Analysis of hyperforin (St. John's wort) action at TRPC6 channel leads to the development of a new class of antidepressant drugs.

St. John's wort is an herb, long used in folk medicine for the treatment of mild depression. Its antidepressant constituent, hyperforin, has properties such as chemical instability and induction of drug-drug interactions that preclude its use for individual pharmacotherapies. Here we identify the transient receptor potential canonical 6 channel (TRPC6) as a druggable target to control anxious and depressive behavior and as a requirement for hyperforin antidepressant action. We demonstrate that TRPC6 deficiency in mice not only results in anxious and depressive behavior, but also reduces excitability of hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Using electrophysiology and targeted mutagenesis, we show that hyperforin activates the channel via a specific binding motif at TRPC6. We performed an analysis of hyperforin action to develop a new antidepressant drug that uses the same TRPC6 target mechanism for its antidepressant action. We synthesized the hyperforin analog Hyp13, which shows similar binding to TRPC6 and recapitulates TRPC6-dependent anxiolytic and antidepressant effects in mice. Hyp13 does not activate pregnan-X-receptor (PXR) and thereby loses the potential to induce drug-drug interactions. This may provide a new approach to develop better treatments for depression, since depression remains one of the most treatment-resistant mental disorders, warranting the development of effective drugs based on naturally occurring compounds.

Investigating the in vitro steatotic mixture effects of similarly and dissimilarly acting test compounds using an adverse outcome pathway-based approach.

Within the EuroMix project, we have previously developed an adverse outcome pathway (AOP)-based in vitro assay toolbox to investigate the combined effects of liver steatosis-inducing compounds in human HepaRG hepatocarcinoma cells. In this study, we applied the toolbox to further investigate mixture effects of combinations, featuring either similarly acting or dissimilarly acting substances. The valproic acid structural analogs 2-propylheptanoic acid (PHP) and 2-propylhexanoic acid (PHX) were chosen for establishing mixtures of similarly acting substances, while a combination with the pesticidal active substance clothianidin (CTD) was chosen for establishing mixtures of dissimilarly acting compounds. We first determined relative potency factors (RPFs) for each compound based on triglyceride accumulation results. Thereafter, equipotent mixtures were tested for nuclear receptor activation in transfected HepG2 cells, while gene expression and triglyceride accumulation were investigated in HepaRG cells, following the proposed AOP for liver steatosis. Dose addition was observed for all combinations and endpoints tested, indicating the validity of the additivity assumption also in the case of the tested mixtures of dissimilarly acting substances. Gene expression results indicate that the existing steatosis AOP can still be refined with respect to the early key event (KE) of gene expression, in order to reflect the diversity of molecular mechanisms underlying the adverse outcome.

More than additive effects on liver triglyceride accumulation by combinations of steatotic and non-steatotic pesticides in HepaRG cells.

The liver is constantly exposed to mixtures of hepatotoxic compounds, such as food contaminants and pesticides. Dose addition is regularly assumed for mixtures in risk assessment, which however might not be sufficiently protective in case of synergistic effects. Especially the prediction of combination effects of substances which do not share a common adverse outcome (AO) might be problematic. In this study, the focus was on the endpoint liver triglyceride accumulation in vitro, an indicator of hepatic fatty acid changes. The hepatotoxic compounds difenoconazole, propiconazole and tebuconazole were chosen which cause hepatic fatty acid changes in vivo, whereas fludioxonil was chosen as a hepatotoxic substance not causing fatty acid changes. Triglyceride accumulation was analyzed for combinations of steatotic and non-steatotic pesticides in human HepaRG hepatocarcinoma cells. Investigations revealed a potentiation of triglyceride accumulation by mixtures of the steatotic compounds with the non-steatotic fludioxonil, as compared to the single compounds. Mathematical modeling of combination effects indicated more than additive effects for the tested combinations if the method by Chou was applied, and a decrease in EC50 values of the steatotic compounds when applied in mixtures. Use of an adverse outcome pathway (AOP)-driven testing strategy for liver steatosis showed interactions of the test compounds with the nuclear receptors AHR, CAR and PXR, as well as a downregulation of ACOX2. An ACOX2-dependent mechanism underlying the observed mixture effect could not be verified using a siRNA approach. By contrast, a toxicokinetic interaction was identified including an inhibition of the metabolic enzyme CYP3A4 by fludioxonil and a decreased metabolic conversion of the CYP3A4 substrate difenoconazole when used in mixture experiments. In conclusion, an interaction by a steatotic and a non-steatotic compound at the toxicokinetic level on the endpoint triglyceride accumulation in vitro was described.

Transcriptomics analysis of hepatotoxicity induced by the pesticides imazalil, thiacloprid and clothianidin alone or in binary mixtures in a 28-day study in female Wistar rats.

Co-occurrence of pesticide residues in food commodities raises a potential safety issue as their mixture effects on human health are largely unknown. In a previous study, we reported the toxicological effects (pathology and histopathology) of imazalil (IMZ), thiacloprid (THI), and clothianidin (CTD) alone and in binary mixtures in a 28-day oral gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg body weight/day) ranging from a typical toxicological reference value to a clear effect dose were applied. In the present study, we undertook a transcriptomics analysis of rat livers by means of total RNA sequencing (RNA-Seq). Bioinformatic data analysis involving Ingenuity Pathway Analysis (IPA) was used to gain mechanistic information on hepatotoxicity-related pathways affected after treatment with the pesticides, alone and in mixtures. Our data show that 2986 genes were differentially regulated by CTD while IMZ and THI had effects on 194 and 225 genes, respectively. All three individual compounds shared a common subset of genes whose network is associated with xenobiotic metabolism and nuclear receptor activation. Similar networks were retrieved for the mixtures. Alterations in the expression of individual genes were in line with the assumption of dose addition. Our results bring new insight into the hepatotoxicity mechanisms of IMZ, THI, and CTD and their mixtures.

Combinations of LXR and RXR agonists induce triglyceride accumulation in human HepaRG cells in a synergistic manner.

Activation of nuclear receptors (NR), for example the retinoid-X-receptors (RXR) or the liver-X-receptors (LXR), plays a crucial role as the molecular initiating event in the adverse outcome pathway for liver steatosis. The downstream biological consequences of NR interactions are still not fully understood, especially with multi-receptor-activating compounds and their mixtures. While the default assumption for mixture risk assessment is dose addition, the potential of combinations of synthetic RXR agonists to exert synergistic effects has been shown in the context of NR activation studies. The fact that RXR and LXR are heterodimerization partners raises the question whether combinations of LXR and RXR agonists may cause synergistic effects. Compounds with defined properties were chosen to examine their interactions regarding the activation of RXR and LXR, as well as the steatosis-related key events target gene activation and triglyceride accumulation, using the human HepaRG liver cell model. Synergistic effects were determined for cellular triglyceride accumulation, especially at high compound concentrations, as evaluated using five different mathematical models. Altered LXRα activation in the presence of RXR agonists was observed, and synergistic effects on LXR target genes were identified as a presumably underlying mechanism of the observed synergistic effect. These findings challenge the general validity of dose addition as the default assumption for mixture effects, and point toward the need for a mode of action-based risk assessment for chemical mixtures.

Uptake and effects of orally ingested polystyrene microplastic particles in vitro and in vivo.

Evidence exists that humans are exposed to plastic microparticles via diet. Data on intestinal particle uptake and health-related effects resulting from microplastic exposure are scarce. Aim of the study was to analyze the uptake and effects of microplastic particles in human in vitro systems and in rodents in vivo. The gastrointestinal uptake of microplastics was studied in vitro using the human intestinal epithelial cell line Caco-2 and thereof-derived co-cultures mimicking intestinal M-cells and goblet cells. Different sizes of spherical fluorescent polystyrene (PS) particles (1, 4 and 10 µm) were used to study particle uptake and transport. A 28-days in vivo feeding study was conducted to analyze transport at the intestinal epithelium and oxidative stress response as a potential consequence of microplastic exposure. Male reporter gene mice were treated three times per week by oral gavage with a mixture of 1 µm (4.55 × 107 particles), 4 µm (4.55 × 107 particles) and 10 µm (1.49 × 106 particles) microplastics at a volume of 10 mL/kg/bw. Effects of particles on macrophage polarization were investigated using the human cell line THP-1 to detect a possible impact on intestinal immune cells. Altogether, the results of the study demonstrate the cellular uptake of a minor fraction of particles. In vivo data show the absence of histologically detectable lesions and inflammatory responses. The particles did not interfere with the differentiation and activation of the human macrophage model. The present results suggest that oral exposure to PS microplastic particles under the chosen experimental conditions does not pose relevant acute health risks to mammals.

In vitro nanoparticle dosimetry for adherent growing cell monolayers covering bottom and lateral walls.

BACKGROUND: Even though a continuously high number of in vitro studies on nanoparticles are being published, the issue of correct dose matter is often not sufficiently taken into account. Due to their size, the diffusion of nanoparticles is slower, as compared to soluble chemicals, and they sediment slowly. Therefore, the administered dose of particles in in vitro experiments is not necessarily the same (effective) dose that comes into contact with the cellular system. This can lead to misinterpretations of experimental toxic effects and disturbs the meaningfulness of in vitro studies. In silico calculations of the effective nanoparticle dose can help circumventing this problem. RESULTS: This study addresses more complex in vitro models like the human intestinal cell line Caco-2 or the human liver cell line HepaRG, which need to be differentiated over a few weeks to reach their full complexity. During the differentiation time the cells grow up the wall of the cell culture dishes and therefore a three-dimensional-based in silico model of the nanoparticle dose was developed to calculate the administered dose received by different cell populations at the bottom and the walls of the culture dish. Moreover, the model can perform calculations based on the hydrodynamic diameter which is measured by light scattering methods, or based on the diffusion coefficient measured by nanoparticle tracking analysis (NTA). This 3DSDD (3D-sedimentation-diffusion-dosimetry) model was experimentally verified against existing dosimetry models and was applied to differentiated Caco-2 cells incubated with silver nanoparticles. CONCLUSIONS: The 3DSDD accounts for the 3D distribution of cells in in vitro cell culture dishes and is therefore suitable for differentiated cells. To encourage the use of dosimetry calculating software, our model can be downloaded from the supporting information.

Protein Corona Analysis of Silver Nanoparticles Links to Their Cellular Effects.

The breadth of applications of nanoparticles and the access to food-associated consumer products containing nanosized materials lead to oral human exposure to such particles. In biological fluids nanoparticles dynamically interact with biomolecules and form a protein corona. Knowledge about the protein corona is of great interest for understanding the molecular effects of particles as well as their fate inside the human body. We used a mass spectrometry-based toxicoproteomics approach to elucidate mechanisms of toxicity of silver nanoparticles and to comprehensively characterize the protein corona formed around silver nanoparticles in Caco-2 human intestinal epithelial cells. Results were compared with respect to the cellular function of proteins either affected by exposure to nanoparticles or present in the protein corona. A transcriptomic data set was included in the analyses in order to obtain a combined multiomics view of nanoparticle-affected cellular processes. A relationship between corona proteins and the proteomic or transcriptomic responses was revealed, showing that differentially regulated proteins or transcripts were engaged in the same cellular signaling pathways. Protein corona analyses of nanoparticles in cells might therefore help in obtaining information about the molecular consequences of nanoparticle treatment.

Dosimetric Quantification of Coating-Related Uptake of Silver Nanoparticles.

The elucidation of mechanisms underlying the cellular uptake of nanoparticles (NPs) is an important topic in nanotoxicological research. Most studies dealing with silver NP uptake provide only qualitative data about internalization efficiency and do not consider NP-specific dosimetry. Therefore, we performed a comprehensive comparison of the cellular uptake of differently coated silver NPs of comparable size in different human intestinal Caco-2 cell-derived models to cover also the influence of the intestinal mucus barrier and uptake-specialized M-cells. We used a combination of the Transwell system, transmission electron microscopy, atomic absorption spectroscopy, and ion beam microscopy techniques. The computational in vitro sedimentation, diffusion, and dosimetry (ISDD) model was used to determine the effective dose of the particles in vitro based on their individual physicochemical characteristics. Data indicate that silver NPs with a similar size and shape show coating-dependent differences in their uptake into Caco-2 cells. The internalization of silver NPs was enhanced in uptake-specialized M-cells while the mucus did not provide a substantial barrier for NP internalization. ISDD modeling revealed a fivefold underestimation of dose-response relationships of NPs in in vitro assays. In summary, the present study provides dosimetry-adjusted quantitative data about the influence of NP coating materials in cellular uptake into human intestinal cells. Underestimation of particle effects in vitro might be prevented by using dosimetry models and by considering cell models with greater proximity to the in vivo situation, such as the M-cell model.

Impact of an Artificial Digestion Procedure on Aluminum-Containing Nanomaterials.

Aluminum has gathered toxicological attention based on relevant human exposure and its suspected hazardous potential. Nanoparticles from food supplements or food contact materials may reach the human gastrointestinal tract. Here, we monitored the physicochemical fate of aluminum-containing nanoparticles and aluminum ions when passaging an in vitro model of the human gastrointestinal tract. Small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), ion beam microscopy (IBM), secondary ion beam mass spectrometry (TOF-SIMS), and inductively coupled plasma mass spectrometry (ICP-MS) in the single-particle mode were employed to characterize two aluminum-containing nanomaterials with different particle core materials (Al0, γAl2O3) and soluble AlCl3. Particle size and shape remained unchanged in saliva, whereas strong agglomeration of both aluminum nanoparticle species was observed at low pH in gastric fluid together with an increased ion release. The levels of free aluminum ions decreased in intestinal fluid and the particles deagglomerated, thus liberating primary particles again. Dissolution of nanoparticles was limited and substantial changes of their shape and size were not detected. The amounts of particle-associated phosphorus, chlorine, potassium, and calcium increased in intestinal fluid, as compared to nanoparticles in standard dispersion. Interestingly, nanoparticles were found in the intestinal fluid after addition of ionic aluminum. We provide a comprehensive characterization of the fate of aluminum nanoparticles in simulated gastrointestinal fluids, demonstrating that orally ingested nanoparticles probably reach the intestinal epithelium. The balance between dissolution and de novo complex formation should be considered when evaluating nanotoxicological experiments.

Impact of food components during in vitro digestion of silver nanoparticles on cellular uptake and cytotoxicity in intestinal cells.

Because of the rising application of nanoparticles in food and food-related products, we investigated the influence of the digestion process on the toxicity and cellular uptake of silver nanoparticles for intestinal cells. The main food components--carbohydrates, proteins and fatty acids--were implemented in an in vitro digestion process to simulate realistic conditions. Digested and undigested silver nanoparticle suspensions were used for uptake studies in the well-established Caco-2 model. Small-angle X-ray scattering was used to estimate particle core size, size distribution and stability in cell culture medium. Particles proved to be stable and showed radii from 3.6 to 16.0 nm. Undigested particles and particles digested in the presence of food components were comparably taken up by Caco-2 cells, whereas the uptake of particles digested without food components was decreased by 60%. Overall, these findings suggest that in vivo ingested poly (acrylic acid)-coated silver nanoparticles may reach the intestine in a nanoscaled form even if enclosed in a food matrix. While appropriate for studies on the uptake into intestinal cells, the Caco-2 model might be less suited for translocation studies. Moreover, we show that nanoparticle digestion protocols lacking food components may lead to misinterpretation of uptake studies and inconclusive results.

3D bioprinting of liver models: A systematic scoping review of methods, bioinks, and reporting quality.

Background: Effective communication is crucial for broad acceptance and applicability of alternative methods in 3R biomedical research and preclinical testing. 3D bioprinting is used to construct intricate biological structures towards functional liver models, specifically engineered for deployment as alternative models in drug screening, toxicological investigations, and tissue engineering. Despite a growing number of reviews in this emerging field, a comprehensive study, systematically assessing practices and reporting quality for bioprinted liver models is missing. Methods: In this systematic scoping review we systematically searched MEDLINE (Ovid), EMBASE (Ovid) and BioRxiv for studies published prior to June 2nd, 2022. We extracted data on methodological conduct, applied bioinks, the composition of the printed model, performed experiments and model applications. Records were screened for eligibility and data were extracted from included articles by two independent reviewers from a panel of seven domain experts specializing in bioprinting and liver biology. We used RAYYAN for the screening process and SyRF for data extraction. We used R for data analysis, and R and Graphpad PRISM for visualization. Results: Through our systematic database search we identified 1042 records, from which 63 met the eligibility criteria for inclusion in this systematic scoping review. Our findings revealed that extrusion-based printing, in conjunction with bioinks composed of natural components, emerged as the predominant printing technique in the bioprinting of liver models. Notably, the HepG2 hepatoma cell line was the most frequently employed liver cell type, despite acknowledged limitations. Furthermore, 51% of the printed models featured co-cultures with non-parenchymal cells to enhance their complexity. The included studies offered a variety of techniques for characterizing these liver models, with their primary application predominantly focused on toxicity testing. Among the frequently analyzed liver markers, albumin and urea stood out. Additionally, Cytochrome P450 (CYP) isoforms, primarily CYP3A and CYP1A, were assessed, and select studies employed nuclear receptor agonists to induce CYP activity. Conclusion: Our systematic scoping review offers an evidence-based overview and evaluation of the current state of research on bioprinted liver models, representing a promising and innovative technology for creating alternative organ models. We conducted a thorough examination of both the methodological and technical facets of model development and scrutinized the reporting quality within the realm of bioprinted liver models. This systematic scoping review can serve as a valuable template for systematically evaluating the progress of organ model development in various other domains. The transparently derived evidence presented here can provide essential support to the research community, facilitating the adaptation of technological advancements, the establishment of standards, and the enhancement of model robustness. This is particularly crucial as we work toward the long-term objective of establishing new approach methods as reliable alternatives to animal testing, with extensive and versatile applications.

Corrigendum: Development of new approach methods for the identification and characterization of endocrine metabolic disruptors-a PARC project.

[This corrects the article DOI: 10.3389/ftox.2023.1212509.].

Adverse outcome pathway-based analysis of liver steatosis in vitro using human liver cell lines.

Here, we present an in vitro test battery to analyze chemicals for their potential to induce liver triglyceride accumulation, a hallmark of liver steatosis. We describe steps for using HepG2 and HepaRG human hepatoma cells in conjunction with a combination of several in vitro assays covering the different molecular initiating events and key events of the respective adverse outcome pathway. This protocol is suitable for assessing single substance effects as well as mixtures allowing their classification as steatotic or non-steatotic. For complete details on the use and execution of this protocol, please refer to Luckert et al. (2018),1 Lichtenstein et al. (2020),2 and Knebel et al. (2019).3.

Unnatural Endotype B PPAPs as Novel Compounds with Activity against Mycobacterium tuberculosis.

Pre-SARS-CoV-2, tuberculosis was the leading cause of death by a single pathogen. Repetitive exposure of Mycobacterium tuberculosis(Mtb) supported the development of multidrug- and extensively drug-resistant strains, demanding novel drugs. Hyperforin, a natural type A polyprenylated polycyclic acylphloroglucinol from St. John's wort, exhibits antidepressant and antibacterial effects also against Mtb. Yet, Hyperforin's instability limits the utility in clinical practice. Here, we present photo- and bench-stable type B PPAPs with enhanced antimycobacterial efficacy. PPAP22 emerged as a lead compound, further improved as the sodium salt PPAP53, drastically enhancing solubility. PPAP53 inhibits the growth of virulent extracellular and intracellular Mtb without harming primary human macrophages. Importantly, PPAP53 is active against drug-resistant strains of Mtb. Furthermore, we analyzed the in vitro properties of PPAP53 in terms of CYP induction and the PXR interaction. Taken together, we introduce type PPAPs as a new class of antimycobacterial compounds, with remarkable antibacterial activity and favorable biophysical properties.

Development of new approach methods for the identification and characterization of endocrine metabolic disruptors-a PARC project.

In past times, the analysis of endocrine disrupting properties of chemicals has mainly been focused on (anti-)estrogenic or (anti-)androgenic properties, as well as on aspects of steroidogenesis and the modulation of thyroid signaling. More recently, disruption of energy metabolism and related signaling pathways by exogenous substances, so-called metabolism-disrupting chemicals (MDCs) have come into focus. While general effects such as body and organ weight changes are routinely monitored in animal studies, there is a clear lack of mechanistic test systems to determine and characterize the metabolism-disrupting potential of chemicals. In order to contribute to filling this gap, one of the project within EU-funded Partnership for the Assessment of Risks of Chemicals (PARC) aims at developing novel in vitro methods for the detection of endocrine metabolic disruptors. Efforts will comprise projects related to specific signaling pathways, for example, involving mTOR or xenobiotic-sensing nuclear receptors, studies on hepatocytes, adipocytes and pancreatic beta cells covering metabolic and morphological endpoints, as well as metabolism-related zebrafish-based tests as an alternative to classic rodent bioassays. This paper provides an overview of the approaches and methods of these PARC projects and how this will contribute to the improvement of the toxicological toolbox to identify substances with endocrine disrupting properties and to decipher their mechanisms of action.

An eight-compound mixture but not corresponding concentrations of individual chemicals induces triglyceride accumulation in human liver cells.

In real life, organisms are exposed to complex mixtures of chemicals at low concentration levels, whereas research on toxicological effects is mostly focused on single compounds at comparably high doses. Mixture effects deviating from the assumption of additivity, especially synergistic effects, are of concern. In an adverse outcome pathway (AOP)-guided manner, we analyzed the accumulation of triglycerides in human HepaRG liver cells by a mixture of eight steatotic chemicals (amiodarone, benzoic acid, cyproconazole, flusilazole, imazalil, prochloraz, propiconazole and tebuconazole), each present below its individual effect concentration at 1-3 µM. Pronounced and significantly enhanced triglyceride accumulation was observed with the mixture, and similar effects were seen at the level of pregnane-X-receptor activation, a molecular initiating event leading to hepatic steatosis. Transcript pattern analysis indicated subtle pro-steatotic changes at low compound concentrations, which did not exert measurable effects on cellular triglycerides. Mathematical modeling of mixture effects indicated potentially more than additive behavior using a model for compounds with similar modes of action. The present data underline the usefulness of AOP-guided in vitro testing for the identification of mixture effects and highlight the need for further research on chemical mixtures and harmonization of data interpretation of mixture effects.

Pesticide mixture effects on liver protein abundance in HepaRG cells.

Toxicological effects of chemicals are mostly tested individually. However, consumers encounter exposure to complex mixtures, for example multiple pesticide residues, by consuming food such as crops, fruits or vegetables. Currently, more than 450 active substances are approved in the European Union, and there is little data on effects after combined exposure to several pesticides. Toxicological animal studies would increase enormously, if pesticide combinations had to be analyzed in vivo. Therefore, in vitro methods addressing this issue are needed. We have developed 32 immunoaffinity-based mass spectrometry assays to investigate the impact of hepatotoxic active substances on liver proteins in human HepaRG cells. Five compounds were selected based on their (dis)similar capability to modulate protein levels, and on their combined use in commercially available formulations. Four binary mixtures were prepared from these five substances and tested in different concentrations over three time points. We applied a novel statistical method to describe deviations from additivity and to detect antagonistic and synergistic effects. The results regarding the abundance of hepatotoxicity-related proteins showed additive behavior for 1323 out of 1427 endpoints tested, while 104 combinatorial effects deviating from additivity, such as antagonism or synergism were observed.

A targeted transcriptomics approach for the determination of mixture effects of pesticides.

While real-life exposure occurs to complex chemical mixtures, toxicological risk assessment mostly focuses on individual compounds. There is an increasing demand for in vitro tools and strategies for mixture toxicity analysis. Based on a previously established set of hepatotoxicity marker genes, we analyzed mixture effects of non-cytotoxic concentrations of different pesticides in exposure-relevant binary mixtures in human HepaRG hepatocarcinoma cells using targeted transcriptomics. An approach for mixture analysis at the level of a complex endpoint such as a transcript pattern is presented, including mixture design based on relative transcriptomic potencies and similarities. From a mechanistic point of view, goal of the study was to evaluate combinations of chemicals with varying degrees of similarity in order to determine whether differences in mechanisms of action lead to different mixtures effects. Using a model deviation ratio-based approach for assessing mixture effects, it was revealed that most data points are consistent with the assumption of dose addition. A tendency for synergistic effects was only observed at high concentrations of some combinations of the test compounds azoxystrobin, cyproconazole, difenoconazole, propiconazole and thiacloprid, which may not be representative of human real-life exposure. In summary, the findings of our study suggest that, for the pesticide mixtures investigated, risk assessment based on the general assumption of dose addition can be considered sufficiently protective for consumers. The way of data analysis presented in this paper can pave the way for a more comprehensive use of multi-gene expression data in experimental studies related to mixture toxicity.

Mixture effects of chemicals: The difficulty to choose appropriate mathematical models for appropriate conclusions.

Many different approaches have been proposed to evaluate and predict mixture effects. From a regulatory perspective, several guidance documents have been recently published and provide a strategy for mixture risk assessment based on valuable frameworks to investigate potential synergistic effects. However, some methodological aspects, e.g. for considering mathematical models, are not sufficiently defined. Therefore, the aim of this study was to examine the usefulness of five main mathematical models for mixture effect interpretation: theoretical additivity (TA), concentration addition (CA), independent action (IA), Chou-Talalay (CT), and a benchmark dose approach (BMD) were tested using a fictional data set depicting scenarios of additivity, synergism and antagonism. The synergism and antagonism scenarios were split in x-axis and y-axis synergism/antagonism, meaning a shift of the curve on x-axis or y-axis. The BMD approach was the only model which showed a perfect correspondence for dose addition. Regarding synergism and antagonism, all approaches correspond well for the x-axis synergism and antagonism with only few exceptions. In contrast, some limitations were observed in the particular scenarios of y-axis synergism and antagonism. Therefore our results show that each model has advantages and disadvantages, and that therefore no single model appears the best one for all kinds of application. We would recommend instead the parallel use of different models to increase confidence in the result of mixture effect evaluation.