Trust your gut: Establishing confidence in gastrointestinal models - An overview of the state of the science and contexts of use.

The webinar series and workshop titled Trust Your Gut: Establishing Confidence in Gastrointestinal Models - An Overview of the State of the Science and Contexts of Use was co-organized by NICEATM, NIEHS, FDA, EPA, CPSC, DoD, and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and hosted at the National Institutes of Health in Bethesda, MD, USA on October 11-12, 2023. New approach methods (NAMs) for assessing issues of gastrointestinal tract (GIT)-related toxicity offer promise in addressing some of the limitations associated with animal-based assessments. GIT NAMs vary in complexity, from two-dimensional monolayer cell line-based systems to sophisticated 3-dimensional organoid systems derived from human primary cells. Despite advances in GIT NAMs, challenges remain in fully replicating the complex interactions and processes occurring within the human GIT. Presentations and discussions addressed regulatory needs, challenges, and innovations in incorporating NAMs into risk assessment frameworks; explored the state of the science in using NAMs for evaluating systemic toxicity, understanding absorption and pharmacokinetics, evaluating GIT toxicity, and assessing potential allergenicity; and discussed strengths, limitations, and data gaps of GIT NAMs as well as steps needed to establish confidence in these models for use in the regulatory setting.

Non-animal methods to assess whether chemicals may be toxic to the human digestive tract promise to complement or improve on animal-based methods. These approaches, which are based on human or animal cells and/or computer models, are faced with their own technical challenges and need to be shown to predict adverse effects in humans. Regulators are tasked with evaluating submitted data to best protect human health and the environment. A webinar series and workshop brought together scientists from academia, industry, military, and regulatory authorities from different countries to discuss how non-animal methods can be integrated into the risk assessment of drugs, food additives, dietary supplements, pesticides, and industrial chemicals for gastrointestinal toxicity.

In vitro toxicity and chemical analysis of e-cigarette aerosol produced amid dry hitting.

Dry hitting, a phenomenon produced by e-cigarettes with refillable cartridges when the liquid in the coil is low, is a common occurrence among regular vapers despite being an unintended consequence of the device. This phenomenon's hazard to public health is still unknown and needs further investigation. Lung cells cultured at the air-liquid interface were exposed to vaped aerosol consisting of 3 % w/v ethyl maltol in propylene glycol for three-second puffs every 30 seconds for 80 total puffs with either dry hit or saturated conditions. Cytotoxicity was measured colorimetrically. The thermal degradation of the heating coils and wicks was visualized using scanning electron microscopy. The chemical byproducts in the aerosol were analyzed using proton nuclear magnetic resonance and inductively coupled plasma mass spectrometry. The results revealed a highly significant increase in cytotoxicity from dry hit treatments. Imaging showed thermal decomposition of the cotton wick after dry hitting, which was confirmed by energy dispersive x-ray spectroscopy with less oxygen in the dry hit cotton. Chemical byproducts were found via unique peaks in the dry hit condensate in the aromatic and alkene regions. Saturated condensate showed higher concentrations of detected metal species than dry-hit condensate. E-cigarette users should avoid dry hitting by refilling tanks or cartridges preemptively or by using disposable coils to avoid increased toxicity during vaping.

Bioaccessibility of Metallic Nickel and Nickel Oxide Nanoparticles in Four Simulated Biological Fluids.

Bioaccessibility of metals from substances and alloys is increasingly used as part of the assessment to predict potential toxicity. However, data are sparse on the metal bioaccessibility from nanoparticle (NP) size metal substances. This study examines nickel ion release from metallic nickel and nickel oxide micron particles (MPs) and NPs in simulated biological fluids at various timepoints including those relevant for specific routes of exposure. The results suggest that MPs of both metallic nickel and nickel oxide generally released more nickel ions in acidic simulated biological fluids (gastric and lysosomal) than NPs of the same substance, with the largest differences being for nickel oxide. In more neutral pH fluids (interstitial and perspiration), nickel metal NPs released more nickel ions than MPs, with nickel oxide results showing a higher release for MPs in interstitial fluid yet a lower release in perspiration fluid. Various experimental factors related to the particle, fluid, and extraction duration were identified that can have an impact on the particle dissolution and release of nickel ions. Overall, the results suggest that based on nickel release alone, nickel NPs are not inherently more hazardous than nickel MPs. Moreover, analyses should be performed on a case-by-case basis with consideration of various experimental factors and correlation with in vivo data.

The impact of surface functionalization of cellulose following simulated digestion and gastrointestinal cell-based model exposure.

Surface-functionalized cellulose materials are developed for various purposes, including food additives and food contact materials. A new biologically relevant testing strategy has been developed based on guidance from the European Food Safety Authority to demonstrate the safety of several next-generation surface-functionalized cellulose materials. This strategy involves a complex three-stage simulated digestion to compare the health effects of thirteen novel different types of cellulose. The physical and chemical properties of surface-functionalized fibrillated celluloses differed depending on the type, amount, and location of functional groups such as sulfonate, TEMPO-oxidized carboxy, and periodate-chlorite oxidized dicarboxylic acid celluloses. Despite exposure to gastrointestinal fluids, the celluloses maintained their physicochemical properties, such as negative surface charges and high length-to-width/thickness aspect ratios. An established intestinal co-culture model was used to measure cytotoxicity, barrier integrity, oxidative stress, and pro-inflammatory response to create a toxicological profile for these unique materials. We conclude that the C6 carboxylated cellulose nanofibrils by TEMPO-oxidation induced the most toxicity in the biological model used in this study and that the observed effects were most prominent at the 4-hour post-exposure time point.

The transcriptomic signature of respiratory sensitizers using an alveolar model.

Environmental contaminants are ubiquitous in the air we breathe and can potentially cause adverse immunological outcomes such as respiratory sensitization, a type of immune-driven allergic response in the lungs. Wood dust, latex, pet dander, oils, fragrances, paints, and glues have all been implicated as possible respiratory sensitizers. With the increased incidence of exposure to chemical mixtures and the rapid production of novel materials, it is paramount that testing regimes accounting for sensitization are incorporated into development cycles. However, no validated assay exists that is universally accepted to measure a substance's respiratory sensitizing potential. The lungs comprise various cell types and regions where sensitization can occur, with the gas-exchange interface being especially important due to implications for overall lung function. As such, an assay that can mimic the alveolar compartment and assess sensitization would be an important advance for inhalation toxicology. Some such models are under development, but in-depth transcriptomic analyses have yet to be reported. Understanding the transcriptome after sensitizer exposure would greatly advance hazard assessment and sustainability. We tested two known sensitizers (i.e., isophorone diisocyanate and ethylenediamine) and two known non-sensitizers (i.e., chlorobenzene and dimethylformamide). RNA sequencing was performed in our in vitro alveolar model, consisting of a 3D co-culture of epithelial, macrophage, and dendritic cells. Sensitizers were readily distinguishable from non-sensitizers by principal component analysis. However, few differentially regulated genes were common across all pair-wise comparisons (i.e., upregulation of genes SOX9, UACA, CCDC88A, FOSL1, KIF20B). While the model utilized in this study can differentiate the sensitizers from the non-sensitizers tested, further studies will be required to robustly identify critical pathways inducing respiratory sensitization.

Regioselectively Carboxylated Cellulose Nanofibril Models from Dissolving Pulp: C6 via TEMPO Oxidation and C2,C3 via Periodate-Chlorite Oxidation.

Regioselective C6 and C2,C3 carboxylated cellulose nanofibrils (CNFs) have been robustly generated from dissolving pulp, a readily available source of unmodified cellulose, via stoichiometrically optimized 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO)-mediated and sequential sodium periodate-sodium chlorite (PC) oxidation coupled with high-speed blending. Both regioselectively optimized carboxylated CNF series possess the widest ranges of comparable charges (0.72-1.48 mmol/g for T-CNFs vs. 0.72-1.10 mmol/g for PC-CNFs), but similar ranges of thickness (1.3-2.4 nm for T-CNF, 1.8-2.7 nm PC-CNF), widths (4.6-6.6 nm T-CNF, 5.5-5.9 nm PC-CNF), and lengths (254-481 nm T-CNF, 247-442 nm PC-CNF). TEMPO-mediated oxidation is milder and one-pot, thus more time and process efficient, whereas the sequential periodate-chlorite oxidation produces C2,C3 dialdehyde intermediates that are amenable to further chemical functionalization or post-reactions. These two well-characterized regioselectively carboxylated CNF series represent coherent cellulose nanomaterial models from a single woody source and have served as references for their safety study toward the development of a safer-by-design substance evaluation tool.

Aqueous exfoliation and dispersion of monolayer and bilayer graphene from graphite using sulfated cellulose nanofibrils.

Amphiphilic sulfated cellulose nanofibrils were synthesized with yields in excess of 99% by sulfation of dissolving pulp cellulose using chlorosulfonic acid in anhydrous N,N-dimethyl formamide followed by high-speed blending. The sulfation level was stoichiometrically tunable to between 1.48 and 2.23 mmol g-1. The optimized SCNF demonstrated the ability to act as an effective dispersant for graphene produced via exfoliation in aqueous media, allowing for the production of aqueous stabilized graphene with 3.9 ± 0.3 wt% graphite to graphene conversion and suspended solids comprised of 19.5 ± 1.5 wt% graphene. Graphene exfoliated with SCNF was observed to consist exclusively of mono- and bilayers, with 42% of sheets being monolayer. Furthermore, it was demonstrated that SCNF defibrillation and graphene exfoliation could be combined into a single one-pot process.

High-throughput screening of respiratory hazards: Exploring lung surfactant inhibition with 20 benchmark chemicals.

As environmental air quality worsens and respiratory health injuries and diseases increase, it is essential to enhance our ability to develop better methods to identify potential hazards. One promising approach in emerging toxicology involves the utilization of lung surfactant as a model that addresses the limitations of conventional in vitro toxicology methods by incorporating the biophysical aspect of inhalation. This study employed a constrained drop surfactometer to assess 20 chemicals for potential surfactant inhibition. Of these, eight were identified as inhibiting lung surfactant function: 1-aminoethanol, bovine serum albumin, maleic anhydride, propylene glycol, sodium glycocholate, sodium taurocholate, sodium taurodeoxycholate, and Triton X-100. These results are consistent with previously reported chemical-induced acute lung dysfunction in vivo. The study provides information on each chemical's minimum and maximum surface tension conditions and corresponding relative area and contact angle values. Isotherms and box plots are reported for selected chemicals across doses, and vector plots are used to summarize and compare the results concisely. This lung surfactant bioassay is a promising non-animal model for hazard identification, with broader implications for developing predictive modeling and decision-making tools.

Modeling mixtures interactions in environmental toxicology.

In the environment, organisms are exposed to mixtures of different toxicants, which may interact in ways that are difficult to predict when only considering each component individually. Adapting and expanding tools from pharmacology, the toxicology field uses analytical, graphical, and computational methods to identify and quantify interactions in multi-component mixtures. The two general frameworks are concentration addition, where components have similar modes of action and their effects sum together, or independent action, where components have dissimilar modes of action and do not interact. Other interaction behaviors include synergism and antagonism, where the combined effects are more or less than the additive sum of individual effects. This review covers foundational theory, methods, an in-depth survey of original research from the past 20 years, current trends, and future directions. As humans and ecosystems are exposed to increasingly complex mixtures of environmental contaminants, analyzing mixtures interactions will continue to become a more critical aspect of toxicological research.

Developmental effects of zebrafish (Danio rerio) embryos after exposure to glyphosate and lead mixtures.

Natural aquatic environments have a heterogeneous composition; therefore, simultaneous exposure to multiple contaminants is relevant and more realistic when assessing exposure and toxicity. This study examines the combinatorial effects of two compounds found ubiquitously in drinking water across the United States: glyphosate and lead acetate. Zebrafish (Danio rerio) embryos were used as a model for investigating developmental delays following controlled exposures. Six different environmentally relevant exposure concentrations of glyphosate, ranging from 0.001 to 10 ppm, and lead acetate, ranging from 0.5 to 4 ppm, were applied first as single exposures and then as co-exposures. The sublethal endpoints of hatching and coagulation were quantified to determine potencies. Results indicate that higher concentrations of the individual chemicals correlate with later hatching with correlation coefficients of 0.71 and 0.40 for glyphosate and lead acetate respectively, while the co-exposure at lower concentrations induced earlier hatching with a correlation coefficient 0.74. In addition, increased levels of coagulation and glutathione reductase activity were observed following co-exposure, as compared to the individual exposures, suggesting potential toxicological interactions. These results support the need for further work assessing the combined potencies of aquatic contaminants rather than individual exposures.

Evaluating Manganese, Zinc, and Copper Metal Toxicity on SH-SY5Y Cells in Establishing an Idiopathic Parkinson's Disease Model.

Parkinson's disease (PD) is a neurodegenerative condition marked by loss of motor coordination and cognitive impairment. According to global estimates, the worldwide prevalence of PD will likely exceed 12 million cases by 2040. PD is primarily associated with genetic factors, while clinically, cases are attributed to idiopathic factors such as environmental or occupational exposure. The heavy metals linked to PD and other neurodegenerative disorders include copper, manganese, and zinc. Chronic exposure to metals induces elevated oxidative stress and disrupts homeostasis, resulting in neuronal death. These metals are suggested to induce idiopathic PD in the literature. This study measures the effects of lethal concentration at 10% cell death (LC10) and lethal concentration at 50% cell death (LC50) concentrations of copper, manganese, and zinc chlorides on SH-SY5Y cells via markers for dopamine, reactive oxygen species (ROS) generation, DNA damage, and mitochondrial dysfunction after a 24 h exposure. These measurements were compared to a known neurotoxin to induce PD, 100 µM 6-hydroxydopamine (6-ODHA). Between the three metal chlorides, zinc was statistically different in all parameters from all other treatments and induced significant dopaminergic loss, DNA damage, and mitochondrial dysfunction. The LC50 of manganese and copper had the most similar response to 6-ODHA in all parameters, while LC10 of manganese and copper responded most like untreated cells. This study suggests that these metal chlorides respond differently from 6-ODHA and each other, suggesting that idiopathic PD utilizes a different mechanism from the classic PD model.

Rapid enzymatic activity model (REAM) to decipher the toxic action of per- and polyfluoroalkyl substances.

Per- and polyfluoroalkyl substances (PFAS) have been identified as emerging contaminants and human exposure to these substances is a rising public health concern. We have developed a rapid enzymatic activity model (REAM), which can serve as a cell-free screening tool that elucidates possible mechanisms of toxic action inexpensively and quickly for these and other environmentally relevant chemicals.

Effects of short-chain per- and polyfluoroalkyl substances (PFAS) on toxicologically relevant gene expression profiles in a liver-on-a-chip model.

Short-chain per- and polyfluoroalkyl substances (PFAS) are highly stable and widely used environmental contaminants that pose potential health risks to humans. Aggregating reliable mechanistic information for safety assessments necessitates physiologically relevant high-throughput screening approaches. Here, we demonstrated the utility of a liver-on-a-chip model to investigate the effects of five short-chain PFAS at low (1 nM) and high (1 µM) concentrations on toxicologically-relevant gene expression profiles using the QuantiGene® Plex Assay. We found that the short-chain PFAS tested in this study modulated the expression of ABCG2, a gene encoding for the breast cancer resistance protein (BCRP), with marked and significant upregulation (up to 4-fold) observed for all but one of the short-chain PFAS tested. PFBS and HFPO-DA repressed SLCO1B3 expression, a gene that encodes for an essential liver-specific organic anion transporter. High concentrations of PFBS, PFHxA, and PFHxS upregulated the expression of genes encCYP1A1,CYP2B6 and CYP2C19 with the same treatments resulting in the repression of the expression of the gene encoding CYP1A2. This dysregulation could have consequences for the clearance of endogenous compounds and xenobiotics. However, we acknowledge that increased expression of genes encoding for transporters and biotransformation enzymes may or may not indicate changes to their protein expression or activity. Overall, our study provides important insights into the effects of short-chain PFAS on liver function and their potential implications for human health. The use of the liver-on-a-chip model in combination with the QuantiGene® Plex Assay may be a valuable tool for future high-throughput screening and gene expression profiling in toxicology studies.

An In Vitro Alveolar Model Allows for the Rapid Assessment of Particles for Respiratory Sensitization Potential.

Dust, both industrial and household, contains particulates that can reach the most distal aspects of the lung. Silica and nickel compounds are two such particulates and have known profiles of poor health outcomes. While silica is well-characterized, nickel compounds still need to be fully understood for their potential to cause long-term immune responses in the lungs. To assess these hazards and decrease animal numbers used in testing, investigations that lead to verifiable in vitro methods are needed. To understand the implications of these two compounds reaching the distal aspect of the lungs, the alveoli, an architecturally relevant alveolar model consisting of epithelial cells, macrophages, and dendritic cells in a maintained submerged system, was utilized for high throughput testing. Exposures include crystalline silica (SiO2) and nickel oxide (NiO). The endpoints measured included mitochondrial reactive oxygen species and cytostructural changes assessed via confocal laser scanning microscopy; cell morphology evaluated via scanning electron microscopy; biochemical reactions assessed via protein arrays; transcriptome assessed via gene arrays, and cell surface activation markers evaluated via flow cytometry. The results showed that, compared to untreated cultures, NiO increased markers for dendritic cell activation, trafficking, and antigen presentation; oxidative stress and cytoskeletal changes, and gene and cytokine expression of neutrophil and other leukocyte chemoattractants. The chemokines and cytokines CCL3, CCL7, CXCL5, IL-6, and IL-8 were identified as potential biomarkers of respiratory sensitization.

Peripheral (lung-to-brain) exposure to diesel particulate matter induces oxidative stress and increased markers for systemic inflammation.

Combustion-derived air pollution is a complex environmental toxicant that has become a global health concern due to urbanization. Air pollution contains pro-inflammatory stimulants such as fine and ultrafine particulate matter, gases, volatile organic compounds, and metals. This study is focused on the particulate phase, which has been shown to induce systemic inflammation after chronic exposure due to its ability to travel to the lower airway, resulting in the activation of local immune cell populations, releasing acute phase reactants to mitigate ongoing inflammation. The systemic response is a potential mechanism for the co-morbidity associated with regions with high pollution and neuropathology. We exposed diesel particulate matter (DPM) to a pulmonary cell-derived in vitro model where macrophages mimic the diffusion of cytokines into the peripheral circulation to microglia. Alveolar macrophages (transformed U937) were inoculated with resuspended DPM in an acute exposure (24-h incubation) and analyzed for MCP-1 expression and acute phase reactants (IL-1ß, IL-6, IL-8, and TNF-α). Post-exposure serum was collected and filtered from cultured alveolar macrophages, introduced to a healthy culture of microglial cells (HMC3), and measured for neurotoxic cytokines, oxidative stress, and pattern recognition receptors. After DPM exposure, the macrophages significantly upregulated all measured acute phase reactants, increased H2O2 production, and increased MCP-1 expression. After collection and filtration to remove excess particulates, microglia cells were incubated with the collected serum for 48 h to allow for cytokine diffusion between the periphery of microglia. Microglia significantly upregulated IL-6, IL-8, and oxidative stress with a moderate increase in IL-1ß and TNF-α. As a marker required for signaling tissue damage, CD14 indicated that compared to direct inoculation of DPM, peripheral exposure resulted in the potent activation of microglia cells. The specificity and potency of the response have implications for neuropathology through lung-to-brain mechanisms after inhalation of environmental pollutants.

A preparation of bacterial outer membrane with osmium tetroxide and uranyl acetate co-stain enables improved structural determination by transmission electron microscopy.

Biological nanoparticles, such as bacterial outer membrane vesicles (OMVs), are routinely characterized through transmission electron microscopy (TEM). In this study, we report a novel method to prepare OMVs for TEM imaging. To preserve vesicular shape and structure, we developed a dual fixation protocol involving osmium tetroxide incubation prior to negative staining with uranyl acetate. Combining osmium tetroxide with uranyl acetate resulted in preservation of sub-50 nm vesicles and improved morphological stability, enhancing characterization of lipid-based nanoparticles by TEM.

Nanoparticle surface coatings produce distinct antibacterial effects that are consistent across diverse bacterial species.

Nanoparticles have been proposed as tunable delivery vehicles for targeted treatments and, in some cases, the active therapeutic agents themselves. Despite the promise of such customizable impacts, little evidence exists to support these claims in the realm of antibiotics. Exploration of the silver and copper nanoparticle antibacterial impacts have been reported with inconsistent results. Here, we investigate the physical, chemical, and bacterial properties of silver and copper core particles stabilized with commonly used surface coatings, namely, polyvinylpyrrolidone (PVP, to confer a neutrally charged surface), cetrimonium bromide (CTAB, positively charged surface), citrate (Cit, negatively charged surface for silver nanoparticles), and ascorbic acid (AA, negatively charged surface for copper nanoparticles. The impacts of these potential antibacterial nanoparticles are measured against three bacterial species spanning deep divisions in the bacterial tree of life and include Escherichia coli, Staphylococcus aureus, and Sphingobacterium multivorum. Varying dose, core composition, surface coating, and bacterial species revealed that nanoparticle surfaces accounted for most of the variation in antibacterial activity. In all experiments, dose produced a linear inhibitory effect. Surprisingly, bacterial species reacted similarly regardless of evolutionary relatedness. There is a high degree of consistency, effectiveness, and efficacy among PVP silver and copper nanoparticle. These findings have implications for the intentional use of nanotechnology in environmental systems.

Nanoparticle surface stabilizing agents influence antibacterial action.

The antibacterial properties of nanoparticles are of particular interest because of their potential to serve as an alternative therapy to combat antimicrobial resistance. Metal nanoparticles such as silver and copper nanoparticles have been investigated for their antibacterial properties. Silver and copper nanoparticles were synthesized with the surface stabilizing agents cetyltrimethylammonium bromide (CTAB, to confer a positive surface charge) and polyvinyl pyrrolidone (PVP, to confer a neutral surface charge). Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and viable plate count assays were used to determine effective doses of silver and copper nanoparticles treatment against Escherichia coli, Staphylococcus aureus and Sphingobacterium multivorum. Results show that CTAB stabilized silver and copper nanoparticles were more effective antibacterial agents than PVP stabilized metal nanoparticles, with MIC values in a range of 0.003 µM to 0.25 µM for CTAB stabilized metal nanoparticles and 0.25 µM to 2 µM for PVP stabilized metal nanoparticles. The recorded MIC and MBC values of the surface stabilized metal nanoparticles show that they can serve as effective antibacterial agents at low doses.

Chronic Exposure to Polystyrene Microplastic Fragments Has No Effect on Honey Bee Survival, but Reduces Feeding Rate and Body Weight.

Microplastics (MPs), in the form of fragments and fibers, were recently found in honey samples collected in Ecuador as well as in honey bees collected from Denmark and China. However, little is known about how MPs impact bee health. To fill this knowledge gap, we investigated the potential toxicity of irregularly shaped polystyrene (PS)-MP fragments on honey bee health. In the first experiment of its kind with honey bees, we chronically exposed bees with a well-established gut microbiome to small (27 ± 17 µm) or large (93 ± 25 µm) PS-MP fragments at varying concentrations (1, 10, 100 µg mL-1) for 14 days. Bee mortality, food consumption, and body weight were all studied. We found that chronic exposure to PS-MP fragments has no effect on honey bee survival, but reduced the feeding rate and body weight, particularly at 10 µg PS-MP fragments per mL, which may have long-term consequences for honey bee health. The findings of this study could assist in the risk assessment of MPs on pollinator health.

Lung surfactant as a biophysical assay for inhalation toxicology.

Lung surfactant (LS) is a mixture of lipids and proteins that forms a thin film at the gas-exchange surfaces of the alveoli. The components and ultrastructure of LS contribute to its biophysical and biochemical functions in the respiratory system, most notably the lowering of surface tension to facilitate breathing mechanics. LS inhibition can be caused by metabolic deficiencies or the intrusion of endogenous or exogenous substances. While LS has been sourced from animals or synthesized for clinical therapeutics, the biofluid mixture has also gained recent interest as a biophysical model for inhalation toxicity. Various methods can be used to evaluate LS function quantitatively or qualitatively after exposure to potential toxicants. A narrative review of the recent literature was conducted. Studies focused whether LS was inhibited by various environmental contaminants, nanoparticles, or manufactured products. A review is also conducted on synthetic lung surfactants (SLS), which have emerged as a promising alternative to conventional animal-sourced LS. The intrinsic advantages and recent advances of SLS make a strong case for more widespread usage in LS-based toxicological assays.