Toxicity and transcriptomic analysis in Hyalella azteca suggests increased exposure and susceptibility of epibenthic organisms to Zinc Oxide Nanoparticles
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ABSTRACT: Abstract: Nanoparticles (NPs) are expected to make their way into the aquatic environment where sedimentation of particles will likely occur, putting benthic organisms at particular risk. Therefore, organisms such as Hyalella azteca, an epibenthic crustacean which forages at the sediment surface, is likely to have a high potential exposure. Here we show that Zinc Oxide (ZnO) NPs are more toxic to H. azteca compared with the corresponding metal ion, Zn2+. Dissolution of ZnO NPs contributes about 50% of the Zn measured in the ZnO NP suspensions, and cannot account for the toxicity of these particles to H. azteca. However, gene expression analysis is unable to distinguish between the ZnO NP exposures and Zinc Sulfate (ZnSO4) exposures at equitoxic concentrations. These results lead us to hypothesize that ZnO NPs provide and an enhanced exposure route for Zn2+ uptake into H. azteca, and possibly other sediment dwelling organisms. Our study supports the prediction that sediment dwelling organisms are highly susceptible to the effects of ZnO NPs and should be considered in the risk assessment of these nanomaterials. This experiment included four different treatments and an untreated control. Each treatment or control, consisted of ten independent replicates of twenty Hyalella azteca. Of these, six were randomly chosen to be used for the microarray analysis.
Project description:Abstract: Nanoparticles (NPs) are expected to make their way into the aquatic environment where sedimentation of particles will likely occur, putting benthic organisms at particular risk. Therefore, organisms such as Hyalella azteca, an epibenthic crustacean which forages at the sediment surface, is likely to have a high potential exposure. Here we show that Zinc Oxide (ZnO) NPs are more toxic to H. azteca compared with the corresponding metal ion, Zn2+. Dissolution of ZnO NPs contributes about 50% of the Zn measured in the ZnO NP suspensions, and cannot account for the toxicity of these particles to H. azteca. However, gene expression analysis is unable to distinguish between the ZnO NP exposures and Zinc Sulfate (ZnSO4) exposures at equitoxic concentrations. These results lead us to hypothesize that ZnO NPs provide and an enhanced exposure route for Zn2+ uptake into H. azteca, and possibly other sediment dwelling organisms. Our study supports the prediction that sediment dwelling organisms are highly susceptible to the effects of ZnO NPs and should be considered in the risk assessment of these nanomaterials.
Project description:Zinc Oxide nanoparticles (ZnO NPs) are being rapidly developed for use in consumer products, wastewater treatment and chemotherapy providing several possible routes for ZnO NP exposure to humans and aquatic organisms. Recent studies have shown that ZnO NPs undergo rapid dissolution to Zn+2, but the relative contribution of Zn+2 to ZnO NP bioavailability and toxicity is not clear. Gene expression profiling of D. magna exposed to ZnO NPs or ZnSO4 at equitoxic concentrations demonstrated that the particles cause toxicity through a distinct mechanism compared with Zn+2. D. magna were also exposed to a SiO NPs as a particle control at equimolar concentrations. The SiO NPs resulted in few differentially expressed genes and there was very little overlap between the genes affected by the ZnO NPs and the SiO NPs, suggesting that ZnO NPs cause a distinct pattern of differentially expressed genes. In the ZnO NP exposures, effects were observed to genes involved in cytoskeletal transport, cellular respiration and reproduction. Three biomarker genes including a multi-cystatin, ferritin and a C1q containing gene were confirmed as differentially expressed in a specific pattern by ZnO NP and provide a suite of biomarkers for identifying environmental exposure to ZnO NP and differentiating between NP and ionic exposure. We exposed Daphnia magna to the 1/10 LC50 and LC25 of ZnO nanoparticles and Zn++ as ZnSO4 for 24-h. For each exposure condition, we performed 3 exposures and 2 technical replicates (as dye swap) for each exposure (6 microarrays total). All exposures were compared to a unexposed laboratory control
Project description:Aquatic organisms are exposed to many toxic chemicals and interpreting the cause and effect relationships between occurrence and impairment is difficult. Toxicity Identification Evaluation (TIE) provides a systematic approach for identifying responsible toxicants. TIE relies on relatively uninformative and potentially insensitive toxicological endpoints. Gene expression analysis may provide needed sensitivity and specificity aiding in the identification of primary toxicants. The current work aims to determine the added benefit of integrating gene expression endpoints into the TIE process. A cDNA library and a custom microarray were constructed for the marine amphipod Ampelisca abdita. Phase 1 TIEs were conducted using 10% and 40% dilutions of acutely toxic sediment. Gene expression was monitored in survivors and controls. An expression-based classifier was developed and evaluated against control organisms, organisms exposed to low or medium toxicity diluted sediment, and chemically selective manipulations of highly toxic sediment. The expression-based classifier correctly identified organisms exposed to toxic sediment even when little mortality was observed, suggesting enhanced sensitivity of the TIE process. The ability of the expression-based endpoint to correctly identify toxic sediment was lost concomitantly with acute toxicity when organic contaminants were removed. Taken together, this suggests that gene expression enhances the performance of the TIE process. Wild-collected Ampelisca abdita were exposed to either control (from sites in Long Island Sound, labeled LIS) sediment, toxic (from site on Elizabeth River, labeled ER) sediment, a series of mixtures of LIS and ER sediment, sediments manipulated to alter toxin bioavailability, or toxicant amended sediments. Lethality was scored, and survivors were subjected to mRNA expression analysis via oligo microarray.
Project description:Hormesis is a dose response phenomenon characterized by a stimulation of an organismal response at low doses of a chemical and inhibition of the response (toxicity) at a higher dose. In the present study, Daphnia magna were exposed to the energetic compound 2,4,6-trinitrotoluene (TNT) for 21 days, and the endpoints survival, growth (length and dry weight), and reproduction (number of neonates per survivor) were assesed, identifying hormetic responses at the lower treatment levels. In order to elucidate the mechanisms leading to hormesis, microarray analysis was performed at 0.004 (hormetic), 0.12 (sometimes hormetic), and 1.85 (toxic) mg/L TNT. Functional and transcriptional benchmark dose analyses performed on differentially expressed genes suggested the involvement of lipid metabolism in hormetic responses and subsequently lipidomic analysis was performed on the same treatments. Lipid analysis supported the hypothesis that TNT exposure affected lipid metabolism, and showed that hormetic effects could be related to increases in some polyunsaturated fatty acids and ecosanoids known to be involved in Daphnia growth and reproduction. Chronic toxicity tests was designed to identify the hormetic range of D. magna responses to TNT. Daphnia magna younger than 24 h old and obtained from in-house cultures at the U.S. Army Engineer Research and Development Center, Environmental Laboratory (Vicksburg, MS) were used to initiate the bioassay. The experimental design consisted of one D. magna per test chamber and 10 test chambers per treatment level. In the microarray mass exposure, nominal treatment levels were 0.004, 0.02, 0.12, 0.44, and 1.85 mg/L. Based on the results of the testing in the 50 ml beakers, organisms from three treatments were selected for analysis of gene expression: 0.004, 0.12, and 1.85 mg/L. These treatments were selected to represent a hormetic concentration (0.004 mg/L), a toxic concentration (1.85 mg/L), and an intermediate concentration (0.12 mg/L). A total of 23 arrays used, with 5-6 replicates per each dose (solvent control, 0.004 mg/L, 0.12 mg/L, and 1.85 mg/L).
Project description:Zinc Oxide nanoparticles (ZnO NPs) are being rapidly developed for use in consumer products, wastewater treatment and chemotherapy providing several possible routes for ZnO NP exposure to humans and aquatic organisms. Recent studies have shown that ZnO NPs undergo rapid dissolution to Zn+2, but the relative contribution of Zn+2 to ZnO NP bioavailability and toxicity is not clear. Gene expression profiling of D. magna exposed to ZnO NPs or ZnSO4 at equitoxic concentrations demonstrated that the particles cause toxicity through a distinct mechanism compared with Zn+2. D. magna were also exposed to a SiO NPs as a particle control at equimolar concentrations. The SiO NPs resulted in few differentially expressed genes and there was very little overlap between the genes affected by the ZnO NPs and the SiO NPs, suggesting that ZnO NPs cause a distinct pattern of differentially expressed genes. In the ZnO NP exposures, effects were observed to genes involved in cytoskeletal transport, cellular respiration and reproduction. Three biomarker genes including a multi-cystatin, ferritin and a C1q containing gene were confirmed as differentially expressed in a specific pattern by ZnO NP and provide a suite of biomarkers for identifying environmental exposure to ZnO NP and differentiating between NP and ionic exposure.
Project description:To determine toxicant specific effects of Ordnance Related Compound (ORC) exposure we performed microarray hybridizations with RNA isolated from Daphnia magna following different ORC exposures at the 1/10 LC50. The gene expression profiles revealed toxicant specific gene expression profiles allowed for the identification of specific biomarkers of exposure. Keywords: ecotoxicogenomic exposure study We exposed Daphnia magna the 1/10 LC50 of different Ordnance Related Compounds (Cu, Zn, Pb, WO4, RDX, TNT, 2-ADNT, 2-ADNT, TNB, DNB, 2,4-DNT, and 2,6-DNT) for 24 hours. For each exposure condition, we performed 3 exposures and 2 technical replicates (as dye swap) for each exposure (6 microarrays total, except TNT and Cu). All exposures were compared to a unexposed laboratory control (MHRW media).
Project description:Zinc oxide nanoparticles (ZnO NPs) represent an important class of commercially applied materials. Recently, adverse effects of ZnO NPs were found in humans and animals following ingestion, although the effects on endocrine system disease remain unclear. In this study, ZnO NPs were orally administered to mice, and at doses of 25 mg/kg bw (body weight) ZnO NPs and above, plasma glucose increased significantly. The genome-wide effects of ZnO NPs were then investigated using RNA-sequencing technology. In the cluster analysis, the most significantly enriched Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways concerned membranes and their close association with endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) generation. Biochemical and gene and protein expression analyses revealed that ZnO NPs activated a xenobiotic biodegradation response and increased the expression of cytochrome P450 (CYP) enzymes in mice livers, leading to ER stress. The ER stress increased ROS generation. The high levels of ROS activated the MAPK and NF-B pathways and induced an inflammation response, resulting in the phosphorylation of insulin receptor substrate 1. Thus, the insulin resistance that developed was the primary mechanism for the increase in the plasma glucose of mice treated orally with ZnO NPs.
Project description:Increasing interest in the hazardous properties of zinc oxide nanoparticles (ZnO NPs), commonly used as ultraviolet filters in sunscreen, has driven efforts to study the percutaneous application of ZnO NPs to diseased skin; however, in-depth studies for epidermal barrier dysfunction remain lacking. Our epidermal barrier dysfunction model develops large gaps between keratinocytes due to keratinocyte-specific Cdc42 knockout, allowing ZnO NPs entry to the stratum basale of the epidermis, impacting melanocytes. ZnO NP application for 14 and 49 consecutive days induced melanoma-like skin lesions, dysregulated melanoma-associated gene expression, increased oxidative injury, inhibited apoptosis, and increased nuclear factor kappa B (NF-κB) p65 and Bcl-2 expression in melanocytes. Exposure to 5.0 µg·mL−1 ZnO NPs for 72 h increased cell viability, decreased apoptosis, and increased Fkbp51 expression in melanocytes, consistent with histological observations. The oxidative stress–mediated mechanism underlying the induction of anti-apoptotic effects was verified using the reactive oxygen species scavenger N-acetylcysteine. The entry of ZnO NPs into the stratum basale of skin with epidermal barrier dysfunction resulted in melanoma-like skin lesions and an anti-apoptotic effect induced by oxidative stress, activating the NF-κB pathway in melanocytes. These findings suggest that ZnO NPs are potentially carcinogenic for skin with a damaged barrier function.
Project description:The goal of the study was to investigate gene expression differences in Hyalella azteca exposed to pyrethroid insecticides and compare a laboratory strain to a wild population believed to be resistant to the pesticides. H. azteca reared in the laboratory at the University of CA, Berkeley (UCB) were exposed to cyfluthrin, a commerical pyrethroid insecticide for 96-h. A wild population collected from Grayson Creek (GC), CA was reared in the laboratory for several days and also exposed to cyfluthrin. Toxicity testing revealed that GC animals were two orders of magnitude less sensitive to cyfluthrin compared to the laboratory animals with the no observed effect concentration (NOEC) for UCB = 0.4 ng/L and GC= 170 ng/L. Unexposed, control animals and animals exposed to 0.4 ng/L (GC and UCB) or 170 ng/L cyfluthrin were collected following 96-h treatments. Differences in gene expression were measured using a custom Hyalella azteca microarray. Gene expression profiles revealed that laboratory H. azteca responded to cyfluthrin through differential expression of genes involved in neurological system processes. In contract, H. azteca from Grayson Creek showed a pattern of oxidative stress through the differential expression of glutathione-S-transferases, heat shock proteins, and other genes involved in oxidation-reduction processes. Four replicate exposures consisting of ten animals were collected for each treatment or control. A one-color hybridization protocol was used so that each sample was labeled with cy3 and only one sample was hybridized to each array.
Project description:ZnO nanoparticles can elicit a range of perturbed cell responses in vitro. The liver is a target for ZnO nanoparticle-, or Zn2+ released from ZnO nanoparticles-induced accumulation and/or impact in vitro and in vivo. The response of human hepatic stellate cells to ZnO nanoparticles has not yet been assessed. We aimed to determine whether the presence of surface coatings could protect human hepatic stellate cells from ZnO nanoparticle-induced cytotoxicity.