Incineration-Generated Polyethylene Micro-Nanoplastics Increase Triglyceride Lipolysis and Absorption in an In Vitro Small Intestinal Epithelium Model.

Despite mounting evidence of micro-nanoplastics (MNPs) in food and drinking water, little is known of the potential health risks of ingested MNPs, and nothing is known of their potential impact on nutrient digestion and absorption. We assessed the effects of environmentally relevant secondary MNPs generated by incineration of polyethylene (PE-I), on digestion and absorption of fat in a high fat food model using a 3-phase in vitro simulated digestion coupled with a tri-culture small intestinal epithelium model. The presence of 400 µg/mL PE-I increased fat digestion by 33% and increased fat absorption by 147 and 145% 1 and 2 h after exposure. Analysis of the PE-I lipid corona during digestion revealed predominantly triacylglycerols with enrichment of fatty acids in the small intestinal phase. Protein corona analysis showed enrichment of triacylglycerol lipase and depletion of ß-casein in the small intestinal phase. These findings suggest digestion of triacylglycerol by lipase on the surface of lipid-coated MNPs as a potential mechanism. Further studies are needed to investigate the mechanisms underlying the greater observed increase in fat absorption, to verify these results in an animal model, and to determine the MNP properties governing their effects on lipid digestion and absorption.

Safer-by-design flame-sprayed silicon dioxide nanoparticles: the role of silanol content on ROS generation, surface activity and cytotoxicity.

BACKGROUND: Amorphous silica nanoparticles (SiO2 NPs) have been regarded as relatively benign nanomaterials, however, this widely held opinion has been questioned in recent years by several reports on in vitro and in vivo toxicity. Surface chemistry, more specifically the surface silanol content, has been identified as an important toxicity modulator for SiO2 NPs. Here, quantitative relationships between the silanol content on SiO2 NPs, free radical generation and toxicity have been identified, with the purpose of synthesizing safer-by-design fumed silica nanoparticles. RESULTS: Consistent and statistically significant trends were seen between the total silanol content, cell membrane damage, and cell viability, but not with intracellular reactive oxygen species (ROS), in the macrophages RAW264.7. SiO2 NPs with lower total silanol content exhibited larger adverse cellular effects. The SAEC epithelial cell line did not show any sign of toxicity by any of the nanoparticles. Free radical generation and surface reactivity of these nanoparticles were also influenced by the temperature of combustion and total silanol content. CONCLUSION: Surface silanol content plays an important role in cellular toxicity and surface reactivity, although it might not be the sole factor influencing fumed silica NP toxicity. It was demonstrated that synthesis conditions for SiO2 NPs influence the type and quantity of free radicals, oxidative stress, nanoparticle interaction with the biological milieu they come in contact with, and determine the specific mechanisms of toxicity. We demonstrate here that it is possible to produce much less toxic fumed silicas by modulating the synthesis conditions.

Analysis of lipid adsorption on nanoparticles by nanoflow liquid chromatography-tandem mass spectrometry.

Nanoparticles (NPs) tend to adsorb matrix molecules like proteins and lipids incubated with biological fluids, forming a biological corona. While the formation and functions of protein corona have been studied extensively, little attention has been paid to lipid adsorption on NPs. However, lipids are also abundantly present in biological fluids and play important roles in processes like cell signaling and angiogenesis. Therefore, in this study, we established the analytical procedure for study of lipid adsorption on three different types of NPs in two matrices: human serum and heavy cream, using nanoflow liquid chromatography-mass spectrometry (nanoflowLC-MS). Serum was chosen to represent the common environment the NPs would be present once entering human body, and heavy cream was the representative food matrix NPs may be added to improve the color or taste. Steps of liquid-liquid extraction were established and optimized to achieve maximum recovery of the adsorbed, standard lipids from the NPs. Then, the LC-MS/MS method was developed to attain base-line separation of the standard lipids that represent the major lipid classes. At last, the lipid adsorption profiles of the three NPs were compared. We found that the lipid adsorption profile on the same type of NP was significantly different between the two matrices. The established method will help us investigate lipid adsorption on additional NPs and reveal how it could be affected by the physiochemical properties of NPs and the presence of proteins and other components in the biological matrix.

Development of high throughput, high precision synthesis platforms and characterization methodologies for toxicological studies of nanocellulose.

Cellulose is one of the most abundant natural polymers, is readily available, biodegradable, and inexpensive. Recently, interest is growing around nanoscale cellulose due to the sustainability of these materials, the novel properties, and the overall low environmental impact. The rapid expansion of nanocellulose uses in various applications makes the study of the toxicological properties of these materials of great importance to public health regulators. However, most of the current toxicological studies are highly conflicting, inconclusive, and contradictory. The major reasons for these discrepancies are the lack of standardized methods to produce industry-relevant reference nanocellulose and relevant characterization that will expand beyond the traditional cellulose characterization for applications. In order to address these issues, industry-relevant synthesis platforms were developed to produce nanocellulose of controlled properties that can be used as reference materials in toxicological studies. Herein, two types of nanocellulose were synthesized, cellulose nanofibrils (CNF) and cellulose nanocrystals (CNC) using the friction grinding platform and an acid hydrolysis approach respectively. The nanocellulose structures were characterized extensively regarding their physicochemical properties, including testing for endotoxins and bacteria contamination.

Immunomodulators targeting MARCO expression improve resistance to postinfluenza bacterial pneumonia.

Downregulation of the alveolar macrophage (AM) receptor with collagenous structure (MARCO) leads to susceptibility to postinfluenza bacterial pneumonia, a major cause of morbidity and mortality. We sought to determine whether immunomodulation of MARCO could improve host defense and resistance to secondary bacterial pneumonia. RNAseq analysis identified a striking increase in MARCO expression between days 9 and 11 after influenza infection and indicated important roles for Akt and Nrf2 in MARCO recovery. In vitro, primary human AM-like monocyte-derived macrophages (AM-MDMs) and THP-1 macrophages were treated with IFNγ to model influenza effects. Activators of Nrf2 (sulforaphane) or Akt (SC79) caused increased MARCO expression and a MARCO-dependent improvement in phagocytosis in IFNγ-treated cells and improved survival in mice with postinfluenza pneumococcal pneumonia. Transcription factor analysis also indicated a role for transcription factor E-box (TFEB) in MARCO recovery. Overexpression of TFEB in THP-1 cells led to marked increases in MARCO. The ability of Akt activation to increase MARCO expression in IFNγ-treated AM-MDMs was abrogated in TFEB-knockdown cells, indicating Akt increases MARCO expression through TFEB. Increasing MARCO expression by targeting Nrf2 signaling or the Akt-TFEB-MARCO pathway are promising strategies to improve bacterial clearance and survival in postinfluenza bacterial pneumonia.

Free actin impairs macrophage bacterial defenses via scavenger receptor MARCO interaction with reversal by plasma gelsolin.

Lung injury can release intracellular actin into the alveolar milieu and is also associated with increased susceptibility to secondary infections. We investigated the effect of free (extracellular) actin on lung macrophage host defense functions. Western blot analysis demonstrated free actin release into the lung lavage fluids of mouse models of ozone injury, influenza infection, and secondary pneumococcal pneumonia and in samples from patients following burn and inhalation injury. Using levels comparable with those observed in lung injury, we found that free actin markedly inhibited murine lung macrophage binding and uptake in vitro of S. pneumoniae, S. aureus, and E. coli, (e.g., S. pneumoniae, mean %inhibition, actin vs. vehicle: 85 ± 0.3 (SD); n = 22, P < .001). Similar effects were observed on the ability of primary human macrophages to bind and ingest fluorescent Saureus (~75% inhibition). Plasma gelsolin (pGSN), a protein that functions to bind and cleave actin, restored bacterial binding and uptake by both murine and human macrophages. Scavenger receptor inhibitors reduced binding of fluorescent actin by murine macrophages [fluorescence index (×10-3) after incubation with vehicle, actin, or actin + polyinosinic acid, respectively: 0.8 ± 0.7, 101.7 ± 50.7, or 52.7 ± 16.9; n = 5-6, P < 0.05]. In addition, actin binding was reduced in a MARCO/SR-AI/II-deficient cell line and by normal AMs obtained from MARCO-/- mice. After release from injured cells during lung injury, free actin likely contributes to impaired host defense by blocking scavenger receptor binding of bacteria. This mechanism for increased risk of secondary infections after lung injury or inflammation may represent another target for therapeutic intervention with pGSN.

Semi-automated scoring of triple-probe FISH in human sperm using confocal microscopy.

Structural and numerical sperm chromosomal aberrations result from abnormal meiosis and are directly linked to infertility. Any live births that arise from aneuploid conceptuses can result in syndromes such as Kleinfelter, Turners, XYY and Edwards. Multi-probe fluorescence in situ hybridization (FISH) is commonly used to study sperm aneuploidy, however manual FISH scoring in sperm samples is labor-intensive and introduces errors. Automated scoring methods are continuously evolving. One challenging aspect for optimizing automated sperm FISH scoring has been the overlap in excitation and emission of the fluorescent probes used to enumerate the chromosomes of interest. Our objective was to demonstrate the feasibility of combining confocal microscopy and spectral imaging with high-throughput methods for accurately measuring sperm aneuploidy. Our approach used confocal microscopy to analyze numerical chromosomal abnormalities in human sperm using enhanced slide preparation and rigorous semi-automated scoring methods. FISH for chromosomes X, Y, and 18 was conducted to determine sex chromosome disomy in sperm nuclei. Application of online spectral linear unmixing was used for effective separation of four fluorochromes while decreasing data acquisition time. Semi-automated image processing, segmentation, classification, and scoring were performed on 10 slides using custom image processing and analysis software and results were compared with manual methods. No significant differences in disomy frequencies were seen between the semi automated and manual methods. Samples treated with pepsin were observed to have reduced background autofluorescence and more uniform distribution of cells. These results demonstrate that semi-automated methods using spectral imaging on a confocal platform are a feasible approach for analyzing numerical chromosomal aberrations in sperm, and are comparable to manual methods. © 2017 International Society for Advancement of Cytometry.

An integrated methodology for assessing the impact of food matrix and gastrointestinal effects on the biokinetics and cellular toxicity of ingested engineered nanomaterials.

BACKGROUND: Engineered nanomaterials (ENMs) are increasingly added to foods to improve their quality, sensory appeal, safety and shelf-life. Human exposure to these ingested ENMs (iENMS) is inevitable, yet little is known of their hazards. To assess potential hazards, efficient in vitro methodologies are needed to evaluate particle biokinetics and toxicity. These methodologies must account for interactions and transformations of iENMs in foods (food matrix effect) and in the gastrointestinal tract (GIT) that are likely to determine nano-biointeractions. Here we report the development and application of an integrated methodology consisting of three interconnected stages: 1) assessment of iENM-food interactions (food matrix effect) using model foods; 2) assessment of gastrointestinal transformations of the nano-enabled model foods using a three-stage GIT simulator; 3) assessment of iENMs biokinetics and cellular toxicity after exposure to simulated GIT conditions using a triculture cell model. As a case study, a model food (corn oil-in-water emulsion) was infused with Fe2O3 (Iron(III) oxide or ferric oxide) ENMs and processed using this three-stage integrated platform to study the impact of food matrix and GIT effects on nanoparticle biokinetics and cytotoxicity . METHODS: A corn oil in phosphate buffer emulsion was prepared using a high speed blender and high pressure homogenizer. Iron oxide ENM was dispersed in water by sonication and combined with the food model. The resulting nano-enabled food was passed through a three stage (mouth, stomach and small intestine) GIT simulator. Size distributions of nano-enabled food model and digestae at each stage were analyzed by DLS and laser diffraction. TEM and confocal imaging were used to assess morphology of digestae at each phase. Dissolution of Fe2O3 ENM along the GIT was assessed by ICP-MS analysis of supernatants and pellets following centrifugation of digestae. An in vitro transwell triculture epithelial model was used to assess biokinetics and toxicity of ingested Fe2O3 ENM. Translocation of Fe2O3 ENM was determined by ICP-MS analysis of cell lysates and basolateral compartment fluid over time. RESULTS: It was demonstrated that the interactions of iENMs with food and GIT components influenced nanoparticle fate and transport, biokinetics and toxicological profile. Large differences in particle size, charge, and morphology were observed in the model food with and without Fe2O3 and among digestae from different stages of the simulated GIT (mouth, stomach, and small intestine). Immunoflorescence and TEM imaging of the cell culture model revealed markers and morphology of small intestinal epithelium including enterocytes, goblet cells and M cells. Fe2O3 was not toxic at concentrations tested in the digesta. In biokinetics studies, translocation of Fe2O3 after 4 h was <1% and ~2% for digesta with and without serum, respectively, suggesting that use of serum proteins alters iENMs biokinetics and raises concerns about commonly-used approaches that neglect iENM - food-GIT interactions or dilute digestae in serum-containing media. CONCLUSIONS: We present a simple integrated methodology for studying the biokinetics and toxicology of iENMs, which takes into consideration nanoparticle-food-GIT interactions. The importance of food matrix and GIT effects on biointeractions was demonstrated, as well as the incorporation of these critical factors into a cellular toxicity screening model. Standardized food models still need to be developed and used to assess the effect of the food matrix effects on the fate and bioactivity of iENMs since commercial foods vary considerably in their compositions and structures.

Advanced computational modeling for in vitro nanomaterial dosimetry.

BACKGROUND: Accurate and meaningful dose metrics are a basic requirement for in vitro screening to assess potential health risks of engineered nanomaterials (ENMs). Correctly and consistently quantifying what cells "see," during an in vitro exposure requires standardized preparation of stable ENM suspensions, accurate characterizatoin of agglomerate sizes and effective densities, and predictive modeling of mass transport. Earlier transport models provided a marked improvement over administered concentration or total mass, but included assumptions that could produce sizable inaccuracies, most notably that all particles at the bottom of the well are adsorbed or taken up by cells, which would drive transport downward, resulting in overestimation of deposition. METHODS: Here we present development, validation and results of two robust computational transport models. Both three-dimensional computational fluid dynamics (CFD) and a newly-developed one-dimensional Distorted Grid (DG) model were used to estimate delivered dose metrics for industry-relevant metal oxide ENMs suspended in culture media. Both models allow simultaneous modeling of full size distributions for polydisperse ENM suspensions, and provide deposition metrics as well as concentration metrics over the extent of the well. The DG model also emulates the biokinetics at the particle-cell interface using a Langmuir isotherm, governed by a user-defined dissociation constant, K(D), and allows modeling of ENM dissolution over time. RESULTS: Dose metrics predicted by the two models were in remarkably close agreement. The DG model was also validated by quantitative analysis of flash-frozen, cryosectioned columns of ENM suspensions. Results of simulations based on agglomerate size distributions differed substantially from those obtained using mean sizes. The effect of cellular adsorption on delivered dose was negligible for K(D) values consistent with non-specific binding (> 1 nM), whereas smaller values (≤ 1 nM) typical of specific high-affinity binding resulted in faster and eventual complete deposition of material. CONCLUSIONS: The advanced models presented provide practical and robust tools for obtaining accurate dose metrics and concentration profiles across the well, for high-throughput screening of ENMs. The DG model allows rapid modeling that accommodates polydispersity, dissolution, and adsorption. Result of adsorption studies suggest that a reflective lower boundary condition is appropriate for modeling most in vitro ENM exposures.

Assessment of Ingested Micro- and Nanoplastic (MNP)-Mediated Genotoxicity in an In Vitro Model of the Small Intestinal Epithelium (SIE).

Micro- and nanoplastics (MNPs) have become ubiquitous contaminants of water and foods, resulting in high levels of human ingestion exposure. MNPs have been found in human blood and multiple tissues, suggesting that they are readily absorbed by the gastrointestinal tract (GIT) and widely distributed. Growing toxicological evidence suggests that ingested MNPs may pose a serious health threat. The potential genotoxicity of MNPs, however, remains largely unknown. In this study, genotoxicity of primary and environmentally relevant secondary MNPs was assessed in a triculture small intestinal epithelium (SIE) model using the CometChip assay. Aqueous suspensions of 25 and 1000 nm carboxylated polystyrene spheres (PS25C and PS1KC), and incinerated polyethylene (PEI PM0.1) were subjected to simulated GIT digestion to create physiologically relevant exposures (digestas), which were applied to the SIE model at final MNP concentrations of 1, 5, and 20 µg/mL for 24 or 48 h. PS25C and PS1KC induced DNA damage in a time- and concentration-dependent manner. To our knowledge, this is one of the first assessment of MNP genotoxicity in an integrated in vitro ingestion platform including simulated GIT digestion and a triculture SIE model. These findings suggest that ingestion of high concentrations of carboxylated PS MNPs could have serious genotoxic consequences in the SIE.

Mechanisms of ingested polystyrene micro-nanoplastics (MNPs) uptake and translocation in an in vitro tri-culture small intestinal epithelium.

Micro and nanoplastics (MNPs) are now ubiquitous contaminants of food and water. Many cellular and animal studies have shown that ingested MNPs can breach the intestinal barrier to reach the circulation. To date however, the cellular mechanisms involved in intestinal absorption of MNPs have not been investigated with physiologically relevant models, and thus remain unknown. We employed in vitro simulated digestion, a tri-culture small intestinal epithelium model, and a panel of inhibitors to assess the contributions of the possible mechanisms to absorption of 26 nm carboxylated polystyrene (PS26C) MNPs. Inhibition of ATP synthesis reduced translocation by only 35 %, suggesting uptake by both active endocytic pathways and passive diffusion. Translocation was also decreased by inhibition of dynamin and clathrin, suggesting involvement of clathrin mediated endocytosis (CME) and fast endophilin-mediated endocytosis (FEME). Inhibition of actin polymerization also significantly reduced translocation, suggesting involvement of macropinocytosis or phagocytosis. However, inhibition of the Na+-H+ exchanger had no effect on translocation, thus ruling out macropinocytosis. Together these results suggest uptake by passive diffusion as well as by active phagocytosis, CME, and FEME pathways. Further studies are needed to assess uptake mechanisms for other environmentally relevant MNPs as a function of polymer, surface chemistry, and size.

Micro- and nanoplastics (MNPs) and their potential toxicological outcomes: State of science, knowledge gaps and research needs.

Plastic waste has been produced at a rapidly growing rate over the past several decades. The environmental impacts of plastic waste on marine and terrestrial ecosystems have been recognized for years. Recently, researchers found that micro- and nanoplastics (MNPs), micron (100 nm - 5 mm) and nanometer (1 - 100 nm) scale particles and fibers produced by degradation and fragmentation of plastic waste in the environment, have become an important emerging environmental and food chain contaminant with uncertain consequences for human health. This review provides a comprehensive summary of recent findings from studies of potential toxicity and adverse health impacts of MNPs in terrestrial mammals, including studies in both in vitro cellular and in vivo mammalian models. Also reviewed here are recently released biomonitoring studies that have characterized the bioaccumulation, biodistribution, and excretion of MNPs in humans. The majority MNPs in the environment to which humans are most likely to be exposed, are of irregular shapes, varied sizes, and mixed compositions, and are defined as secondary MNPs. However, the MNPs used in most toxicity studies to date were commercially available primary MNPs of polystyrene (PS), polyethylene (PE), polyvinyl chloride (PVC), polyethylene terephthalate (PET), and other polymers. The emerging in vitro and in vivo evidence reviewed here suggests that MNP toxicity and bioactivity are largely determined by MNP particle physico-chemical characteristics, including size, shape, polymer type, and surface properties. For human exposure, MNPs have been identified in human blood, urine, feces, and placenta, which pose potential health risks. The evidence to date suggests that the mechanisms underlying MNP toxicity at the cellular level are primarily driven by oxidative stress. Nonetheless, large knowledge gaps in our understanding of MNP toxicity and the potential health impacts of MNP exposures still exist and much further study is needed to bridge those gaps. This includes human population exposure studies to determine the environmentally relevant MNP polymers and exposure concentrations and durations for toxicity studies, as well as toxicity studies employing environmentally relevant MNPs, with surface chemistries and other physico-chemical properties consistent with MNP particles in the environment. It is especially important to obtain comprehensive toxicological data for these MNPs to understand the range and extent of potential adverse impacts of microplastic pollutants on humans and other organisms.

Ingested Polystyrene Nanospheres Translocate to Placenta and Fetal Tissues in Pregnant Rats: Potential Health Implications.

Recent studies in experimental animals found that oral exposure to micro- and nano-plastics (MNPs) during pregnancy had multiple adverse effects on outcomes and progeny, although no study has yet identified the translocation of ingested MNPs to the placenta or fetal tissues, which might account for those effects. We therefore assessed the placental and fetal translocation of ingested nanoscale polystyrene MNPs in pregnant rats. Sprague Dawley rats (N = 5) were gavaged on gestational day 19 with 10 mL/kg of 250 µg/mL 25 nm carboxylated polystyrene spheres (PS25C) and sacrificed after 24 h. Hyperspectral imaging of harvested placental and fetal tissues identified abundant PS25C within the placenta and in all fetal tissues examined, including liver, kidney, heart, lung and brain, where they appeared in 10-25 µm clusters. These findings demonstrate that ingested nanoscale polystyrene MNPs can breach the intestinal barrier and subsequently the maternal-fetal barrier of the placenta to access the fetal circulation and all fetal tissues. Further studies are needed to assess the mechanisms of MNP translocation across the intestinal and placental barriers, the effects of MNP polymer, size and other physicochemical properties on translocation, as well as the potential adverse effects of MNP translocation on the developing fetus.

Cellulose Nanofiber Platform for Pesticide Sequestration in the Gastrointestinal Tract.

Exploitation of nature-derived materials is an important approach to promote environmental sustainability. Among these materials, cellulose is of particular interest due to its abundance and relative ease of access. As a food ingredient, cellulose nanofibers (CNFs) have found interesting applications as emulsifiers and modulators of lipid digestion and absorption. In this report, we show that CNFs can also be modified to modulate the bioavailability of toxins, such as pesticides, in the gastrointestinal tract (GIT) by forming inclusion complexes and promoting interaction with surface hydroxyl groups. CNFs were successfully functionalized with (2-hydroxypropyl)-ß-cyclodextrin (HPBCD) using citric acid as a crosslinker via esterification. Functionally, the potential for pristine and functionalized CNFs (FCNFs) to interact with a model pesticide, boscalid, was tested. Based on direct interaction studies, adsorption of boscalid saturated at around 3.09% on CNFs and at 12.62% on FCNFs. Using an in vitro GIT simulation platform, the adsorption of boscalid on CNFs/FCNFs was also studied. The presence of a high-fat food model was found to have a positive effect in binding boscalid in a simulated intestinal fluid environment. In addition, FCNFs were found to have a greater effect in retarding triglyceride digestion than CNFs (61% vs 30.6%). Overall, FCNFs were demonstrated to evoke synergistic effects of reducing fat absorption and pesticide bioavailability through inclusion complex formation and the additional binding of the pesticide onto surface hydroxyl groups on HPBCD. By adopting food-compatible materials and processes for production, FCNFs have the potential to be developed into a functional food ingredient for modulating food digestion and the uptake of toxins.

Semi-automated scoring of triple-probe FISH in human sperm: methods and further validation.

Although the frequency and consequence of sperm chromosomal abnormalities are considerable, few epidemiologic studies in large samples have been conducted to investigate etiologic risk factors. This is, in part, attributable to the labor intensive demands of manual sperm fluorescence in situ hybridization (FISH) scoring. As part of an epidemiologic study investigating environmental risk factors for aneuploidy among men attending a hospital-based fertility clinic, a semi-automated method of slide scoring was further validated and used to estimate sex chromosome sperm disomy frequency in a large number of samples. Multiprobe FISH for chromosomes X, Y, and 18 was used to determine sex chromosome disomy in sperm nuclei. Semi-automated scoring methods were used to quantify X disomy (sperm FISH genotype XX18), Y disomy (YY18), and XY disomy (XY18). The semi-automated results were compared with the results from manual scoring in 10 slides. The semi-automated method was then used to estimate sex chromosome disomy frequency in 60 men. Of 10 slides scored, significant differences between the manual and semi-automated results were seen primarily in one slide that was of poor quality because of over swollen nuclei. Among 60 men analyzed using the semi-automated method, median total sex chromosome disomy frequency was 1.65%, which is higher than seen among normal men but within range with reports from fertility clinic populations. These results further validate that semi-automated methods can be used to score sperm disomy with results comparable to manual methods. This is the largest study to date to provide estimates of sex chromosome disomy among men attending fertility clinics. These methods should be replicated in larger clinic populations to arrive at stable estimates of aneuploidy frequency in men who are members of subfertile couples. © 2011 International Society for Advancement of Cytometry.

Co-exposure to boscalid and TiO2 (E171) or SiO2 (E551) downregulates cell junction gene expression in small intestinal epithelium cellular model and increases pesticide translocation.

A recent published study showed that TiO2 (E171) and SiO2 (E551), two widely used nano-enabled food additives, increased the translocation of the commonly used pesticide boscalid by 20% and 30% respectively. Such increased absorption of pesticides due to the presence of engineered nanomaterials (ENMs) in food raises health concerns for these food additives. In this companion study, mRNA expression of genes related to cell junctions in a small intestinal epithelial cellular model after exposure to simulated digestas of fasting food model (phosphate buffer) containing boscalid (150 ppm) with or without either TiO2 or SiO2 (1% w/w) were analyzed. Specific changes in cell barrier function underlying or contributing to the increased translocation of boscalid observed in the previous study were assessed. Results showed that exposure to boscalid alone has no significant effect on cell junction genes, however, co-exposure to boscalid and TiO2 significantly regulated expression of cell-matrix junction focal adhesion-related genes, e.g., downregulating Cav1 (- 1.39-fold, p<0.05), upregulating Cav3 (+ 3.30-fold, p<0.01) and Itga4 (+ 3.30-fold, p<0.05). Similarly, co-exposure to boscalid and SiO2 significantly downregulated multiple cell-cell junction genes, including tight junction genes (Cldn1, Cldn11, Cldn16, Cldn18, and Jam3), adherens junction genes (Notch1, Notch3, Pvrl1) and gap junction genes (Gja3 and Gjb2), as well as cell-matrix junction focal adhesion genes (Itga4, Itga6, Itga7). Together, these findings suggest that co-ingestion of boscalid with TiO2 (E171) or SiO2 (E551) could cause weakening of cell junctions and intercellular adhesion, which could result in dysregulation of paracellular transport, and presumably contributed to the previously observed increased translocation of boscalid at the presence of these ENMs. This novel finding raises health safety concerns for such popular food additives.

Co-exposure to boscalid and TiO2 (E171) or SiO2 (E551) downregulates cell junction gene expression in small intestinal epithelium cellular model and increases pesticide translocation.

A recent published study showed that TiO2 (E171) and SiO2 (E551), two widely used nano-enabled food additives, increased the translocation of the commonly used pesticide boscalid by 20% and 30% respectively. Such increased absorption of pesticides due to the presence of engineered nanomaterials (ENMs) in food raises health concerns for these food additives. In this companion study, mRNA expression of genes related to cell junctions in a small intestinal epithelial cellular model after exposure to simulated digestas of fasting food model (phosphate buffer) containing boscalid (150 ppm) with or without either TiO2 or SiO2 (1% w/w) were analyzed. Specific changes in cell barrier function underlying or contributing to the increased translocation of boscalid observed in the previous study were assessed. Results showed that exposure to boscalid alone has no significant effect on cell junction genes, however, co-exposure to boscalid and TiO2 significantly regulated expression of cell-matrix junction focal adhesion-related genes, e.g., downregulating Cav1 (-1.39-fold, p < 0.05), upregulating Cav3 (+ 3.30-fold, p < 0.01) and Itga4 (+ 3.30-fold, p < 0.05). Similarly, co-exposure to boscalid and SiO2 significantly downregulated multiple cell-cell junction genes, including tight junction genes (Cldn1, Cldn11, Cldn16, Cldn18, and Jam3), adherens junction genes (Notch1, Notch3, Pvrl1) and gap junction genes (Gja3 and Gjb2), as well as cell-matrix junction focal adhesion genes (Itga4, Itga6, Itga7). Together, these findings suggest that co-ingestion of boscalid with TiO2 (E171) or SiO2 (E551) could cause weakening of cell junctions and intercellular adhesion, which could result in dysregulation of paracellular transport, and presumably contributed to the previously observed increased translocation of boscalid at the presence of these ENMs. This novel finding raises health safety concerns for such popular food additives.

Effects of ingested nanocellulose and nanochitosan materials on carbohydrate digestion and absorption in an in vitro small intestinal epithelium model.

Nanoscale materials derived from natural biopolymers like cellulose and chitosan have many potentially useful agri-food and oral drug delivery applications. Because of their large and potentially bioactive surface areas and other unique physico-chemical properties, it is essential when evaluating their toxicological impact to assess potential effects on the digestion and absorption of co-ingested nutrients. Here, the effects of cellulose nanofibers (CNF), cellulose nanocrystals (CNC), and chitosan nanoparticles (Chnp) on the digestion and absorption of carbohydrates were studied. Starch digestion was assessed by measuring maltose released during simulated digestion of starch solutions. Glucose absorption was assessed by measuring translocation from the resulting digestas across an in vitro transwell tri-culture model of the small intestinal epithelium and calculating the area under the curve increase in absorbed glucose, analogous to the glycemic index. At 1% w/w, CNF and Chnp had small but significant effects (11% decrease and 14% increase, respectively) and CNC had no effect on starch hydrolysis during simulated digestion of a 1% w/w rice starch solution. In addition, at 2% w/w CNC had no effect on amylolysis in 1% solutions of either rice, corn, or wheat starch. Similarly, absorption of glucose from digestas of starch solutions (i.e., from maltose), was unaffected by 1% w/w CNF or CNC, but was slightly increased (10%, p<0.05) by 1% Chnp, possibly due to the slightly higher maltose concentration in the Chnp-containing digestas. In contrast, all of the test materials caused sharp increases (~1.2, 1.5, and 1.6 fold for CNC, CNF, and Chnp, respectively) in absorption of glucose from starch-free digestas spiked with free glucose at a concentration corresponding to complete hydrolysis of 1% w/w starch. The potential for ingested cellulose and chitosan nanomaterials to increase glucose absorption could have important health implications. Further studies are needed to elucidate the mechanisms underlying the observed increases and to evaluate the potential glycemic effects in an intact in vivo system.

Toxicity, uptake, and nuclear translocation of ingested micro-nanoplastics in an in vitro model of the small intestinal epithelium.

Despite mounting evidence of increasing micro- and nanoplastics (MNPs) in natural environments, food, and drinking water, little is known of the potential health hazards of MNPs ingestion. We assessed toxicity and uptake of environmentally relevant MNPs in an in vitro small intestinal epithelium (SIE). Test MNPs included 25 and 1000 nm polystyrene (PS) microspheres (PS25 and PS1K); 25, 100, and 1000 nm carboxyl modified PS spheres (PS25C, PS100C, and PS1KC), and secondary MNPs from incinerated polyethylene (PEI). MNPs were subjected to 3-phase digestion to mimic transformations in the gastrointestinal tract (GIT) and digestas applied to the SIE. Carboxylated MNPs significantly reduced viability and increased permeability to 3 kD dextran. Uptake of carboxyl PS materials was size dependent, with significantly greater uptake of PS25C. Fluorescence confocal imaging showed some PS25C agglomerates entering cells independent of endosomes (suggesting diffusion), others within actin shells (suggesting phagocytosis), and many free within the epithelial cells, including agglomerates within nuclei. Pre-treatment with the dynamin inhibitor Dyngo partially reduced PS25 translocation, suggesting a potential role for endocytosis. These findings suggest that ingestion exposures to MNPs could have serious health consequences and underscore the urgent need for additional detailed studies of the potential hazards of ingested MNPs.

Effects of Ingested Nanomaterials on Tissue Distribution of Co-ingested Zinc and Iron in Normal and Zinc-Deficient Mice.

Cellulose nanofibers (CNF) reduced serum triglyceride levels in rats when co-administered with heavy cream by gavage. Do CNF and other nanomaterials (NMs) alter the tissue distribution and retention of co-administered metal ions? We evaluated whether 5 different NMs affected tissue distribution of co-ingested 65Zn++ and 59Fe+++ in zinc-replete versus zinc-deficient mice. Male C57BL/6J mice were fed either zinc-replete or zinc-deficient diets for 3 weeks, followed by gavage with NM suspensions in water containing both 65ZnCl2 and 59FeCl3. Urine and feces were measured for 48 h post-gavage. Mice were euthanized and samples of 22 tissues were collected and analyzed for 65Zn and 59Fe in a gamma counter. Our data show that zinc deficiency alters the tissue distribution of 65Zn but not of 59Fe, indicating that zinc and iron homeostasis are regulated by distinct mechanisms. Among the tested NMs, soluble starch-coated chitosan nanoparticles, cellulose nanocrystals, and TiO2 reduced Zn and Fe tissue retention in zinc-deficient but not in zinc-replete animals.