Joseph H. Bisesi

Tracking and quantification of single-walled carbon nanotubes in fish using near infrared fluorescence.

Detection of SWCNTs in complex matrices presents a unique challenge as common techniques lack spatial resolution and specificity. Near infrared fluorescence (NIRF) has emerged as a valuable tool for detecting and quantifying SWCNTs in environmental samples by exploiting their innate fluorescent properties. The objective of this study was to optimize NIRF-based imaging and quantitation methods for tracking and quantifying SWCNTs in an aquatic vertebrate model in conjunction with assessing toxicological end points. Fathead minnows (Pimephales promelas) were exposed by single gavage to SWCNTs and their distribution was tracked using a custom NIRF imaging system for 7 days. No overt toxicity was observed in any of the SWCNT treated fish; however, histopathology observations from gastrointestinal (GI) tissue revealed edema within the submucosa and altered mucous cell morphology. NIRF images showed strong SWCNT-derived fluorescence signals in whole fish and excised intestinal tissues. Fluorescence was not detected in other tissues examined, indicating that no appreciable intestinal absorption occurred. SWCNTs were quantified in intestinal tissues using a NIRF spectroscopic method revealing values that were consistent with the pattern of fluorescence observed with NIRF imaging. Results of this work demonstrate the utility of NIRF imaging as a valuable tool for examining uptake and distribution of SWCNTs in aquatic vertebrates.

Fate of single walled carbon nanotubes in wetland ecosystems

We report here the first studies addressing fate and transport of single walled carbon nanotubes (SWNTs) in aquatic mesocosms. The experimental design was structured to study the impact of nanomaterials within a tightly controlled and highly instrumented wetland ecosystems (aka mesocosm) and to address questions including fate and transport, effect on community structure, effects on biogeochemical function, and effects on productivity of the ecosystem. We added well-dispersed CoMoCat SWNTs (cSWNT,0 = 2.5 mg L−1) to the water column of a wetland mesocosm and examined the resulting phase distribution over time. Rapid settling of SWNTs from the water column was observed within a period of 2 days (Cw,t/Cw,0 < 0.01) after spiking. Samples from all mesocosm compartments (e.g. aquatic/semi aquatic plants, biofilm, mosquitofish and sediment) were analyzed to evaluate the transport and fate of SWNTs in the ecosystem. SWNTs were quantified in organism and sediment extracts using near-infrared fluorescence spectroscopy (NIRF). This technique can be used to quantitatively detect SWNTs in sediment and biotic matrices at environmentally relevant concentrations (MDLwater 5 μg L−1 MDLsediment 0.5 μg g−1 MDLbiota 5 μg g−1 wet weight) and qualitatively characterize SWNT samples before and after the studies. Results indicated that rapid aggregation and settling of SWNT resulted in accumulation of SWNT in surficial sediment. Sediment concentrations were spatially variable across the mesocosm, and thus estimates of SWNT mass balance within the mesocosm ranged from 7–48%. No bioaccumulation of SWNT in aquatic plants or vertebrates was observed over the 10 month incubation. However, NIRF imaging analysis suggested that mosquitofish ingested SWNT-laden particles but that burdens of SWNTs were confined to gut contents and may have been rapidly eliminated.

Single-walled carbon nanotubes increase pandemic influenza A H1N1 virus infectivity of lung epithelial cells.

BackgroundAirborne exposure to nanomaterials from unintended occupational or environmental exposures or as a consequence of product use may lead to adverse health effects. Numerous studies have focused on single-walled carbon nanotubes (SWCNTs) and their ability to cause pulmonary injury related to fibrosis, and cancer; however few studies have addressed their impact on infectious agents, particularly viruses that are known for causing severe disease. Here we have demonstrated the ability of pristine SWCNTs of diverse electronic structure to increase the susceptibility of small airway epithelial cells (SAEC) to pandemic influenza A H1N1 infection and discerned potential mechanisms of action driving this response.MethodsSmall airway epithelial cells (SAEC) were exposed to three types of SWCNTs with varying electronic structure (SG65, SG76, CG200) followed by infection with A/Mexico/4108/2009 (pH1N1). Cells were then assayed for viral infectivity by immunofluorescence and viral titers. We quantified mRNA and protein levels of targets involved in inflammation and anti-viral activity (INFβ1, IL-8, RANTES/CCL5, IFIT2, IFIT3, ST3GAL4, ST6GAL1, IL-10), localized sialic acid receptors, and assessed mitochondrial function. Hyperspectral imaging analysis was performed to map the SWCNTs and virus particles in fixed SAEC preparations. We additionally performed characterization analysis to monitor SWCNT aggregate size and structure under biological conditions using dynamic light scattering (DLS), static light scattering (SLS).ResultsBased on data from viral titer and immunofluorescence assays, we report that pre-treatment of SAEC with SWCNTs significantly enhances viral infectivity that is not dependent on SWCNT electronic structure and aggregate size within the range of 106 nm – 243 nm. We further provide evidence to support that this noted effect on infectivity is not likely due to direct interaction of the virus and nanoparticles, but rather a combination of suppression of pro-inflammatory (RANTES) and anti-viral (IFIT2, IFIT3) gene/protein expression, impaired mitochondrial function and modulation of viral receptors by SWCNTs.ConclusionsResults of this work reveal the potential for SWCNTs to increase susceptibility to viral infections as a mechanism of adverse effect. These data highlight the importance of investigating the ability of carbon-nanomaterials to modulate the immune system, including impacts on anti-viral mechanisms in lung cells, thereby increasing susceptibility to infectious agents.

Examination of Single-Walled Carbon Nanotubes Uptake and Toxicity from Dietary Exposure: Tracking Movement and Impacts in the Gastrointestinal System

Previous studies indicate that exposure of fish to pristine single-walled carbon nanotubes (SWCNTs) by oral gavage, causes no overt toxicity, and no appreciable absorption has been observed. However, in the environment, SWCNTs are likely to be present in dietary sources, which may result in differential impacts on uptake and biological effects. Additionally, the potential of these materials to sorb nutrients (proteins, carbohydrates, and lipids) while present in the gastrointestinal (GI) tract may lead to nutrient depletion conditions that impact processes such as growth and reproduction. To test this phenomenon, fathead minnows were fed a commercial diet either with or without SWCNTs for 96 h. Tracking and quantification of SWCNTs using near-infrared fluorescence (NIRF) imaging during feeding studies showed the presence of food does not facilitate transport of SWCNTs across the intestinal epithelia. Targeting genes shown to be responsive to nutrient depletion (peptide transporters, peptide hormones, and lipases) indicated that pept2, a peptide transporter, and cck, a peptide hormone, showed differential mRNA expression by 96 h, a response that may be indicative of nutrient limitation. The results of the current study increase our understanding of the movement of SWCNTs through the GI tract, while the changes in nutrient processing genes highlight a novel mechanism of sublethal toxicity in aquatic organisms.

Dynamism of Stimuli-Responsive Nanohybrids: Environmental Implications

Nanomaterial science and design have shifted from generating single passive nanoparticles to more complex and adaptive multi-component nanohybrids. These adaptive nanohybrids (ANHs) are designed to simultaneously perform multiple functions, while actively responding to the surrounding environment. ANHs are engineered for use as drug delivery carriers, in tissue-engineered templates and scaffolds, adaptive clothing, smart surface coatings, electrical switches and in platforms for diversified functional applications. Such ANHs are composed of carbonaceous, metallic or polymeric materials with stimuli-responsive soft-layer coatings that enable them to perform such switchable functions. Since ANHs are engineered to dynamically transform under different exposure environments, evaluating their environmental behavior will likely require new approaches. Literature on polymer science has established a knowledge core on stimuli-responsive materials. However, translation of such knowledge to environmental health and safety (EHS) of these ANHs has not yet been realized. It is critical to investigate and categorize the potential hazards of ANHs, because exposure in an unintended or shifting environment could present uncertainty in EHS. This article presents a perspective on EHS evaluation of ANHs, proposes a principle to facilitate their identification for environmental evaluation, outlines a stimuli-based classification for ANHs and discusses emerging properties and dynamic aspects for systematic EHS evaluation.

Effect of natural organic matter on the photo‐induced toxicity of titanium dioxide nanoparticles

Nano-titanium dioxide (TiO2 ) is the most widely used form of nanoparticles in commercial industry and comes in 2 main configurations: rutile and anatase. Rutile TiO2 is used in ultraviolet (UV) screening applications, whereas anatase TiO2 crystals have a surface defect that makes them photoreactive. There are numerous reports in the literature of photo-induced toxicity to aquatic organisms following coexposure to anatase nano-TiO2 and UV. All natural freshwater contains varying amounts of natural organic matter (NOM), which can drive UV attenuation and quench reactive oxygen species (ROS) in aquatic ecosystems. The present research examined how NOM alters the photo-induced toxicity of anatase nano-TiO2 . Daphnia magna neonates were coexposed to NOM and photoexcited anatase nano-TiO2 for 48 h. Natural organic matter concentrations as low as 4 mg/L reduced anatase nano-TiO2 toxicity by nearly 100%. These concentrations of NOM attenuated UV by <10% in the exposure system. However, ROS production measured using a fluorescence assay was significantly reduced in a NOM concentration--dependent manner. Taken together, these data suggest that NOM reduces anatase nano-TiO2 toxicity via an ROS quenching mechanism and not by attenuation of UV. Environ Toxicol Chem 2017;36:1661-1666.

Influence of the Gastrointestinal Environment on the Bioavailability of Ethinyl Estradiol Sorbed to Single-Walled Carbon Nanotubes

Recent evidence suggests that, because of their sorptive nature, if single-walled carbon nanotubes (SWCNTs) make their way into aquatic environments, they may reduce the toxicity of other waterborne contaminants. However, few studies have examined whether contaminants remain adsorbed following ingestion by aquatic organisms. The objective of this study was to examine the bioavailability and bioactivity of ethinyl estradiol (EE2) sorbed onto SWCNTs in a fish gastrointestinal (GI) tract. Sorption experiments indicated that SWCNTs effectively adsorbed EE2, but the chemical was still able to bind and activate soluble estrogen receptors (ERs) in vitro. However, centrifugation to remove SWCNTs and adsorbed EE2 significantly reduced ER activity compared to that of EE2 alone. Additionally, the presence of SWCNTs did not reduce the extent of EE2-driven induction of vitellogenin 1 in vivo compared to the levels in organisms exposed to EE2 alone. These results suggest that while SWCNTs adsorb EE2 from aqueous solutions, under biological conditions EE2 can desorb and retain bioactivity. Additional results indicate that interactions with gastrointestinal proteins may decrease the level of adsorption of estrogen to SWCNTs by 5%. This study presents valuable data for elucidating how SWCNTs interact with chemicals that are already present in our aquatic environments, which is essential for determining their potential health risk.

Differential recruitment of co-regulatory proteins to the human estrogen receptor 1 in response to xenoestrogens.

The diverse biological effects of xenoestrogens may be explained by their ability to differentially recruit co-regulatory proteins to the estrogen receptor (ER). We employed high-throughput receptor affinity binding and co-regulatory protein recruitment screening assays based on fluorescence polarization and time resolved florescence resonance energy transfer (TR-FRET), respectively, to assess xenoestrogen-specific binding and co-regulatory protein recruitment to the ER. Then we used a functional proteomic assay based on co-immunoprecipitation of ER-bound proteins to isolate and identify intact co-regulatory proteins recruited to a ligand-activated ER. Through these approaches, we revealed differential binding affinity of bisphenol-A (BPA) and genistein (GEN) to the human ERα (ESR1) and ligand-dependent recruitment of SRC-1 and SRC-3 peptides. Recruitment profiles were variable for each ligand and in some cases were distinct compared to 17β-estradiol (E2). For example, E2 and GEN recruited both SRC-1 and -3 peptides whereas BPA recruited only SRC-1 peptides. Results of the functional proteomic assay showed differential recruitment between ligands where E2 recruited the greatest number of proteins followed by BPA then GEN. A number of proteins share previously identified relationships with ESR1 as determined by STRING analysis. Although there was limited overlap in proteins identified between treatments, all ligands recruited proteins involved in cell growth as determined by subnetwork enrichment analysis (p<0.05). A comparative, in silico analysis revealed that fewer interactions exist between zebrafish (Danio rerio) esr1 and zebrafish orthologs of proteins identified in our functional proteomic analysis. Taken together these results identify recruitment of known and previously unknown co-regulatory proteins to ESR1 and highlight new methods to assay recruitment of low abundant and intact, endogenous co-regulatory proteins to ESR1 or other nuclear receptors, in both human and aquatic species.

Oral bioavailability and sex specific tissue partitioning of quantum dots in fathead minnows, Pimephales promelas

The number of potential applications for manufactured nanomaterials (NMs) is growing exponentially around the world as is concomitant research into the possible consequences of inadvertent or purposeful releases into the environment. Fish and other aquatic organisms reside in bodies of water where many NMs may potentially be deposited, as these environments act as terminal sinks for many contaminants. A growing body of evidence suggests that some NMs, depending on their composition, size, and/or surface functionalization, can affect fish health by adversely interacting with gill function or by entering circulation through the digestive tract. The goal of this study was to investigate the role surface functionalization plays on oral bioavailability of NMs, using quantum dots (QDs) as a model. Three different surface functional groups, amino, carboxyl, and PEG were investigated. Additionally, two different exposure scenarios, a single dose or 5 sequential doses over 2 weeks, were used to determine which tissues were the sites of greatest accumulation over time. Results show QDs are able to enter the blood stream after ingestion, and accumulate in the intestine, liver, gonads, and other organs in female and male fathead minnows. Data from a repeated dosing experiment indicated that QDs were retained and accumulated in most tissues in a surface functionalization and sex specific manner. The carboxyl and amino QDs were found to be most readily taken up and the carboxyl QDs were found to be in the greatest concentration in the most number of tissues including the gonad, spleen and kidneys in both males and females.

The Gut Microbiome and Aquatic Toxicology: An Emerging Concept for Environmental Health

The microbiome plays an essential role in the health and onset of diseases in all animals, including humans. The microbiome has emerged as a central theme in environmental toxicology because microbes interact with the host immune system in addition to its role in chemical detoxification. Pathophysiological changes in the gastrointestinal tissue caused by ingested chemicals and metabolites generated from microbial biodegradation can lead to systemic adverse effects. The present critical review dissects what we know about the impacts of environmental contaminants on the microbiome of aquatic species, with special emphasis on the gut microbiome. We highlight some of the known major gut epithelium proteins in vertebrate hosts that are targets for chemical perturbation, proteins that also directly cross-talk with the microbiome. These proteins may act as molecular initiators for altered gut function, and we propose a general framework for an adverse outcome pathway that considers gut dysbiosis as a major contributing factor to adverse apical endpoints. We present 2 case studies, nanomaterials and hydrocarbons, with special emphasis on the Deepwater Horizon oil spill, to illustrate how investigations into the microbiome can improve understanding of adverse outcomes. Lastly, we present strategies to functionally relate chemical-induced gut dysbiosis with adverse outcomes because this is required to demonstrate cause-effect relationships. Further investigations into the toxicant-microbiome relationship may prove to be a major breakthrough for improving animal and human health. Environ Toxicol Chem 2018;37:2758-2775.