Maria S. Sepúlveda

Toxicological studies on silver nanoparticles: challenges and opportunities in assessment, monitoring and imaging

Silver nanoparticles (Ag NPs) are becoming increasingly prevalent in consumer products as antibacterial agents. The increased use of Ag NP-enhanced products may lead to an increase in toxic levels of environmental silver, but regulatory control over the use or disposal of such products is lagging due to insufficient assessment on the toxicology of Ag NPs and their rate of release into the environment. In this article we discuss recent research on the transport, activity and fate of Ag NPs at the cellular and organismic level, in conjunction with traditional and recently established methods of nanoparticle characterization. We include several proposed mechanisms of cytotoxicity based on such studies, as well as new opportunities for investigating the uptake and fate of Ag NPs in living systems.

Trans-Synaptic Axonal Degeneration in the Visual Pathway in Multiple Sclerosis

To evaluate the association between the damage to the anterior and posterior visual pathway as evidence of the presence of retrograde and anterograde trans‐synaptic degeneration in multiple sclerosis (MS).

Agricultural Contributions of Antimicrobials and Hormones on Soil and Water Quality

Detection of many emerging chemicals of concern, including antimicrobials and steroid hormones, in the environment has increased in the past decade with the advancement of analytical techniques. There are several potential sources of these inputs, including municipal wastewater discharge, municipal biosolids, pharmaceutical production, and agriculture‐related activities. However, the heavy use of antibiotics in the livestock industry and the dramatic shift in recent years toward more highly concentrated animal feeding operations (CAFOs), thus a concomitant increase in the volume of animal wastes per unit of land, has drawn attention to the role of animal waste‐borne antimicrobials, antibiotic‐resistant bacteria, and steroid hormones on ecosystem and human health. Antimicrobials, although frequently detected, are typically present in water at concentrations in orders of magnitude below what would be considered inhibitory to most biota. Most antibiotics have a high affinity for soil and sediment, thus residual soil concentrations are usually much higher than noted in water but still often below concentrations of concern. The focal point with antibiotic use in animal production is the development of antibiotic‐resistant bacteria. Although there is a growing body of evidence of the presence of numerous antibiotic‐resistant genes in animal wastes, in soils where wastes are land applied, and in water bodies receiving runoff from manure‐amended fields or discharges from aquacultures, conclusive evidence of animal‐derived antibiotic‐resistant pathogens compromising human health is lacking. In contrast to antibiotics, hormones and related chemicals can cause significant biological responses at very low concentrations. CAFO discharges will include a variety of estrogens, natural and synthetic androgens and progesterones, and phytoestrogens associated with animal feed. Measurable concentrations of many of these hormones have been detected in soil, and ground and surface waters receiving runoff from fields fertilized with animal manure and downstream from farm animal operations. Overall, hormones appear to be moderately to highly sorbed and to dissipate quickly in an aerobic soil environment, but quantitative information on hormone persistence in manure‐applied fields and subsequent effects of hormone loads from CAFOs to the aquatic environment is lacking. Research directed toward evaluating the facilitated transport processes with regards to antimicrobial and hormone inputs from manure‐amended fields is in its infancy. With the advances in analytical techniques and what has already been learned with regards to transport of nutrients (nitrogen, phosphorus, and carbon) and pesticides from agricultural fields, a reasonable evaluation of CAFOs and associated activities (land application of animal wastes) should be forthcoming in the next decade. Meanwhile, implementation of management practices that optimize reduction in already regulated nutrient releases from CAFOs should also help to minimize the release of antimicrobials and hormones.

A review of studies on androgen and estrogen exposure in fish early life stages: effects on gene and hormonal control of sexual differentiation.

Teleost fish are unique among vertebrates in that phenotypic sex or onset of sex inversion can be easily manipulated by hormonal treatments. In recent years, researchers have begun reporting concentrations of synthetic and natural hormones in the environment. Although concentrations are very low (in the parts per trillion to low parts per billion), they are still of concern because of the high potency of synthetic hormones and the enhanced susceptibility of teleost fishes, especially early life stages, to hormonal exposures. In this review, we will focus on sex differentiation in teleost fishes and how these processes in fish early life stages may be impacted by environmental hormones which are known to contaminate aquatic environments. We will start by reviewing information on sources and concentrations of hormones in the environment and continue by summarizing the state of knowledge of sex differentiation in teleost gonochoristic fishes, including information on genes involved (e.g. cyp19, dmrt1, sox9 and foxl2). We will end our review with a summary of studies that have examined the effects of androgens and estrogens on fish sex differentiation after exposure of fish embryos and larvae and with ideas for future research. Copyright

Review of recent proteomic applications in aquatic toxicology.

Over the last decade, the environmental sciences have witnessed an incredible movement towards the utilization of high-throughput molecular tools that are capable of detecting simultaneous changes of hundreds, and even thousands, of molecules and molecular components after exposure of organisms to different environmental stressors. These techniques have received a great deal of attention because they not only offer the potential to unravel novel mechanisms of physiological and toxic action but are also amenable to the discovery of biomarkers of exposure and effects. In this article, we review the state of knowledge of one of these tools in ecotoxicological research: proteomics. We summarize the state of proteomics research in fish, and follow with studies conducted with aquatic invertebrates. A brief discussion on proteomic methods is also presented. We conclude with some ideas for future proteomic studies with fish and aquatic invertebrates.

An evaluation of biomarkers of reproductive function and potential contaminant effects in Florida largemouth bass (Micropterus salmoides floridanus) sampled from the St. Johns River

The objective of this study was to describe and compare several reproductive parameters for Florida largemouth bass (Micropterus salmoides floridanus) inhabiting the St. Johns River and exposed to different types and/or degrees of contamination. Welaka was selected as the reference site in this study because of its low urban and agricultural development, Palatka is in close proximity to a paper mill plant, the Green Cove site is influenced by marine shipping activities and Julington Creek site receives discharges of domestic wastewater and storm water runoff from recreational boating marinas. For this study, bass were sampled both prior to (September 1996) and during the spawning season (February 1997). In order to characterize chemical exposure, bass livers were analyzed for up to 90 trace organics and 11 trace metal contaminants. Reproductive parameters measured included gonadosomatic index (GSI), histological evaluation of gonads and plasma concentrations of vitellogenin (VTG), 17beta-estradiol (E2) and 11-ketotestosterone (11-KT). In general, the sum of organic chemicals was highest in livers from Palatka bass and bass from Green Cove and Julington Creek had higher hepatic concentrations of low molecular polycyclic aromatic hydrocarbons and polychlorinated biphenyls when compared to fish from Welaka. Metals were more variable across sites, with highest mean concentrations found in bass from either Julington Creek (Ag, As, Cr, Cu, Zn) or Welaka (Cd, Hg, Pb, Se, Tn). Female bass from Palatka and Green Cove had lower concentrations of E2, VTG and lower GSI in relation to Welaka. Males from Palatka and Green Cove showed comparable declines in 11-KT in relation to males from Julington Creek and GSI were decreased only in Palatka males. These results indicate a geographical trend in reproductive effects, with changes being most pronounced at the site closest to the paper mill (Palatka) and decreasing as the St. Johns River flows downstream. Since reproductive alterations were most evident in bass sampled from the site closest to the paper mill discharge, it is possible that exposure to these effluents might explain at least some of the results reported here. However, the presence of reproductive alterations in fish sampled at a considerable distance from the mill discharge (Green Cove, 40 km) would suggest exposure to chemicals released from sources other than the paper mill plant. It is clear that additional studies are needed to evaluate the potential impact of these reproductive changes in populations of Florida largemouth bass inhabiting the St. Johns River.

Development of GCxGC/TOF-MS metabolomics for use in ecotoxicological studies with invertebrates

The majority of metabolomic studies used in ecotoxicology have implemented (1)H NMR analysis. Despite constant improvement, major limitations of NMR-based techniques include relatively low sensitivity that results in an examination of a limited number of metabolites. An alternative approach is the use of liquid or gas chromatography (GC) for separation of metabolites and mass spectrometry (MS) for their quantification and identification. The objective of our study was to develop a two dimensional GC coupled with time of flight MS (GCxGC/TOF-MS) coupled with multivariate analysis to compare metabolite profiles of Diporeia under different environmental conditions. We compared metabolite profiles between Diporeia collected from Lake Michigan (declining populations) to those residing in Lake Superior (stable populations), and also between Diporeia exposed to a chemical stressor (atrazine) and controls. Overall, 76 and 302 total metabolites were detected from the lake comparison and atrazine studies, respectively. Many of the identified metabolites included fatty acids, amino acids, and hydrocarbons. Furthermore, we observed unique and almost non-overlapping metabolite profiles in both studies. In conclusion, we established the feasibility of using GCxGC/TOF-MS for detecting metabolites as well as developed software to align and merge chromatographic peaks to compare metabolite differences between invertebrate groups sampled under different environmental conditions. This ability to detect unique metabolite profiles under different environmental conditions will increase our undertsanding on the physiological processes and whole-organism reponses occuring as a result of exposure to different environmental stressors.

Gene expression responses in male fathead minnows exposed to binary mixtures of an estrogen and antiestrogen

BackgroundAquatic organisms are continuously exposed to complex mixtures of chemicals, many of which can interfere with their endocrine system, resulting in impaired reproduction, development or survival, among others. In order to analyze the effects and mechanisms of action of estrogen/anti-estrogen mixtures, we exposed male fathead minnows (Pimephales promelas) for 48 hours via the water to 2, 5, 10, and 50 ng 17α-ethinylestradiol (EE2)/L, 100 ng ZM 189,154/L (a potent antiestrogen known to block activity of estrogen receptors) or mixtures of 5 or 50 ng EE2/L with 100 ng ZM 189,154/L. We analyzed gene expression changes in the gonad, as well as hormone and vitellogenin plasma levels.ResultsSteroidogenesis was down-regulated by EE2 as reflected by the reduced plasma levels of testosterone in the exposed fish and down-regulation of genes in the steroidogenic pathway. Microarray analysis of testis of fathead minnows treated with 5 ng EE2/L or with the mixture of 5 ng EE2/L and 100 ng ZM 189,154/L indicated that some of the genes whose expression was changed by EE2 were blocked by ZM 189,154, while others were either not blocked or enhanced by the mixture, generating two distinct expression patterns. Gene ontology and pathway analysis programs were used to determine categories of genes for each expression pattern.ConclusionOur results suggest that response to estrogens occurs via multiple mechanisms, including canonical binding to soluble estrogen receptors, membrane estrogen receptors, and other mechanisms that are not blocked by pure antiestrogens.

Multiple origins of pyrethroid insecticide resistance across the species complex of a nontarget aquatic crustacean, Hyalella azteca

Significance The crustacean, Hyalella azteca, is commonly used in environmental monitoring to test the toxicity of water or sediment. We show that among three laboratory cultures and seven wild populations of H. azteca, there is a more than 550-fold variation in sensitivity to widely used pyrethroid insecticides. Some individuals have attained resistance by mutations in the voltage-gated sodium channel, the target site for pyrethroid toxicity. Similar mutations have been found in agricultural pests targeted by pyrethroids, but this study indicates that runoff of terrestially applied urban and agricultural pesticides has been sufficient to induce resistance in a nontarget aquatic species on multiple, independent occasions. Our results have far-reaching implications for biomonitoring programs in general and especially those relying on H. azteca. Use of pesticides can have substantial nonlethal impacts on nontarget species, including driving evolutionary change, often with unknown consequences for species, ecosystems, and society. Hyalella azteca, a species complex of North American freshwater amphipods, is widely used for toxicity testing of water and sediment and has frequently shown toxicity due to pyrethroid pesticides. We demonstrate that 10 populations, 3 from laboratory cultures and 7 from California water bodies, differed by at least 550-fold in sensitivity to pyrethroids. The populations sorted into four phylogenetic groups consistent with species-level divergence. By sequencing the primary pyrethroid target site, the voltage-gated sodium channel, we show that point mutations and their spread in natural populations were responsible for differences in pyrethroid sensitivity. At least one population had both mutant and WT alleles, suggesting ongoing evolution of resistance. Although nonresistant H. azteca were susceptible to the typical neurotoxic effects of pyrethroids, gene expression analysis suggests the mode of action in resistant H. azteca was not neurotoxicity but was oxidative stress sustained only at considerably higher pyrethroid concentrations. The finding that a nontarget aquatic species has acquired resistance to pesticides used only on terrestrial pests is troubling evidence of the impact of chronic pesticide transport from land-based applications into aquatic systems. Our findings have far-reaching implications for continued uncritical use of H. azteca as a principal species for monitoring and environmental policy decisions.

Transcriptome alterations following developmental atrazine exposure in zebrafish are associated with disruption of neuroendocrine and reproductive system function, cell cycle, and carcinogenesis

Atrazine, a herbicide commonly applied to agricultural areas and a common contaminant of potable water supplies, is implicated as an endocrine-disrupting chemical (EDC) and potential carcinogen. Studies show that EDCs can cause irreversible changes in tissue formation, decreased reproductive potential, obesity, and cancer. The U.S. Environmental Protection Agency considers an atrazine concentration of ≤ 3 ppb in drinking water safe for consumption. The specific adverse human health effects associated with a developmental atrazine exposure and the underlying genetic mechanisms of these effects are not well defined. In this study, zebrafish embryos were exposed to a range of atrazine concentrations to establish toxicity. Morphological, transcriptomic, and protein alterations were then assessed at 72h postfertilization following developmental atrazine exposure at 0, 0.3, 3, or 30 ppb. A significant increase in head length was observed in all three atrazine treatments. Transcriptomic profiles revealed 21, 62, and 64 genes with altered expression in the 0.3, 3, and 30 ppb atrazine treatments, respectively. Altered genes were associated with neuroendocrine and reproductive system development, function, and disease; cell cycle control; and carcinogenesis. There was a significant overlap (42 genes) between the 3 and 30 ppb differentially expressed gene lists, with two of these genes (CYP17A1 and SAMHD1) present in all three atrazine treatments. Increased transcript levels were translated to significant upregulation in protein expression. Overall, this study identifies genetic and molecular targets altered in response to a developmental atrazine exposure to further define the biological pathways and mechanisms of toxicity.