Amelia Romoser

The relationship between pH and zeta potential of ∼ 30 nm metal oxide nanoparticle suspensions relevant to in vitro toxicological evaluations

Abstract Zeta potential measurements are common in nanotoxicology. This research probes the effects of pH and time on nanoparticle zeta potential, agglomerate size, and cellular viability. The nanoparticles TiO2, Fe2O3, Al2O3, ZnO, and CeO2, were titrated from pH 12.0–2.0. The isoelectric points (IEP) of the nanoparticles were near neutral with the exception of TiO2 (IEP = 5.19) and Fe2O3 (IEP = 4.24). Nanoparticle agglomerates were largest at the IEP. TiO2 and Fe2O3 increased in zeta potential and agglomerate size over time; while Al2O3 and ZnO displayed little change. CeO2 increased in zeta potential; however, the net charge remained negative. Cytotoxicity studies revealed that TiO2 and Fe2O3 caused decreasing cellular viability over 48 h. Al2O3, ZnO, and CeO2 cellular viability remained similar to control. Results indicate that alterations in the pH have a large effect on zeta potential and agglomerate size which may be used as a predictive measure of nanotoxicity.

Quantum dots trigger immunomodulation of the NFκB pathway in human skin cells.

The immunological effects of quantum dots are dependent on a variety of factors including, but not limited to, exposure time and dosing concentrations. In this study, we investigated the influence of 15 nm CdSe/ZnS-COOH quantum dot nanocrystals (QDs) on cell density, viability, and morphology in human epidermal keratinocytes (HEK) and human dermal fibroblasts (HDF). Furthermore, inflammatory and non-inflammatory immune responses were measured using protein and real time PCR array analysis from HDF cells exposed to predetermined sub-lethal concentrations of QDs. CdSe/ZnS-COOH QDs caused concentration-dependent (1-120 nM exposure concentrations) and time-dependent (8 h or 48 h) cell death, as evidenced by metabolic activity and morphological changes. QD exposure induced upregulation of apoptotic, inflammatory and immunoregulatory proteins such as TNF-α, IL-1B and IL-10. HMOX1, an indicator of stress due to reactive oxygen intermediates (ROIs) and/or metals, was upregulated at the later time point as well. QDs also caused modulation of genes known to be associated with inflammatory (IL1-β, CCL2, IRAK-2), immune (IL-1, IL-6, PGLYRP1, SERPINA1, IL-10), stress due to ROIs and/or heavy metals (HMOX1), and apoptotic (CASP1, ADORA2A) responses. Cellular effects from QD exposure were found to primarily follow the NFκB pathway. In addition, QDs induced a differential cytotoxicity in keratinocytes and fibroblasts at different exposure concentrations and time points, even at physiologically relevant dosing concentrations, thus emphasizing the need to investigate potential mechanisms of action among different cell types within the same target organ.

Comparative cytological responses of lung epithelial and pleural mesothelial cells following in vitro exposure to nanoscale SiO2.

Due to unique surface chemistries and the ability to easily functionalize their surface, amorphous silica nanoparticles are being assimilated into medicinal and consumer products at an increasing rate. Subsequently, there is an emergent need to understand the interactions of these particulates with biological systems in an attempt to mitigate toxicity. The identification of susceptible or resistant cell types of the pulmonary system remains a critical step in the development of toxicity assessments for nanoparticle-based platforms. Specific to this study, the cellular responses of A549 lung epithelial and MeT-5A pleural mesothelial cell lines as a means of detecting nanoparticle-induced oxidative stress were examined. Basal expression and cellular antioxidant activity, including SOD, CAT, and GSH, were examined prior to H(2)O(2) and ~30 nm SiO(2) (0.01-100mg/L) exposures. Dose-response observations were made regarding oxidant production, cytotoxicity, GSH depletion and NRF2 transcription factor activation. Results indicated that, while both cell types exhibited susceptibility to H(2)O(2) and SiO(2)-induced oxidative stress and damage, the A549 cell line was relatively more resilient.

Mitigation of Quantum Dot Cytotoxicity by Microencapsulation

When CdSe/ZnS-polyethyleneimine (PEI) quantum dots (QDs) are microencapsulated in polymeric microcapsules, human fibroblasts are protected from acute cytotoxic effects. Differences in cellular morphology, uptake, and viability were assessed after treatment with either microencapsulated or unencapsulated dots. Specifically, QDs contained in microcapsules terminated with polyethylene glycol (PEG) mitigate contact with and uptake by cells, thus providing a tool to retain particle luminescence for applications such as extracellular sensing and imaging. The microcapsule serves as the “first line of defense” for containing the QDs. This enables the individual QD coating to be designed primarily to enhance the function of the biosensor.

Calcium montmorillonite clay reduces AFB1 and FB1 biomarkers in rats exposed to single and co‐exposures of aflatoxin and fumonisin

Aflatoxins (AFs) and fumonisins (FBs) can co‐contaminate foodstuffs and have been associated with hepatocellular and esophageal carcinomas in humans at high risk for exposure. One strategy to reduce exposure (and toxicity) from contaminated foodstuffs is the dietary inclusion of a montmorillonite clay (UPSN) that binds AFs and FBs in the gastrointestinal tract. In this study, the binding capacity of UPSN was evaluated for AFB1, FB1 and a combination thereof in Fischer 344 rats. Rats were pre‐treated with different dietary levels of UPSN (0.25% or 2%) for 1 week. Rats were gavaged with a single dose of either 0.125 mg AFB1 or 25 mg FB1 per kg body weight and a combination thereof in the presence and absence of an aqueous solution of UPSN. The kinetics of mycotoxin excretion were monitored by analyzing serum AFB1‐albumin, urinary AF (AFM1) and FB1 biomarkers over a period of 72 h. UPSN decreased AFM1 excretion by 88–97%, indicating highly effective binding. FB1 excretion was reduced, to a lesser extent, ranging from 45% to 85%. When in combination, both AFB1 and FB1 binding occurred, but capacity was decreased by almost half. In the absence of UPSN, the combined AFB1 and FB1 treatment decreased the urinary biomarkers by 67% and 45% respectively, but increased levels of AFB1‐albumin, presumably by modulating its cytochrome metabolism. UPSN significantly reduced bioavailability of both AFB1 and FB1 when in combination; suggesting that it can be utilized to reduce levels below their respective thresholds for affecting adverse biological effects. Copyright

The effect of aflatoxin-B1 on red drum (Sciaenops ocellatus) and assessment of dietary supplementation of NovaSil for the prevention of aflatoxicosis.

Aflatoxin B1 (AFB1) is a potent carcinogen that causes growth stunting, immunosuppression and liver cancer in multiple species. The recent trend of replacing fishmeal with plant-based proteins in fish feed has amplified the AFB1 exposure risk in farm-raised fish. NovaSil (NS), a calcium montmorillonite clay, has previously been shown to reduce AFB1 bioavailability safely and efficaciously in several mammalian species. This study was designed to: (1) evaluate AFB1 impact on cultured red drum, Sciaenops ocellatus, over the course of seven weeks; and (2) assess NS supplementation as a strategy to prevent aflatoxicosis. Fish were fed diets containing 0, 0.1, 0.25, 0.5, 1, 2, 3, or 5 ppm AFB1. Two additional treatment groups were fed either 5 ppm AFB1 + 1% NS or 5 ppm AFB1 + 2% NS. Aflatoxin B1 negatively impacted red drum weight gain, survival, feed efficiency, serum lysozyme concentration, hepatosomatic index (HSI), whole-body lipid levels, liver histopathological scoring, as well as trypsin inhibition. NovaSil inclusion in AFB1-contaminated diets improved weight gain, feed efficiency, serum lysozyme concentration, muscle somatic index, and intraperitoneal fat ratios compared to AFB1-treated fish. Although not significant, NS reduced AFB1-induced histopathological changes in the liver and decreased Proliferating Cell Nuclear Antigen (PCNA) staining. Importantly, NS supplementation improved overall health of AFB1-exposed red drum.

Distinct immunomodulatory effects of a panel of nanomaterials in human dermal fibroblasts

There are many efforts in understanding the effects of nanoparticles on cell viability and metabolism, however, not much is known regarding the distinct molecular mechanisms of inflammation and cellular stress using low dosing concentrations. To address this gap in the literature, we utilized a novel experimental design that specifically probes the effects of a panel of commonly studied engineered nanomaterials along immunomodulatory pathways, including NF-κB. The panel of particles selected for this study included quantum dot nanocrystals, titanium dioxide, hydroxylated fullerenes, and silver nanoparticles. Cell viability, antioxidant activity, select messenger RNA, and protein modulation were studied in primary human dermal fibroblasts (HDF) and NF-κB knockdown HDF cells. Inflammatory and non-inflammatory immune responses were measured using protein and real-time PCR array analysis from HDF cells exposed to sub-lethal concentrations of nanoparticles. Differences in cellular response to nanoparticles in protein and antioxidant experiments were evident in NF-κB knockdown cells. The methods used in the study, along with the resultant data sets, serve as a potential model for studying the complex pathway-specific biochemical responses in cell and tissue systems associated with nanoparticle exposures.

Modified Hydra Bioassay to Evaluate the Toxicity of Multiple Mycotoxins and Predict the Detoxification Efficacy of a Clay-Based Sorbent

Food shortages and a lack of food supply regulation in developing countries often leads to chronic exposure of vulnerable populations to hazardous mixtures of mycotoxins, including aflatoxin B1 (AFB1) and fumonisin B1 (FB1). A refined calcium montmorillonite clay [i.e. uniform particle size NovaSil (UPSN)] has been reported to tightly bind these toxins, thereby decreasing bioavailability in humans and animals. Hence, our objectives in the present study were to examine the ability of UPSN to bind mixtures of AFB1 and FB1 at gastrointestinally relevant pH in vitro, and to utilize a rapid in vivo bioassay to evaluate AFB1 and FB1 toxicity and UPSN efficacy. Isothermal sorption data indicated tight AFB1 binding to UPSN surfaces at both pH 2.0 and 6.5, but substantially more FB1 bound at pH 2.0 than 6.5. Site‐specific competition occurred between the toxins when exposed to UPSN in combination. Importantly, treatment with UPSN resulted in significant protection to mycotoxin‐exposed hydra maintained at pH 6.9–7.0. Hydra were exposed to FB1, AFB1 and FB1/AFB1 combinations with and without UPSN. A toxic response over 92 h was rated based on morphology and mortality. Hydra assay results indicated a minimum effective concentration (MEC) of 20 µg ml–1 for AFB1, whereas the MEC for FB1 was not reached. The MEC for co‐exposure was 400 µg ml–1 FB1 + 10 µg ml–1 AFB1. This study demonstrates that UPSN sorbs both mycotoxins tightly at physiologically relevant pH levels, resulting in decreased bioavailability, and that a modified hydra bioassay can be used as an initial screen in vivo to predict efficacy of toxin‐binding agents. Copyright

Effects of calcium montmorillonite clay and aflatoxin exposure on dry matter intake, milk production, and milk composition

Fifteen primiparous crossbred dairy cows that were 114±14d in milk and weighed 533±56kg were used in a replicated 5×5 Latin square to test the efficacy of a calcium montmorillonite clay, NovaSil Plus (NSP; BASF Corp., Ludwigshaven, Germany), for the reduction of aflatoxin (AF) metabolite (AFM1) in milk and the effect of NSP on milk composition. Cows were housed in a freestall barn, fed once a day and milked twice a day. The experiment consisted of five 14-d periods: d 1 through 7 were considered for data collection, and d 8 through 14 were considered a wash-out phase. In each period, cows were randomly assigned to 1 of 5 dietary treatments: (1) control (CON), consisting of a basal total mixed ration (TMR); (2) high-dose NSP diet (NSP-1%), consisting of TMR plus 230 g of NSP; (3) aflatoxin diet (AFD), consisting of the TMR plus AF challenge; (4) low-dose NSP with AF (NSP-0.5%+AFD), composed of TMR plus 115 g of NSP and AF challenge; and (5) high-dose NSP with AF (NSP-1%+AFD), consisting of TMR plus 230 g of NSP and AF challenge. The AF challenge consisted of top dressing a daily dose of 100 µg/kg estimated dry matter intake (DMI); similarly, NSP was fed at 1.0 or 0.5% of estimated DMI. Milk yield and DMI were similar across treatments averaging 21.1±1.33 kg/d and 19.7±0.56 kg/d, respectively. Concentration of milk fat, protein, and lactose were similar across treatments with averages of 4.91±0.20%, 3.85±0.10%, and 4.70±0.06%, respectively. Concentration of vitamin A averaged 0.28±0.03 µg/mL and riboflavin concentration averaged 1.57±0.13 µg/mL across treatments. The concentration of minerals in milk were similar for all treatments. Cows fed CON and NSP-1% yielded the lowest concentration of AFM1 in milk with 0.03 and 0.01±0.06 µg/L. Addition of NSP reduced milk AFM1 from 1.10±0.06 µg/L with the AF diet to 0.58 and 0.32±0.06 µg/L with the NSP-0.5%+AF and NSP-1%+AF diets, respectively. Excretion of AFM1 was reduced by NSP; mean values were 24.38, 11.86, 7.38, 0.64, and 0.23, ± 1.71 µg/d, for AFD, NSP-0.5%+AFD, NSP-1%+AFD, NSP-1%, and CON, respectively. More specifically, 1.07±0.08% of the daily AF intake was transferred to the milk of cows consuming the AFD, whereas the AF transfer rates in milk from cows that consumed the NSP-0.5%+AFD and NSP-1%+AFD were 0.52 and 0.32±0.08%. Results from this research demonstrate that feeding NSP to lactating cows is an effective method to reduce the transfer and excretion of AFM1 in milk with no negative effects on dry matter intake, milk production, and composition.

Short-Term Safety and Efficacy of Calcium Montmorillonite Clay (UPSN) in Children

Recently, an association between childhood growth stunting and aflatoxin (AF) exposure has been identified. In Ghana, homemade nutritional supplements often consist of AF-prone commodities. In this study, children were enrolled in a clinical intervention trial to determine the safety and efficacy of Uniform Particle Size NovaSil (UPSN), a refined calcium montmorillonite known to be safe in adults. Participants ingested 0.75 or 1.5 g UPSN or 1.5 g calcium carbonate placebo per day for 14 days. Hematological and serum biochemistry parameters in the UPSN groups were not significantly different from the placebo-controlled group. Importantly, there were no adverse events attributable to UPSN treatment. A significant reduction in urinary metabolite (AFM1) was observed in the high-dose group compared with placebo. Results indicate that UPSN is safe for children at doses up to 1.5 g/day for a period of 2 weeks and can reduce exposure to AFs, resulting in increased quality and efficacy of contaminated foods.