xenobiotic fate in fish is important for the regulatory assessment of chemicals under current legislation. Trout hepatocyte spheroids are a promising in vitro model for this assessment. In this investigation, the gene expression and function for xenobiotic metabolism and cellular efflux were characterised. Using fluorescence, transport and real time PCR analysis, the expression and functionality of a variety of genes related to xenobiotic metabolism and drug efflux were assessed in a range of trout hepatocyte culture preparations. Significantly greater levels of expression of genes involved in xenobiotic metabolism and efflux were measured in spheroids (which have been shown to remain viable in excess of 30 days), compared to hepatocytes cultured using conventional suspension and monolayer culture techniques. A transient decline in the expression of genes related to both xenobiotic metabolism and transport was determined during spheroid development, with a subsequent recovery in older spheroids. The most mature spheroids also exhibited an expression profile most comparable to that reported in vivo. Functionality of efflux transporters in spheroids was also demonstrated using fluorescent markers and specific inhibitors. In conclusion, the more physiologically relevant architecture in spheroid cultures provides a high functional integrity in relation to xenobiotic metabolism and efflux. Together with the enhanced gene expression and longevity of the model, hepatocytes in spheroid culture may prove to be an accurate alternative model to study the mechanisms of these processes in fish liver and provide an assay to determine the bioaccumulation potential of environmental contaminants.
[Show abstract][Hide abstract] ABSTRACT: The high resolution, accurate mass, and fast scanning features of the Orbitrap(TM) mass spectrometer, combined with the separation power of ultrahigh-performance liquid chromatography were applied for the first time to study the metabolic profiles of several organic flame retardants (FRs) present in indoor dust. To mimic real-life exposure, in vitro cultured HepG2 human hepatocyte cell lines were exposed simultaneously to various FRs in an indoor dust extract for 24 h. Target parent FRs, hexabromocyclododecanes (α-, β-, and γ-HBCDs), tris-2-chloroethyl phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCIPP), and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), were separated in a single run for the first time using alternating positive and negative heated ESI source. Further metabolite separation and identification was achieved using full scan (70,000 full width at half maximum (FWHM)), accurate mass (up to 1 ppm) spectrometry. Structural confirmation was performed via all ion fragmentation (AIF) spectra using the optional higher collisional dissociation (HCD) cell and MS/MS analysis. First insights into human metabolism of HBCDs revealed several hydroxylated and debrominated phase I metabolites, in addition to conjugated phase II glucuronides. Furthermore, various hydroxylated, oxidized, and conjugated metabolites of chlorinated phosphorous FRs were identified, leading to the suggestion of α-oxidation as a significant metabolic pathway for these compounds.
Full-text · Article · Jan 2015 · Analytical and Bioanalytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: To elucidate the effects of chemicals on populations of different species in the environment, efficient testing and modeling approaches are needed that consider multiple stressors and allow reliable extrapolation of responses across species. An adverse outcome pathway (AOP) is a concept that provides a framework for organizing knowledge about the progression of toxicity events across scales of biological organization that lead to adverse outcomes relevant for risk assessment. In this paper, we focus on exploring how the AOP concept can be used to guide research aimed at improving both our understanding of chronic toxicity, including delayed toxicity as well as epigenetic and transgenerational effects of chemicals, and our ability to predict adverse outcomes. A better understanding of the influence of subtle toxicity on individual and population fitness would support a broader integration of sublethal endpoints into risk assessment frameworks. Detailed mechanistic knowledge would facilitate the development of alternative testing methods as well as help prioritize higher tier toxicity testing. We argue that targeted development of AOPs supports both of these aspects by promoting the elucidation of molecular mechanisms and their contribution to relevant toxicity outcomes across biological scales. We further discuss information requirements and challenges in application of AOPs for chemical- and site-specific risk assessment and for extrapolation across species. We provide recommendations for potential extension of the AOP framework to incorporate information on exposure, toxicokinetics and situation-specific ecological contexts, and discuss common interfaces that can be employed to couple AOPs with computational modeling approaches and with evolutionary life history theory. The extended AOP framework can serve as a venue for integration of knowledge derived from various sources, including empirical data as well as molecular, quantitative and evolutionary-based models describing species responses to toxicants. This will allow a more efficient application of AOP knowledge for quantitative chemical- and site-specific risk assessment as well as for extrapolation across species in the future.
[Show abstract][Hide abstract] ABSTRACT: Adverse outcome pathways (AOPs) organize knowledge on the progression of toxicity through levels of biological organization. By determining the linkages between toxicity events at different levels, AOPs lay the foundation for mechanism-based alternative testing approaches to hazard assessment. Here, we focus on growth impairment in fish to illustrate the initial stages in the process of AOP development for chronic toxicity outcomes. Growth is an apical endpoint commonly assessed in chronic toxicity tests for which a replacement is desirable. Based on several criteria, we identified reduction in food intake to be a suitable key event for initiation of middle-out AOP development. To start exploring the upstream and downstream links of this key event, we developed three AOP case studies, for pyrethroids, selective serotonin reuptake inhibitors (SSRIs) and cadmium. Our analysis showed that the effect of pyrethroids and SSRIs on food intake is strongly linked to growth impairment, while cadmium causes a reduction in growth due to increased metabolic demands rather than changes in food intake. Locomotion impairment by pyrethroids is strongly linked to their effects on food intake and growth, while for SSRIs their direct influence on appetite may play a more important role. We further discuss which alternative tests could be used to inform on the predictive key events identified in the case studies. In conclusion, our work demonstrates how the AOP concept can be used in practice to assess critically the knowledge available for specific chronic toxicity cases and to identify existing knowledge gaps and potential alternative tests.
[Show abstract][Hide abstract] ABSTRACT: Molecular responses to acute toxicant exposure can be effective biomarkers, however responses to chronic exposure are less well characterised. The aim of this study was to determine chronic molecular responses to environmental mixtures in a controlled laboratory setting, free from the additional variability encountered with environmental sampling of wild organisms. Flounder fish were exposed in mesocosms for seven months to a contaminated estuarine sediment made by mixing material from the Forth (high organics) and Tyne (high metals and tributyltin) estuaries (FT) or a reference sediment from the Ythan estuary (Y). Chemical analyses demonstrated that FT sediment contained significantly higher concentrations of key environmental pollutants (including polycyclic aromatic hydrocarbons (PAHs), chlorinated biphenyls and heavy metals) than Y sediment, but that chronically exposed flounder showed a lack of differential accumulation of contaminants, including heavy metals. Biliary 1-hydroxypyrene concentration and erythrocyte DNA damage increased in FT-exposed fish. Transcriptomic and (1)H NMR metabolomic analyses of liver tissues detected small but statistically significant alterations between fish exposed to different sediments. These highlighted perturbance of immune response and apoptotic pathways, but there was a lack of response from traditional biomarker genes. Gene-chemical association annotation enrichment analyses suggested that polycyclic aromatic hydrocarbons were a major class of toxicants affecting the molecular responses of the exposed fish. This demonstrated that molecular responses of sentinel organisms can be detected after chronic mixed toxicant exposure and that these can be informative of key components of the mixture.
[Show abstract][Hide abstract] ABSTRACT: α-,β- and γ-HBCDs were subjected to in vitro biotransformation experiments with rat and trout liver S9 fractions for different incubation times at 2 concentration levels (1 and 10 µM). The metabolic degradation of target HBCDs followed first order kinetics. While β-HBCD undergoes rapid biotransformation (t0.5 = 6.4 and 38.1 minutes in rat and trout respectively), α-HBCD appears the most resistant to metabolic degradation (t0.5 = 17.1 and 134.9 minutes). The biotransformation rate in trout was slower than rat. Investigation of HBCD degradation profiles revealed the presence of 3 pentabromocyclododecene(PBCD) isomers and 2 tetrabromocyclododecadiene (TBCD) isomers indicating reductive debromination as a metabolic pathway for HBCDs. Both mono- and di- hydroxyl metabolites were identified for parent HBCDs, while only mono hydroxyl metabolites were detected for PBCDs and TBCDs. Interestingly, δ-HBCD was detected only in trout S9 fraction assays indicating metabolic interconversion of HBCDs during biotransformation in trout. Finally, enantioselective analysis showed significant enrichment of the (-)-α-HBCD enantiomer(EF = 0.321 and 0.419after 60 minutes incubationin rat and trout respectively). The greater enrichment of (-)-α-HBCD in rat than in trout underlines the species-specific differences in HBCD metabolism and the need for caution when extending similar results from animal studies to humans.
[Show abstract][Hide abstract] ABSTRACT: Zebrafish (Danio rerio) is one of a number of teleost fish species frequently employed in toxicology. Toxico-genomics determines global transcriptomic responses to chemical exposures and can predict their effects. It has been applied successfully within aquatic toxicology to assist in chemical testing, determination of mechanisms and environmental monitoring. Moreover, the related field of toxico-epigenomics, that determines chemical-induced changes in DNA methylation, histone modifications and micro-RNA expression, is emerging as a valuable contribution to understanding mechanisms of both adaptive and adverse responses. Zebrafish has proven a useful and convenient model species for both transcriptomic and epigenetic toxicological studies. Despite zebrafish's dominance in other areas of fish biology, alternative fish species are used extensively in toxico-genomics. The main reason for this is that environmental monitoring generally focuses on species native to the region of interest. We are starting to see advances in the integration of high-throughput screening, omics techniques and bioinformatics together with more traditional indicator endpoints that are relevant to regulators. Integration of such approaches with high-throughput testing of zebrafish embryos, leading to the discovery of adverse outcome pathways, promises to make a major contribution to ensuring the safety of chemicals in the environment.
Full-text · Article · Jan 2014 · Briefings in functional genomics
[Show abstract][Hide abstract] ABSTRACT: Both genetic and epigenetic responses of organisms to environmental factors, including chemical exposures, influence adaptation, susceptibility to toxicity and biodiversity. In model organisms, it is established that epigenetic alterations, including changes to the methylome, can create a memory of the received signal. This is partly evidenced through the analysis of epigenetic differences that develop between identical twins throughout their lifetime. The epigenetic marks induce alterations to the gene expression profile, which, in addition to mediating homeostatic responses, have the potential to promote an abnormal physiology either immediately or at a later stage of development, for example leading to an adult onset of disease. Although this has been well established, epigenetic mechanisms are not considered in chemical risk assessment or utilised in the monitoring of the exposure and effects of chemicals and environmental change. In this review, epigenetic factors, specifically DNA methylation, are highlighted as mechanisms of adaptation and response to environmental factors and which, if persistent, have the potential, retrospectively, to reflect previous stress exposures. Thus, it is proposed that epigenetic "foot-printing" of organisms could identify classes of chemical contaminants to which they have been exposed throughout their lifetime. In some cases, the potential for persistent transgenerational modification of the epigenome may also inform on parental germ cell exposures. It is recommended that epigenetic mechanisms, alongside genetic mechanisms, should eventually be considered in environmental toxicity safety assessments and in biomonitoring studies. This will assist in determining the mode of action of toxicants, no observed adverse effect level and identification of biomarkers of toxicity for early detection and risk assessment in toxicology but there are critical areas that remain to be explored before this can be achieved.
Full-text · Article · Oct 2013 · Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis