Daniel L. Starnes

Toxicogenomic responses of the model organism Caenorhabditis elegans to gold nanoparticles.

We used Au nanoparticles (Au-NPs) as a model for studying particle-specific effects of manufactured nanomaterials (MNMs) by examining the toxicogenomic responses in a model soil organism, Caenorhabditis elegans . Global genome expression for nematodes exposed to 4-nm citrate-coated Au-NPs at the LC(10) level (5.9 mg·L(-1)) revealed significant differential expression of 797 genes. The levels of expression for five genes (apl-1, dyn-1, act-5, abu-11, and hsp-4) were confirmed independently with qRT-PCR. Seven common biological pathways associated with 38 of these genes were identified. Up-regulation of 26 pqn/abu genes from noncanonical unfolded protein response (UPR) pathway and molecular chaperones (hsp-16.1, hsp-70, hsp-3, and hsp-4) were observed and are likely indicative of endoplasmic reticulum stress. Significant increase in sensitivity to Au-NPs in a mutant from noncanonical UPR (pqn-5) suggests possible involvement of the genes from this pathway in a protective mechanism against Au-NPs. Significant responses to Au-NPs in endocytosis mutants (chc-1 and rme-2) provide evidence for endocytosis pathway being induced by Au-NPs. These results demonstrate that Au-NPs are bioavailable and cause adverse effects to C. elegans by activating both general and specific biological pathways. The experiments with mutants further support involvement of several of these pathways in Au-NP toxicity and/or detoxification.

A micro-sized model for the in vivo study of nanoparticle toxicity: what has Caenorhabditis elegans taught us?

Environmental context. The ability of the soil nematode Caenorhabditis elegans to withstand a wide range of environmental conditions makes it an idea model for studying the bioavailability and effects of engineered nanomaterials. We critically review what has been learned about the environmental fate of engineered nanoparticles, their effects and their mechanisms of toxicity using this model organism. Future systematic manipulation of nanoparticle properties and environmental variables should elucidate how their interaction influences toxicity and increase the predictive power of nanomaterial toxicity studies.

In planta engineering of gold nanoparticles of desirable geometries by modulating growth conditions: an environment-friendly approach.

Unique properties of gold nanoparticles (AuNPs) can be achieved by manipulating their geometries. However, it is not known if the shapes and sizes of AuNPs can be modulated in planta. Here, we evaluated the accumulation of gold across taxonomically diverse plant species (alfalfa, cucumber, red clover, ryegrass, sunflower, and oregano). Significant variations were detected in the uptake of gold in the roots ranging from 500 ppm (ryegrass) to 2500 ppm (alfalfa). Alfalfa was selected for subsequent studies due to its ability to accumulate relatively large quantities of gold in its roots. Temporal analysis revealed that most of the AuNPs formed within 6 h of treatment, and the majority of them fall within the size range of 10-30 nm. Spherical AuNPs (1-50 nm) were detected ubiquitously across different treatments. To elucidate the effects of growth variables on the geometries of in planta synthesized AuNPs, alfalfa was subjected to KAuCl(4) (50 ppm) treatment for 3d under different pH, temperature, and light regimes. Interestingly, manipulation of growth conditions triggered a noticeable shift in the relative abundance of spherical, triangular, hexagonal, and rectangular AuNPs providing empirical evidence toward the feasibility of their in planta engineering.

Distinct transcriptomic responses of Caenorhabditis elegans to pristine and sulfidized silver nanoparticles.

Manufactured nanoparticles (MNP) rapidly undergo aging processes once released from products. Silver sulfide (Ag2S) is the major transformation product formed during the wastewater treatment process for Ag-MNP. We examined toxicogenomic responses of pristine Ag-MNP, sulfidized Ag-MNP (sAg-MNP), and AgNO3 to a model soil organism, Caenorhabditis elegans. Transcriptomic profiling of nematodes which were exposed at the EC30 for reproduction for AgNO3, Ag-MNP, and sAg-MNP resulted in 571 differentially expressed genes. We independently verified expression of 4 genes (numr-1, rol-8, col-158, and grl-20) using qRT-PCR. Only 11% of differentially expressed genes were common among the three treatments. Gene ontology enrichment analysis also revealed that Ag-MNP and sAg-MNP had distinct toxicity mechanisms and did not share any of the biological processes. The processes most affected by Ag-MNP relate to metabolism, while those processes most affected by sAg-MNP relate to molting and the cuticle, and the most impacted processes for AgNO3 exposed nematodes was stress related. Additionally, as observed from qRT-PCR and mutant experiments, the responses to sAg-MNP were distinct from AgNO3 while some of the effects of pristine MNP were similar to AgNO3, suggesting that effects from Ag-MNP is partially due to dissolved silver ions.

Effect of natural organic matter on dissolution and toxicity of sulfidized silver nanoparticles to Caenorhabditis elegans

The objectives of this study were to evaluate the effect of natural organic matter (NOM) on the dissolution and the toxicity of sulfidized AgNPs (sAgNPs) to a model soil organism, Caenorhabditis elegans in two distinct exposure media. This study demonstrated that the aggregation and dissolution of sAgNPs (75% Ag2S) was influenced by media composition, including inorganic composition and natural organic matter (NOM) concentration. Dissolution of sAgNPs was low (∼0.5%) but increased over time in all tested media (2 weeks). The presence of NOM either inhibited or enhanced Ag dissolution. Pony lake fulvic acid increased while Suwanee river and Pahokee peat fulvic acid (PLFA) decreased release of dissolved Ag from sAgNPs. Mortality of C. elegans exposed to sAgNPs was influenced by the inorganic composition of the media: with LC50 values of 8.15 mg Ag L−1 and >15 mg Ag L−1 in moderately hard reconstituted water and soil solution pore water. Toxicity was totally rescued by the presence of all tested NOM types and concentrations, despite the increase of dissolved Ag in the media with PLFA. Overall, these results showed that the toxicity induced by a partly sulfidized AgNPs in C. elegans is low and negligible in the presence of NOM regardless of NOM influence on dissolution.

The effects of manufactured nanomaterial transformations on bioavailability, toxicity and transcriptomic responses of Caenorhabditis elegans

OF DISSERTATION THE EFFECTS OF MANUFACTURED NANOMATERIAL TRANSFORMATIONS ON BIOAVAILABILITY, TOXICITY AND TRANSCRIPTOMIC RESPONSES OF CAENORHABDITIS ELEGANS In recent decades, there has been a rapid expansion in the use of manufactured nanoparticles (MNPs). Experimental evidence and material flow models predict that MNPs enter wastewater treatment plants and partition to sewage sludge and majority of that sludge is land applied as biosolids. During wastewater treatment and after land application, MNPs undergo biogeochemical transformations (aging). The primary transformation process for silver MNPs (Ag-MNPs) is sulfidation, while zinc oxide MNPs (ZnO-MNPs) most likely undergo phosphatation and sulfidation. Our overall goal was to assess bioavailability and toxicogenomic impacts of both pristine, defined assynthesized, and aged Agand ZnO-MNPs, as well as their respective ions, to a model organism, the soil nematode Caenorhabditis elegans. We first investigated the toxicity of pristine Ag-MNPs, sulfidized Ag-MNPs (sAgMNPs), and AgNO3 to identify the most sensitive ecologically relevant endpoint in C. elegans. We identified reproduction as the most sensitive endpoint for all treatments with sAg-MNPs being about 10-fold less toxic than pristine Ag-MNPs. Using synchrotron xray microspectroscopy we demonstrated that AgNO3 and pristine Ag-MNPs had similar bioavailability while aged sAg-MNPs caused toxicity without being taken up by C. elegans. Comparisons of the genomic impacts of both MNPs revealed that Ag-MNPs and sAg-MNPs have transcriptomic profiles distinct from each other and from AgNO3. The toxicity mechanisms of sAg-MNPs are possibly associated with damaging effects to cuticle. We also investigated the effects pristine zinc oxide MNPs (ZnO-MNPs) and aged ZnOMNPs, including phosphatated (pZnO-MNPs) and sulfidized (sZnO-MNPs), as well as ZnSO4 have on C. elegans using a toxicogenomic approach. Aging of ZnO-MNPs reduced toxicity nearly 10-fold. Toxicity of pristine ZnO-MNPs was similar to the toxicity caused by ZnSO4 but less than 30% of responding genes was shared between these two treatments. This suggests that some of the effects of pristine ZnO-MNPs are also particle-specific. The genomic results showed that based on Gene Ontology and induced biological pathways all MNP treatments shared more similarities than any MNP treatment did with ZnSO4. This dissertation demonstrates that the toxicity of Agand ZnO-MNPs to C. elegans is reduced and operates through different mechanisms after transformation during the wastewater treatment process.