Simulating Nanomaterial Transformation in Cascaded Biological Compartments to Enhance the Physiological Relevance of In Vitro Dosing Regimes: Optional or Required?

Would an engineered nanomaterial (ENM) still have the same identity once it reaches a secondary target tissue after a journey through several physiological compartments? Probably not. Does it matter? ENM pre-treatments may enhance the physiological relevance of in vitro testing via controlled transformation of the ENM identity. The implications of material transformation upon reactivity, cytotoxicity, inflammatory, and genotoxic potential of Ag and SiO2 ENM on advanced gastro-intestinal tract cell cultures and 3D liver spheroids are demonstrated. Pre-treatments are recommended for certain ENM only.

Model Complexity as Determining Factor for In Vitro Nanosafety Studies: Effects of Silver and Titanium Dioxide Nanomaterials in Intestinal Models.

With the rising interest in the effects of orally ingested engineered nanomaterials (ENMs), much effort is undertaken to develop and advance intestinal in vitro models. The cytotoxic, proinflammatory, and DNA damaging properties of polyvinylpyrrolidone-capped silver (Ag-PVP) and titanium dioxide (TiO2 , P25) ENM in four in vitro models of increasing complexity-from proliferating Caco-2 and HT29-MTX-E12 monocultures to long-term transwell triple cultures including THP-1 macrophages to reproduce the human intestine in healthy versus inflamed-like state-are studied. Results are compared against in vivo effects of the same ENM through intestinal tissue analysis from 28-day oral exposure studies in mice. Adverse responses are only observed in monocultures and suggest toxic potential for both ENM, typically showing stronger effects for Ag-PVP than for TiO2 . By contrast, no adverse effects are observed in either the transwell cultures or the analyzed murine tissues. The data provide further support that monoculture models represent a cost and time efficient tool for early-phase hazard assessment. However, the observed similarities in morphology and ENM effects in murine intestinal tissue and the in vitro triple culture model suggest that advanced multifacetted research questions concerning oral ENM exposure are more adequately addressed by the more complex and time intensive models.

Stem Cells for Next Level Toxicity Testing in the 21st Century.

The call for a paradigm change in toxicology from the United States National Research Council in 2007 initiates awareness for the invention and use of human-relevant alternative methods for toxicological hazard assessment. Simple 2D in vitro systems may serve as first screening tools, however, recent developments infer the need for more complex, multicellular organotypic models, which are superior in mimicking the complexity of human organs. In this review article most critical organs for toxicity assessment, i.e., skin, brain, thyroid system, lung, heart, liver, kidney, and intestine are discussed with regards to their functions in health and disease. Embracing the manifold modes-of-action how xenobiotic compounds can interfere with physiological organ functions and cause toxicity, the need for translation of such multifaceted organ features into the dish seems obvious. Currently used in vitro methods for toxicological applications and ongoing developments not yet arrived in toxicity testing are discussed, especially highlighting the potential of models based on embryonic stem cells and induced pluripotent stem cells of human origin. Finally, the application of innovative technologies like organs-on-a-chip and genome editing point toward a toxicological paradigm change moves into action.

Investigations of acute effects of polystyrene and polyvinyl chloride micro- and nanoplastics in an advanced in vitro triple culture model of the healthy and inflamed intestine.

The continuous degradation of plastic waste in the environment leads to the generation of micro- and nanoplastic fragments and particles. Due to the ubiquitous presence of plastic particles in natural habitats as well as in food, beverages and tap water, oral exposure of the human population with plastic particles occurs worldwide. We investigated acute toxicological effects of polystyrene (PS) and polyvinyl chloride (PVC) micro- and nanoparticles in an advanced in vitro triple culture model (Caco-2/HT29-MTX-E12/THP-1) mimicking the healthy and inflamed human intestine to study the effect of inflammatory processes on plastic particle toxicity. We monitored barrier integrity, cytotoxicity, cell layer integrity, DNA damage, the release of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8 and TNF-α) and mucus distribution after 24 h of particle exposure. In addition, we investigated cytotoxicity, DNA damage and IL-1ß release in monocultures of the three cell lines. Amine-modified polystyrene nanoparticles (PS-NH2) served as a positive control for particle-induced toxicity. No acute effects in the investigated endpoints were observed in the model of the healthy intestine after PS or PVC exposure. However, during active inflammatory processes, exposure to PVC particles was found to augment the release of IL-1ß and to cause a loss of epithelial cells. Our results suggest that prevalent intestinal inflammation might be an important factor to consider when assessing the hazard of ingested micro- and nanoplastic particles.

Advanced In Vitro Testing Strategies and Models of the Intestine for Nanosafety Research.

There is growing concern about the potential adverse effects of oral exposure to engineered nanomaterials (ENM). Recent years have witnessed major developments in and advancement of intestinal in vitro models for nanosafety evaluation. The present paper reviews the key factors that should be considered for inclusion in nonanimal alternative testing approaches to reliably reflect the in vivo dynamics of the physicochemical properties of ENM as well the intestinal physiology and morphology. Currently available models range from simple cell line-based monocultures to advanced 3D systems and organoids. In addition, in vitro approaches exist to replicate the mucous barrier, digestive processes, luminal flow, peristalsis, and interactions of ENM with the intestinal microbiota. However, while the inclusion of a multitude of individual factors/components of particle (pre)treatment, exposure approach, and cell model approximates in vivo-like conditions, such increasing complexity inevitably affects the system's robustness and reproducibility. The selection of the individual modules to build the in vitro testing strategy should be driven and justified by the specific purpose of the study and, not least, the intended or actual application of the investigated ENM. Studies that address health hazards of ingested ENM likely require different approaches than research efforts to unravel the fundamental interactions or toxicity mechanisms of ENM in the intestine. Advanced reliable and robust in vitro models of the intestine, especially when combined in an integrated testing approach, offer great potential to further improve the field of nanosafety research.

Silver Nanoparticles and Metallic Silver Interfere with the Griess Reaction: Reduction of Azo Dye Formation via a Competing Sandmeyer-Like Reaction.

Silver (Ag) is the most common nanomaterial (NM) in consumer products. Much research has been focused on elucidating the potential impact of Ag-containing NMs on human health, e.g., cytotoxicity, genotoxicity, or proinflammatory responses. In the case of proinflammatory responses, a frequently used end point is the induction of nitric oxide (NO), which is indirectly quantified as nitrite (NO2-) with the Griess reaction. After preliminary studies in a macrophage-like cell culture system showed anomalous false negative results in the presence of silver nanoparticles (Ag NPs), we studied the influence of Ag on the detection of NO2- in a cell-free environment. Solutions containing a known concentration of NaNO2 were prepared in H2O, PBS, or complete cell culture medium (CCM) and analyzed using the Griess reaction in the presence of Ag in its metallic or ionic state. In Milli-Q H2O, the impact of salts on the detection was investigated using NaCl and KBr. After completion of the Griess reaction, the samples were analyzed spectrophotometrically or chromatographically. It was found that the presence of metallic but not ionic Ag interfered with the quantification of NO2-. The effect was more pronounced in PBS and H2O containing NaCl or KBr. The chromatographical analysis provided evidence of a competing reaction consuming the intermediate diazonium salt, which is critical to the Griess reaction. These findings demonstrate yet another substantial interference of NMs with a frequently used in vitro assay. If gone unnoticed, this interference might cause false negative results and an impaired hazard assessment of Ag NMs.

Investigating nanoplastics toxicity using advanced stem cell-based intestinal and lung in vitro models.

Plastic particles in the nanometer range-called nanoplastics-are environmental contaminants with growing public health concern. As plastic particles are present in water, soil, air and food, human exposure via intestine and lung is unavoidable, but possible health effects are still to be elucidated. To better understand the Mode of Action of plastic particles, it is key to use experimental models that best reflect human physiology. Novel assessment methods like advanced cell models and several alternative approaches are currently used and developed in the scientific community. So far, the use of cancer cell line-based models is the standard approach regarding in vitro nanotoxicology. However, among the many advantages of the use of cancer cell lines, there are also disadvantages that might favor other approaches. In this review, we compare cell line-based models with stem cell-based in vitro models of the human intestine and lung. In the context of nanoplastics research, we highlight the advantages that come with the use of stem cells. Further, the specific challenges of testing nanoplastics in vitro are discussed. Although the use of stem cell-based models can be demanding, we conclude that, depending on the research question, stem cells in combination with advanced exposure strategies might be a more suitable approach than cancer cell lines when it comes to toxicological investigation of nanoplastics.

Electrical polarity-dependent gating and a unique subconductance of RyR2 induced by S-adenosyl methionine via the ATP binding site.

S-Adenosyl-L-methionine (SAM) was used to probe the functional effects exerted via the cardiac RyR isoform (RyR2) adenine nucleotide binding site. Single channel experiments revealed that SAM applied to the cytoplasmic face of RyR2 had complex voltage dependent effects on channel gating and conductance. At positive transmembrane holding potentials, SAM caused a striking reduction in channel openings and a reduced channel conductance. In contrast, at negative potentials, SAM promoted a clearly resolved subconductance state. At membrane potentials between -75 and -25 mV, the open probability of the subconductance state was independent of voltage. ATP, but not the non-adenosine-based ryanodine receptor (RyR) activator 4-chloro-m-cresol, interfered with the effects of SAM at both negative and positive potentials. This suggests that ATP and SAM interact with a common binding site. Molecular docking showed SAM bound to the adenine nucleotide binding site and formed a hydrogen bond to Glu4886 in the C-terminal end of the S6 alpha helix. In this configuration, SAM may alter the conformation of the RyR2 ion conduction pathway. This work provides novel insightGraphical Abstract into potential functional outcomes of ligand binding to the RyR adenine nucleotide binding site.

An inverted in vitro triple culture model of the healthy and inflamed intestine: Adverse effects of polyethylene particles.

As environmental pollution with plastic waste is increasing, numerous reports show the contamination of natural habitats, food and drinking water with plastic particles in the micro- and nanometer range. Since oral exposure to these particles is virtually unavoidable, health concerns towards the general population have been expressed and risk assessment regarding ingested plastic particles is of great interest. To study the intestinal effects of polymeric particles with a density of <1 g/cm³ in vitro, we spatially inverted a triple culture transwell model of the healthy and inflamed intestine (Caco-2/HT29-MTX-E12/THP-1), which allows contact between buoyant particles and cells. We validated the inverted model against the original model using the enterotoxic, non-steroidal anti-inflammatory drug diclofenac and subsequently assessed the cytotoxic and pro-inflammatory effects of polyethylene (PE) microparticles. The results show that the inverted model exhibits the same distinct features as the original model in terms of barrier development and inflammatory parameters. Treatment with 2 mM diclofenac causes severe cytotoxicity, DNA damage and complete barrier disruption in both models. PE particles induced cytotoxicity and pro-inflammatory effects in the inverted model, which would have remained undetected in conventional in vitro approaches, as no effect was observed in non-inverted control cultures.

Effects of dietary exposure to the engineered nanomaterials CeO2, SiO2, Ag, and TiO2 on the murine gut microbiome.

Rodent studies on the effects of engineered nanomaterials (ENM) on the gut microbiome have revealed contradictory results. Our aim was to assess the effects of four well-investigated model ENM using a realistic exposure scenario. Two independent ad libitum feeding studies were performed. In study 1, female mice from the local breeding facility received feed pellets containing 1% CeO2 or 1% SiO2 for three weeks. In study 2, both female and male mice were purchased and exposed to 0.2% Ag-PVP or 1% TiO2 for four weeks. A next generation 16S rDNA sequencing-based approach was applied to assess impacts on the gut microbiome. None of the ENM had an effect on the α- or ß-diversity. A decreased relative abundance of the phylum Actinobacteria was observed in SiO2 exposed mice. In female mice, the relative abundance of the genus Roseburia was increased with Ag exposure. Furthermore, in study 2, a sex-related difference in the ß-diversity was observed. A difference in the ß-diversity was also shown between the female control mice of the two studies. We did not find major effects on the gut microbiome. This contrast to other studies may be due to variations in the study design. Our investigation underlined the important role of the sex of test animals and their microbiome composition prior to ENM exposure initiation. Hence, standardization of microbiome studies is strongly required to increase comparability. The ENM-specific effects on Actinobacteria and Roseburia, two taxa pivotal for the human gut homeostasis, warrant further research on their relevance for health.

Ingested Engineered Nanomaterials Affect the Expression of Mucin Genes-An In Vitro-In Vivo Comparison.

The increasing use of engineered nanomaterials (ENM) in food has fueled the development of intestinal in vitro models for toxicity testing. However, ENM effects on intestinal mucus have barely been addressed, although its crucial role for intestinal health is evident. We investigated the effects of ENM on mucin expression and aimed to evaluate the suitability of four in vitro models of increasing complexity compared to a mouse model exposed through feed pellets. We assessed the gene expression of the mucins MUC1, MUC2, MUC5AC, MUC13 and MUC20 and the chemokine interleukin-8 in pre-confluent and confluent HT29-MTX-E12 cells, in stable and inflamed triple cultures of Caco-2, HT29-MTX-E12 and THP-1 cells, and in the ileum of mice following exposure to TiO2, Ag, CeO2 or SiO2. All ENM had shared and specific effects. CeO2 downregulated MUC1 in confluent E12 cells and in mice. Ag induced downregulation of Muc2 in mice. Overall, the in vivo data were consistent with the findings in the stable triple cultures and the confluent HT29-MTX-E12 cells but not in pre-confluent cells, indicating the higher relevance of advanced models for hazard assessment. The effects on MUC1 and MUC2 suggest that specific ENM may lead to an elevated susceptibility towards intestinal infections and inflammations.

S-adenosyl-L-methionine regulation of the cardiac ryanodine receptor involves multiple mechanisms.

Cardiac contraction is triggered by the release of Ca(2+) via the ryanodine receptor (RyR2), a sarcoplasmic reticulum (SR) resident ion channel. RyR2 channel activity is modulated through ligand binding and posttranslational regulatory mechanisms. S-Adenosyl-l-methionine (SAM), the primary methyl group donor for enzyme-mediated methylation of proteins and other biological targets, activates RyR2 via an unknown mechanism. Here we show that the SAM-induced increase in cardiac SR (CSR) vesicle [(3)H]ryanodine binding is unaffected by methyltransferase inhibitors and immunoprecipitation of RyR2 from S-adenosyl-l-[methyl-(3)H]methionine ([(3)H]SAM) pretreated CSR indicates that RyR2 is not a target of SAM-mediated protein methylation. Because SAM contains an adenosine moiety and RyR2 is activated by ATP, we investigated whether SAM exerts its effects through the adenine nucleotide binding sites on the RyR2 channel. In support of this hypothesis, the SAM and ATP concentration dependence of CSR vesicle [(3)H]ryanodine binding virtually overlaps. Furthermore, ryanodine binding assays show that SAM competes with adenine nucleotide activation of RyR2, and the effects of SAM on mean channel open and closed times follow similar trends as those observed for ATP. Interestingly, SAM but not ATP activation of RyR2 was associated with a marked increase in the percent of channel openings to a subconductance level approximately 60% of the maximal single channel conductance. This work highlights the complexity underlying SAM regulation of RyR2 and suggests ligand binding is among the multiple mechanisms responsible for SAM regulation of RyR2.

Ongoing inflammation enhances the toxicity of engineered nanomaterials: Application of an in vitro co-culture model of the healthy and inflamed intestine.

Chronic inflammatory conditions can negatively impact intestinal barrier function and affect the epithelium's interaction with nano-sized materials. We demonstrate the application of a Caco-2/THP-1 co-culture mimicking the intestine in healthy (i.e. stable) or inflamed state in nanotoxicological research. The co-cultures were exposed to non-toxic concentrations of silver nanoparticles (AgNPs) or silver nitrate (AgNO3) for 24 h. The barrier integrity and cytokine release as well as necrotic and apoptotic cell death were investigated. AgNPs and AgNO3 most strongly affected the inflamed co-culture. Higher concentrations of AgNPs induced a significant increase in barrier integrity in the inflamed but not the stable co-culture. Necrotic and apoptotic cell death was detected in both conditions but were significantly more pronounced in the inflamed condition. The exposure to AgNO3 affected barrier integrity in all experimental set-ups, but caused nuclear condensation only in the Caco-2 monoculture and the inflamed co-culture. AgNPs reduced the release of monocyte chemoattractant protein-1 in the stable model. Clear differences were observed in the effects of AgNPs and AgNO3 in relation to the model's health status. The results suggest an increased vulnerability of the inflamed epithelial barrier towards AgNPs underlining the importance to consider the intestinal health status in the safety assessment of nanomaterials.

S-Adenosyl-l-methionine activates the cardiac ryanodine receptor.

S-Adenosyl-l-methionine (SAM) is the biological methyl-group donor for the enzymatic methylation of numerous substrates including proteins. SAM has been reported to activate smooth muscle derived ryanodine receptor calcium release channels. Therefore, we examined the effects of SAM on the cardiac isoform of the ryanodine receptor (RyR2). SAM increased cardiac sarcoplasmic reticulum [(3)H]ryanodine binding in a concentration-dependent manner by increasing the affinity of RyR2 for ryanodine. Activation occurred at physiologically relevant concentrations. SAM, which contains an adenosine moiety, enhanced ryanodine binding in the absence but not in the presence of an ATP analogue. S-Adenosyl-l-homocysteine (SAH) is the product of the loss of the methyl-group from SAM and inhibits methylation reactions. SAH did not activate RyR2 but did inhibit SAM-induced RyR2 activation. SAH did not alter adenine nucleotide activation of RyR2. These data suggest SAM activates RyR2 via a site that interacts with, but is distinct from, the adenine nucleotide binding site.

Recovery nutrition: timing and composition after endurance exercise.

Consumption of macronutrients, particularly carbohydrate (CHO) and possibly a small amount of protein, in the early recovery phase after endurance exercise can enhance muscle glycogen resynthesis rates. A target of at least 1.2 g x kg body weight(-1) x h(-1) CHO (over several hours) is suggested. This rate of CHO intake could be sustained with liquid, gel, or solid food rich in CHO for maximizing muscle glycogen. Whether the coingestion of protein with CHO compared with isocaloric CHO results in meaningful differences in glycogen replenishment that translate into subsequent performance enhancement is equivocal. Advantages of added protein with CHO in reducing true muscle damage from endurance exercise remain to be verified. There are, however, no apparent contraindications for using milk or specialty CHO/protein/amino acid products either. Future investigations that examine signaling mechanisms within muscle should be conducted in parallel with translational evidence in humans.

Development of an in vitro co-culture model to mimic the human intestine in healthy and diseased state.

The intestine forms the largest interface between the environment and the human organism. Its integrity and functioning are crucial for the uptake of nutrients while preventing access of harmful antigens. Inflammatory conditions can significantly change the normal functioning of the intestine. In vitro models that adequately reproduce both healthy and inflamed intestinal tissue could provide a useful tool for studying the mechanisms of intestinal inflammation and investigating new therapeutic drugs. We established a co-culture of Caco-2 and PMA-differentiated THP-1 cells that mimics the intestine in healthy and controlled inflamed states. In homoeostatic conditions without stimulation, Caco-2 and THP-1 cells were co-cultured for 48h without affecting the barrier integrity and with no increase in the release of cytokines, nitric oxide or lactate dehydrogenase. To simulate the inflamed intestine, the Caco-2 barrier was primed with IFN-γ and THP-1 cells were pre-stimulated with LPS and IFN-γ. In these conditions a significant but temporary reduction in barrier integrity was measured, and large concentrations of pro-inflammatory cytokines and cytotoxicity markers detected. With its ability to feature numerous hallmarks of intestinal inflammation the presented co-culture model of epithelial cells and macrophages offers a unique possibility to study exposure effects in relation to the health status of the intestine.