David S. Barber

Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms

Metallic nanoparticles are among the most widely used types of engineered nanomaterials; however, little is known about their environmental fate and effects. To assess potential environmental effects of engineered nanometals, it is important to determine which species are sensitive to adverse effects of various nanomaterials. In the present study, zebrafish, daphnids, and an algal species were used as models of various trophic levels and feeding strategies. To understand whether observed effects are caused by dissolution, particles were characterized before testing, and particle concentration and dissolution were determined during exposures. Organisms were exposed to silver, copper, aluminum, nickel, and cobalt as both nanoparticles and soluble salts as well as to titanium dioxide nanoparticles. Our results indicate that nanosilver and nanocopper cause toxicity in all organisms tested, with 48-h median lethal concentrations as low as 40 and 60 microg/L, respectively, in Daphnia pulex adults, whereas titanium dioxide did not cause toxicity in any of the tests. Susceptibility to nanometal toxicity differed among species, with filter-feeding invertebrates being markedly more susceptible to nanometal exposure compared with larger organisms (i.e., zebrafish). The role of dissolution in observed toxicity also varied, being minor for silver and copper but, apparently, accounting for most of the toxicity with nickel. Nanoparticulate forms of metals were less toxic than soluble forms based on mass added, but other dose metrics should be developed to accurately assess concentration-response relationships for nanoparticle exposures.

Effects of micronutrients on metal toxicity.

There is growing evidence that micronutrient intake has a significant effect on the toxicity and carcinogenesis caused by various chemicals. This paper examines the effect of micronutrient status on the toxicity of four nonessential metals: cadmium, lead, mercury, and arsenic. Unfortunately, few studies have directly examined the effect of dietary deficiency or supplementation on metal toxicity. More commonly, the effect of dietary alteration must be deduced from the results of mechanistic studies. We have chosen to separate the effect of micronutrients on toxic metals into three classes: interaction between essential micronutrients and toxic metals during uptake, binding, and excretion; influence of micronutrients on the metabolism of toxic metals; and effect of micronutrients on secondary toxic effects of metals. Based on data from mechanistic studies, the ability of micronutrients to modulate the toxicity of metals is indisputable. Micronutrients interact with toxic metals at several points in the body: absorption and excretion of toxic metals; transport of metals in the body; binding to target proteins; metabolism and sequestration of toxic metals; and finally, in secondary mechanisms of toxicity such as oxidative stress. Therefore, people eating a diet deficient in micronutrients will be predisposed to toxicity from nonessential metals.

Molecular Mechanisms of 4-Hydroxy-2-nonenal and Acrolein Toxicity: Nucleophilic Targets and Adduct Formation

Acrolein and 4-hydroxy-2-nonenal (HNE) are byproducts of lipid peroxidation and are thought to play central roles in various traumatic injuries and disease states that involve cellular oxidative stress, for example, spinal cord trauma, diabetes, and Alzheimers disease. In this review, we will discuss the chemical attributes of acrolein and HNE that determine their toxicities. Specifically, these aldehydes are classified as type 2 alkenes and are characterized by an alpha,beta-unsaturated carbonyl structure. This structure is a conjugated system that contains mobile pi-electrons. The carbonyl oxygen atom is electronegative and can promote the withdrawal of mobile electron density from the beta-carbon atom causing regional electron deficiency. On the basis of this type of electron polarizability, both acrolein and HNE are considered to be soft electrophiles that preferentially form 1,4-Michael type adducts with soft nucleophiles. Proteomic, quantum mechanical, and kinetic data will be presented, indicating that cysteine sulfhydryl groups are the primary soft nucleophilic targets of acrolein and HNE. This is in contrast to nitrogen groups on harder biological nucleophiles such as lysine or histidine residues. The toxicological outcome of adduct formation is not only dependent upon residue selectivity but also the importance of the targeted amino acid in protein function or structure. In attempting to discern the toxicological significance of a given adduct, we will consider the normal roles of cysteine, lysine, and histidine residues in proteins and the relative merits of corresponding adducts in the manifestations of diseases or toxic states. Understanding the molecular actions of acrolein and HNE could provide insight into many pathogenic conditions that involve initial cellular oxidative stress and could, thereby, offer new efficacious avenues of pharmacological defense.

Application of the Hard and Soft, Acids and Bases (HSAB) Theory to Toxicant–Target Interactions

Many chemical toxicants and/or their active metabolites are electrophiles that cause cell injury by forming covalent bonds with nucleophilic targets on biological macromolecules. Covalent reactions between nucleophilic and electrophilic reagents are, however, discriminatory since there is a significant degree of selectivity associated with these interactions. Over the course of the past few decades, the theory of Hard and Soft, Acids and Bases (HSAB) has proven to be a useful tool in predicting the outcome of such reactions. This concept utilizes the inherent electronic characteristic of polarizability to define, for example, reacting electrophiles and nucleophiles as either hard or soft. These HSAB definitions have been successfully applied to chemical-induced toxicity in biological systems. Thus, according to this principle, a toxic electrophile reacts preferentially with biological targets of similar hardness or softness. The soft/hard classification of a xenobiotic electrophile has obvious utility in discerning plausible biological targets and molecular mechanisms of toxicity. The purpose of this perspective is to discuss the HSAB theory of electrophiles and nucleophiles within a toxicological framework. In principle, covalent bond formation can be described by using the properties of their outermost or frontier orbitals. Because these orbital energies for most chemicals can be calculated using quantum mechanical models, it is possible to quantify the relative softness (σ) or hardness (η) of electrophiles or nucleophiles and to subsequently convert this information into useful indices of reactivity. This atomic level information can provide insight into the design of corroborative laboratory research and thereby help investigators discern corresponding molecular sites and mechanisms of toxicant action. The use of HSAB parameters has also been instrumental in the development and identification of potential nucleophilic cytoprotectants that can scavenge toxic electrophiles. Clearly, the difficult task of delineating molecular sites and mechanisms of toxicant action can be facilitated by the application of this quantitative approach.

Influence of Suwannee River humic acid on particle properties and toxicity of silver nanoparticles

Adsorption of natural organic matter (NOM) on nanoparticles can have dramatic impacts on particle dispersion resulting in altered fate and transport as well as bioavailability and toxicity. In this study, the adsorption of Suwannee River humic acid (SRHA) on silver nanoparticles (nano-Ag) was determined and showed a Langmuir adsorption at pH 7 with an adsorption maximum of 28.6 mg g(-1) nano-Ag. It was also revealed that addition of <10 mg L(-1) total organic carbon (TOC) increased the total Ag content suspended in the aquatic system, likely due to increased dispersion. Total silver content decreased with concentrations of NOM greater than 10mg TOCL(-1) indicating an increase in nanoparticle agglomeration and settling above this concentration. However, SRHA did not have any significant effect on the equilibrium concentration of ionic Ag dissolved in solution. Exposure of Daphnia to nano-Ag particles (50 μg L(-1) and pH 7) produced a linear decrease in toxicity with increasing NOM. These results clearly indicate the importance of water chemistry on the fate and toxicity of nanoparticulates.

Phenol-oxidizing laccases from the termite gut.

cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Laccase transcripts and phenoloxidase activity were most abundant in symbiont-free salivary gland and foregut tissue, verifying that the genes and activities are host-derived. Using a baculovirus-insect expression system, the two isoforms were functionally expressed with histidine tags and purified to near homogeneity. ICP-MS (inductively coupled plasma - mass spectrometry) analysis of RfLacA identified bound metals consisting mainly of copper (∼4 copper molecules per laccase protein molecule and ∼3 per histidine tag) with lesser amounts of calcium, manganese and zinc. Both recombinant enzyme preparations showed strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by the recombinant RfLacA preparation was also observed. These findings provide evidence that R. flavipes gut laccases are evolutionarily distinct, host-derived, produced in the salivary gland, secreted into the foregut, bind copper, and play a role in lignocellulose digestion. These findings contribute to a better understanding of termite digestion and gut physiology, and will assist future translational studies that examine the contributions of individual termite enzymes in lignocellulose digestion.

NsaRS is a cell-envelope-stress-sensing two-component system of Staphylococcus aureus

Staphylococcus aureus possesses 16 two-component systems (TCSs), two of which (GraRS and NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the firmicutes. NsaRS has recently been documented as being important for nisin resistance in S. aureus. In this study, we present a characterization of NsaRS and reveal that, as with other IM-HK TCSs, it responds to disruptions in the cell envelope. Analysis using a lacZ reporter-gene fusion demonstrated that nsaRS expression is upregulated by a variety of cell-envelope-damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, carbonyl cyanide m-chlorophenylhydrazone and penicillin G. Additionally, we reveal that NsaRS regulates a downstream transporter NsaAB during nisin-induced stress. NsaS mutants also display a 200-fold decreased ability to develop resistance to the cell-wall-targeting antibiotic bacitracin. Microarray analysis reveals that the transcription of 245 genes is altered in an nsaS mutant, with the vast majority being downregulated. Included within this list are genes involved in transport, drug resistance, cell envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using inductively coupled plasma-MS we observed a decrease in intracellular divalent metal ions in an nsaS mutant when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants have alterations in cell envelope structure. Finally, a variety of virulence-related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus, NsaRS is important in sensing cell damage in S. aureus and functions to reprogram gene expression to modify cell envelope architecture, facilitating adaptation and survival.

Distribution of silver nanoparticles in pregnant mice and developing embryos

Abstract The objective of this study was to evaluate the distribution of silver nanoparticles (NPs) in pregnant mice and their developing embryos. Silver NPs (average diameter 50 nm) were intravenously injected into pregnant CD-1 mice on gestation days (GDs) 7, 8, and 9 at dose levels of 0, 35, or 66 μg Ag/mouse. Mice were euthanised on GD10, and tissue samples were collected and analysed for silver content. Compared with control animals injected with citrate buffer vehicle, silver content was significantly increased (p < 0.05) in nearly all tissues from silver NP-treated mice. Silver accumulation was significantly higher in liver, spleen, lung, tail (injection site), visceral yolk sac, and endometrium compared with other organs from silver NP-treated mice. Furthermore, silver NPs were identified in vesicles in endodermal cells of the visceral yolk sac. In summary, the results demonstrated that silver NPs distributed to most maternal organs, extra-embryonic tissues, and embryos, but did not accumulate significantly in embryos.

Uptake, retention and internalization of quantum dots in Daphnia is influenced by particle surface functionalization.

Nanomaterials are a diverse group of compounds whose inevitable release into the environment warrants study of the fundamental processes that govern the ingestion, uptake and accumulation in aquatic organisms. Nanomaterials have the ability to transfer to higher trophic levels in aquatic ecosystems, and recent evidence suggests that the surface chemistry of both the nanoparticle and biological membrane can influence uptake kinetics. Therefore, our study investigates the effect of surface functionalization on uptake, internalization and depuration in Daphnia spp. Uncharged (polyethylene glycol; PEG), positively charged (amino-terminated: NH2) and negatively charged (carboxyl-modified; COOH) cadmium selenide/zinc sulfide quantum dots were used to monitor ingestion, uptake and depuration of nanometals in Daphnia magna and Ceriodaphnia dubia over 24h of exposure. These studies demonstrated that particles with higher negative charge (COOH quantum dots) were taken up to a greater extent by Daphnia (259.17±17.70 RFU/20 Daphnia) than either the NH2 (150.01±18.91) or PEG quantum dots (95.17±9.78), however this is likely related to the functional groups attached to the nanoparticles as there were no real differences in zeta potential. Whole body fluorescence associates well with fluorescent microscopic images obtained at the 24h timepoint. Confocal and electron microscopic analysis clearly demonstrated that all three types of quantum dots could cross the intestinal epithelial barrier and be translocated to other cells. Upon cessation of exposure, elimination of all three materials was biphasic with rapid initial clearance that likely represents elimination of material remaining in the GI tract followed by a much slower elimination phase that likely represents elimination of internalized material. These studies demonstrate that daphnids can take up intact nanomaterial from the water column and that this uptake is strongly influenced by particle surface functionalization. In addition, the usefulness of using quantum dots as a proxy for other nanometals (no acute toxicity, clear visualization in electron microscopy), in conjunction with several different imaging techniques in assessing uptake and accumulation of nanoparticles in daphnids was demonstrated.

Chronic Nanoparticulate Silver Exposure Results in Tissue Accumulation and Transcriptomic Changes in Zebrafish

Increasing utilization of metallic nanomaterials in recent years implies an increasing rate of release to the environment, with potentially serious adverse effects on environmentally important species. Previously, we demonstrated that exposure to nanoparticulate silver for 24-48 h results in dramatic alterations in global gene expression patterns and increased tissue burdens in zebrafish gills. The present study reports outcomes associated with chronic exposure to nanoparticulate silver in zebrafish. Adult female Danio rerio were exposed to 5, 15, 25, or 50 μg/L nanoparticulate silver in a time course up to 28 days. A soluble silver treatment (5 μg/L) was also included. Results indicate that use of flow-through systems for chronic nanometal studies is a viable concept; measured concentrations of approximately 60% of nominal values over the course of the 28-day exposure were observed. Dissolution of nanoparticulate silver was measured twice weekly throughout the exposure ranging between 0.5 and 1.0 μg/L, and was relatively consistent between nanoparticulate silver tanks, with no differences between treatments. Gill samples from the 28-day time point were analyzed for global gene expression patterns and histopathology. Tissue accumulation in both gill and eviscerated carcass was dose-dependent, and remained elevated 4 days after the silver was removed. Microarray analysis also revealed a dose-dependent response pattern, with the largest number of genes affected in the 50 μg/L AgNP exposure. Pathway analysis of affected genes identified a number of GO terms that were significantly over-represented in the high AgNP dataset. These terms are associated with DNA damage repair, cellular restructuring, and developmental processes.