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[
International Worm Meeting,
2013]
Zinc oxide (ZnO) nano-particles (NPs) are extensively used in cosmetics, sunscreens, food products, paints, drugs, ground water remediation and water treatment because of their unique physico-chemical properties. The enormous usage of NPs demands risk assessments on health and environment. In this regard the safety assessment of different sizes of ZnO NPs was examined in the soil nematode Caenorhabditis elegans. C. elegans were maintained on nematode growth medium with E.coli OP50 as food source. 50nm and 100nm ZnO NPs (SIGMA) along with bulk was tested in the present study. The NPs were characterized in the exposure medium using TEM and Zeta-sizer. Dose response in L4 stage worms was estimated for all the particles, followed by oxidative stress (ROS and MTT assay) at sub-lethal concentrations. Physiological end-points such as growth, behavior and reproduction were also analyzed. Quantitative PCR for genes involved in stress response pathway followed by biochemical assay and/or transgenic GFP strain screening was also carried out to investigate relationship between toxicity and size of ZnO NPs. Our studies revealed that lethality on exposure to ZnO NPs was both dose as well as size dependent. Further in comparison to bulk the ZnO NPs showed (1) more prominent adverse effects on physiology of nematodes, (2) increase in ROS levels, reduced glutathione peroxidase and reduced glutathione level, (3) enhanced lipid peroxidation, protein carbonylation and DNA damage, (4) suppression of Insulin/IGF- like signaling pathway and activation of stress response pathway through up regulation of genes which are controlled by DAF-16/HSF-1/SKN-1transcription factors and (5) reduced acetylcholine enzyme level suggesting hampered neuro-transmission. Therefore we conclude that ZnO NPs have size-related eco-toxic potentials in C. elegans. Further our study also provide evidenced that ZnO NPs are genotoxic and disrupts various physiological processes at very low concentration (0.23ug/mL) in C. elegans.
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Tsyusko, O., Starnes, Daniel L, Bertsch, P., Collin, B., Smith, J., Oostveen, E., Unrine, J., Starnes, C.
[
International Worm Meeting,
2013]
There are over 2000 consumer products containing manufactured nanomaterials (MNMs) available today. One of the MNMs of major concern is silver nanoparticles (AgNPs). During wastewater treatment, AgNPs can undergo transformations resulting in partially or fully sulfidized AgNPs. Our objective is to understand the bioavailability and toxicity of polyvinylpyrrolidone (PVP) coated AgNPs (Ag-PVP) and fully sulfidized AgNPs (Ag-S) to a model organism Caenorhabditis elegans using a toxicogenomic approach. Since AgNP toxicity can also be determined by dissolution and release of Ag+, to differentiate between particle and ion-specific toxicity we included AgNO3 as an additional treatment. Our results showed that for AgNPs, Ag-PVP are more toxic to C. elegans than Ag-S due in part to a greater bioavailability and uptake as evidenced by synchrotron-based x-ray microscopy. Evidence also suggests that the observed toxicity is partially particle specific for both Ag-PVP and Ag-S because nematodes exposed to particle free supernatants showed very low mortality. Among endpoints screened, reproduction was the most sensitive and was used for the microarray study. The transcriptomic data indicate that each treatment produced a distinct genomic response. Ag+ had the largest number of differentially expressed genes (312) followed by Ag-S (223), and lastly by Ag-PVP (136). Of the total number of significant differentially expressed genes only 3% were shared among all of the treatments and 86% were uniquely expressed for the respective treatments. The genomic data support the hypothesis that the observed responses are partially particle specific, since both Ag-PVP and Ag-S have a unique set of genes that are not shared with the Ag+ treatment. In addition, our results demonstrate that C. elegans transcriptomic responses to the Ag-S are distinct from those to Ag-PVP resulting in their different toxicities.
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[
International Worm Meeting,
2017]
Studies in the nematode Caenorhabditis elegans have shown that exposure to as-synthesized and transformed (sulfidized) silver nanoparticles (sAg-NPs) can cause various toxic effects, but genomic effects that can be induced by prolonged exposure over multiple generations have not been investigated. A previous multigenerational study of continuous exposure of C. elegans to AgNO3, Ag-NPs and sAg-NPs showed that increased sensitivity, in terms of reproductive toxicity from Ag, occurred as early as the second generation for AgNO3 and Ag-NPs, but not sAg-NPs. This suggested that Ag-NPs may cause mutations or epi-mutations. In this study, we used wild type N2 C. elegans as a model organism to determine if mutations and/or epi-mutations contribute to the observed multigenerational effects. Exposure was carried out using sub-lethal concentrations (EC30 for reproduction) of AgNO3, Ag-NPs and sAg-NPs in simulated soil pore water for 10 generations with the parent generation (F0) unexposed for all groups. Four replicates were used per treatment, from which DNA was extracted from an entire brood of a single worm for whole genome DNA sequencing by NextSeq. Analysis of sequencing data revealed no significant differences in the total number of mutations (SNPs, deletions or insertions) from the controls. This suggests that induced germline mutations may not be responsible for the increased sensitivity observed in multigenerational exposures. We hypothesized that epigenetic mechanisms are involved. Increased DNA methylation (6-methyladenine), an epigenetic marker, which was recently discovered in C. elegans, has been shown to negatively impact reproduction over multiple generations. We are currently investigating DNA methylation as a potential mechanism by which reproductive toxicity can be passed on to subsequent generations after exposure to Ag-NPs. Preliminary experiments using slot blots show that there is a potential increase in DNA methylation after 2 generations of exposure to pristine Ag-NPs. Interestingly, there seems to be a decrease in DNA methylation for AgNO3 exposed worms. In addition to confirming these results and testing sAg-NPs, we will also examine if the higher levels of DNA methylation persist in subsequent generations after cessation of exposure.
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[
International Worm Meeting,
2015]
The increasing use of silver nanoparticles (AgNP) in consumer products greatly increases the potential for the environmental release of these particles. Studies have shown that exposure of the Caenorhabditis elegans to NP has adverse effects. Decreased reproduction, alteration in gene expression, and increased mortality has been observed in different studies involving the exposure of C. elegans to AgNPs . Some of these effects have been shown to be multigenerational. The short generation time for C. elegans makes it an ideal model for studying multigenerational effects of silver NP. In this study, C. elegans were exposed to AgNP in synthetic soil pore water continuously over 10 generations at an environmentally relevant concentration. Effects on reproduction were measured by counting the offspring of individual nematodes. Individual variability at five microsatellite loci was also assessed using PCRs and DNA fragment analysis. AgNP release silver ions into exposure medium which accounts for some toxicity. Biotransformation such as sulfidation of AgNP occurs in the environment. AgNO3 and sulfidized AgNP were used as controls. No effects on reproduction were observed over 10 generations. There were also no observed differences for the microsatellite loci that were tested. This was probably due to the low concentration that was used to mimic environmental concentrations of NP and/or selection of the loci used for this experiment. A preliminary experiment involving the continuous exposure for over five generations has been carried out using EC30 (1.3mg/L) for reproduction. This is a much higher concentration compared to the concentration used in the previous experiment (0.3mg/L). Unlike the previous experiment, both control and exposed generations originated from the same lineage. DNA extractions were carried out from an entire brood of a single nematode. Whole genome sequencing was carried out using MiSeq. Sequencing data obtained from the F0 and F5 generations for control and exposed nematodes were aligned to a reference genome. A conservative approach was used for mutations' detection using SnpEff program. Mutations involved missense, deletion and insertions. The mutations were observed in coding, regulatory 5' and 3' untranslated regions, intergenic, introns, and microsatellite regions. Mutations within coding regions as well as immediately upstream and downstream of coding regions will be examined. Some of these mutations may have consequences in the function of proteins as well as regulation of gene expression. A full scale experiment, involving all of the treatments discussed for the first experiment will be carried out to further examine multigenerational effects at the genomic level.