Cytotoxicity Endpoints Research Articles

ToxProfiler: A novel human-based reporter assay for in vitro chemical safety assessment

In vitro chemical safety assessment often relies on simple and general cytotoxicity endpoint measurements and fails to adequately predict human toxicity. To improve the in vitro chemical safety assessment, it is important to understand the underlying mechanisms of toxicity. Here we introduce ToxProfiler, a novel human-based reporter assay that quantifies the chemical-induced stress responses at a single-cell level and reveals the toxicological mode-of-action (MoA) of novel drugs and chemicals. The assay accurately measures the activation of seven major cellular stress response pathways (oxidative stress, cell cycle stress, endoplasmic reticulum stress, ion stress, protein stress, autophagy and inflammation) that play a role in the adaptive responses prior to cellular toxicity. To assess the applicability of the assay in predicting the toxicity MoA of chemicals, we tested a set of 100 chemicals with well-known in vitro and in vivo toxicological profiles. Concentration response modeling and point-of-departure estimation for each reporter protein allowed for chemical potency ranking and revealed the primary toxicological MoA of chemicals. Furthermore, the assay could effectively group chemicals based on their shared toxicity signatures and link them to specific toxicological targets, e.g. mitochondrial toxicity and genotoxicity, and different human pathologies, including liver toxicity and cardiotoxicity. Overall, ToxProfiler is a quantitative in vitro reporter assay that can accurately provide insight into the toxicological MoA of compounds, thereby assisting in the future mechanism-based safety assessment of chemicals.

Predicting Acute Oral Toxicity Using AcutoX: An Animal Product-Free and Metabolically Relevant Human Cell-Based Test.

AcutoX is a human in vitro test method for the evaluation of acute oral toxicity, developed using a library of 67 curated test chemicals. These chemicals cover a wide variety of chemistries, industrial sectors, rodent toxicities, and all EPA and GHS hazard categories. The test uses two different cytotoxicity endpoints (Neutral Red uptake and MTT metabolism), performed both in the presence and absence of a pooled human liver extract (S9), to produce four EC50 values. The EC50 values are used in prediction models to assign a "highly toxic" and "low toxicity" category for both EPA and GHS classification, which can be further refined to assign a hazard category. The binary "highly toxic" / "low toxicity" prediction model has an accuracy of 73.8% and 63.1% for EPA and GHS, respectively, with the subsequent hazard categorization offering a protective prediction (correct or higher category) in 90.0% and 93.3% of cases, respectively. Moreover, the AcutoX test can identify chemicals activated or detoxified by liver metabolism.

Alleviation effects of dexmedetomidine on myocardial ischemia/reperfusion injury through fatty acid metabolism pathway via Elovl6

Dexmedetomidine (Dex) is widely used in the sedation in intensive care units and as an anesthetic adjunct. Considering the anti-inflammatory and antioxidant properties of Dex, we applied in vivo rat model as well as in vitro cardiomyocyte models (embryonic rat cardiomyocytes H9c2 cells and neonatal rat cardiomyocytes, NRCMs) to evaluate the effects of Dex against myocardial ischemia reperfusion (I/R) injury. Transcriptomic sequencing for gene expression in heart tissues from control rats and Dex-treated rats identified that genes related to fatty acid metabolism were significantly regulated by Dex. Among these genes, the elongation of long-chain fatty acids (ELOVL) family member 6 (Elovl6) was most increased upon Dex-treatment. By comparing the effects of Dex on both wild type and Elovl6-knockdown H9c2 cells and NRCMs under oxygen-glucose deprivation/reoxygenation (OGD/R) challenge, we found that Elovl6 knockdown attenuated the protection efficiency of Dex, which was supported by the cytotoxicity endpoints (cell viability and lactate dehydrogenase release) and apoptosis as well as key gene expressions. These results indicate that Dex exhibited the protective function against myocardial I/R injury via fatty acid metabolism pathways and Elovl6 plays a key role in the process, which was further confirmed using palmitate exposure in both cells, as well as in an in vivo rat model. Overall, this study systematically evaluates the protective effects of Dex on the myocardial I/R injury and provides better understanding on the fatty acid metabolism underlying the beneficial effects of Dex.

Inter-continental variability in the relationship of oxidative potential and cytotoxicity with PM2.5 mass

Most fine ambient particulate matter (PM2.5)-based epidemiological models use globalized concentration-response (CR) functions assuming that the toxicity of PM2.5 is solely mass-dependent without considering its chemical composition. Although oxidative potential (OP) has emerged as an alternate metric of PM2.5 toxicity, the association between PM2.5 mass and OP on a large spatial extent has not been investigated. In this study, we evaluate this relationship using 385 PM2.5 samples collected from 14 different sites across 4 different continents and using 5 different OP (and cytotoxicity) endpoints. Our results show that the relationship between PM2.5 mass vs. OP (and cytotoxicity) is largely non-linear due to significant differences in the intrinsic toxicity, resulting from a spatially heterogeneous chemical composition of PM2.5. These results emphasize the need to develop localized CR functions incorporating other measures of PM2.5 properties (e.g., OP) to better predict the PM2.5-attributed health burdens.

A proposal for new genotoxic and cytotoxic endpoints to assess chemical effects on the red algae Ceramium tenuicorne.

Macroalgae provide key contributions to aquatic ecosystems, including their role as primary producers and the provision of biotopes for marine organisms, fish spawning, and fish nurseries. The aim of this study was to evaluate the feasibility of a micronucleus test and a comet assay in Ceramium tenuicorne, a red macroalga. This alga is widely distributed in marine ecosystems and brackish waters, and is therefore a potential bioindicator of the effects of anthropogenic pollution in these ecosystems. Unfortunately, the two genotoxicity tests evaluated were not suitable for this alga because the nuclei are generally very small (between 2 and 10µm), are variable in size, and there may be several nuclei in each cell (between 1 and 5 in our observations). However, the present study allowed us to define conditions for observing these algal cells and optimizing the choice of DNA dye (orcein), cell fixation solution (Carnoy's solution), and hydrolysis step (HCl, 1200mmol/L solution for 1min). This research allowed us to propose two genotoxicity and cytotoxicity endpoints for assessing chemical effects on the algal cells: counting of nuclei in cortical cell areas, and the frequency of axial cells in mitosis. These two criteria were measured after exposing C. tenuicorne to two reference substances: cadmium chloride and maleic hydrazide, and we highlight the effects from 1 × 10-5M of CdCl2 and 5 × 10-5M of maleic hydrazide.

Control compounds for preclinical drug-induced liver injury assessment: Consensus-driven systematic review by the ProEuroDILI network

Idiosyncratic drug-induced liver injury (DILI) is a complex and unpredictable event caused by drugs, and herbal or dietary supplements. Early identification of human hepatotoxicity at preclinical stages remains a major challenge, in which the selection of validated invitro systems and test drugs has a significant impact. In this systematic review, we analyzed the compounds used in hepatotoxicity assays and established a list of DILI-positive and -negative control drugs for validation of invitro models of DILI, supported by literature and clinical evidence and endorsed by an expert committee from the COST Action ProEuroDILI Network (CA17112). Following 2020 PRISMA guidelines, original research articles focusing on DILI which used invitro human models and performed at least one hepatotoxicity assay with positive and negative control compounds, were included. Bias of the studies was assessed by a modified 'Toxicological Data Reliability Assessment Tool'. A total of 51 studies (out of 2,936) met the inclusion criteria, with 30 categorized as reliable without restrictions. Although there was a broad consensus on positive compounds, the selection of negative compounds lacked clarity. 2D monoculture, short exposure times and cytotoxicity endpoints were the most tested, although there was no consensus on drug concentrations. Extensive analysis highlighted the lack of agreement on control compounds for invitro DILI assessment. Following comprehensive invitro and clinical data analysis together with input from the expert committee, an evidence-based consensus-driven list of 10 positive and negative control drugs for validation of invitro models of DILI is proposed. Prediction of human toxicity early in the drug development process remains a major challenge, necessitating the development of more physiologically relevant liver models and careful selection of drug-induced liver injury (DILI)-positive and -negative control drugs to better predict the risk of DILI associated with new drug candidates. Thus, this systematic study has crucial implications for standardizing the validation of new invitro models of DILI. By establishing a consensus-driven list of positive and negative control drugs, the study provides a scientifically justified framework for enhancing the consistency of preclinical testing, thereby addressing a significant challenge in early hepatotoxicity identification. Practically, these findings can guide researchers in evaluating safety profiles of new drugs, refining invitro models, and informing regulatory agencies on potential improvements to regulatory guidelines, ensuring a more systematic and efficient approach to drug safety assessment.

Concordance between In Vitro and In Vivo Relative Toxic Potencies of Diesel Exhaust Particles from Different Biodiesel Blends.

Diesel exhaust particles (DEPs) contribute to air pollution exposure-related adverse health impacts. Here, we examined in vitro, and in vivo toxicities of DEPs from a Caterpillar C11 heavy-duty diesel engine emissions using ultra-low-sulfur diesel (ULSD) and biodiesel blends (20% v/v) of canola (B20C), soy (B20S), or tallow-waste fry oil (B20T) in ULSD. The in vitro effects of DEPs (DEPULSD, DEPB20C, DEPB20S, and DEPB20T) in exposed mouse monocyte/macrophage cells (J774A.1) were examined by analyzing the cellular cytotoxicity endpoints (CTB, LDH, and ATP) and secreted proteins. The in vivo effects were assessed in BALB/c mice (n = 6/group) exposed to DEPs (250 µg), carbon black (CB), or saline via intratracheal instillation 24 h post-exposure. Bronchoalveolar lavage fluid (BALF) cell counts, cytokines, lung/heart mRNA, and plasma markers were examined. In vitro cytotoxic potencies (e.g., ATP) and secreted TNF-α were positively correlated (p < 0.05) with in vivo inflammatory potency (BALF cytokines, lung/heart mRNA, and plasma markers). Overall, DEPULSD and DEPB20C appeared to be more potent compared to DEPB20S and DEPB20T. These findings suggested that biodiesel blend-derived DEP potencies can be influenced by biodiesel sources, and inflammatory process- was one of the potential underlying toxicity mechanisms. These observations were consistent across in vitro and in vivo exposures, and this work adds value to the health risk analysis of cleaner fuel alternatives.

In vitro inflammation and toxicity assessment of pre- and post-incinerated organomodified nanoclays to macrophages using high-throughput screening approaches

BackgroundOrganomodified nanoclays (ONC), two-dimensional montmorillonite with organic coatings, are increasingly used to improve nanocomposite properties. However, little is known about pulmonary health risks along the nanoclay life cycle even with increased evidence of airborne particulate exposures in occupational environments. Recently, oropharyngeal aspiration exposure to pre- and post-incinerated ONC in mice caused low grade, persistent lung inflammation with a pro-fibrotic signaling response with unknown mode(s) of action. We hypothesized that the organic coating presence and incineration status of nanoclays determine the inflammatory cytokine secretary profile and cytotoxic response of macrophages. To test this hypothesis differentiated human macrophages (THP-1) were acutely exposed (0–20 µg/cm2) to pristine, uncoated nanoclay (CloisNa), an ONC (Clois30B), their incinerated byproducts (I-CloisNa and I-Clois30B), and crystalline silica (CS) followed by cytotoxicity and inflammatory endpoints. Macrophages were co-exposed to lipopolysaccharide (LPS) or LPS-free medium to assess the role of priming the NF-κB pathway in macrophage response to nanoclay treatment. Data were compared to inflammatory responses in male C57Bl/6J mice following 30 and 300 µg/mouse aspiration exposure to the same particles.ResultsIn LPS-free media, CloisNa exposure caused mitochondrial depolarization while Clois30B exposure caused reduced macrophage viability, greater cytotoxicity, and significant damage-associated molecular patterns (IL-1α and ATP) release compared to CloisNa and unexposed controls. LPS priming with low CloisNa doses caused elevated cathepsin B/Caspage-1/IL-1β release while higher doses resulted in apoptosis. Clois30B exposure caused dose-dependent THP-1 cell pyroptosis evidenced by Cathepsin B and IL-1β release and Gasdermin D cleavage. Incineration ablated the cytotoxic and inflammatory effects of Clois30B while I-CloisNa still retained some mild inflammatory potential. Comparative analyses suggested that in vitro macrophage cell viability, inflammasome endpoints, and pro-inflammatory cytokine profiles significantly correlated to mouse bronchioalveolar lavage inflammation metrics including inflammatory cell recruitment.ConclusionsPresence of organic coating and incineration status influenced inflammatory and cytotoxic responses following exposure to human macrophages. Clois30B, with a quaternary ammonium tallow coating, induced a robust cell membrane damage and pyroptosis effect which was eliminated after incineration. Conversely, incinerated nanoclay exposure primarily caused elevated inflammatory cytokine release from THP-1 cells. Collectively, pre-incinerated nanoclay displayed interaction with macrophage membrane components (molecular initiating event), increased pro-inflammatory mediators, and increased inflammatory cell recruitment (two key events) in the lung fibrosis adverse outcome pathway.

Combined use of multiparametric high-content-screening and in vitro circadian reporter assays in neurotoxicity evaluation.

Accumulating evidence indicates that chronic circadian rhythm disruption is associated with the development of neurodegenerative diseases induced by exposure to neurotoxic chemicals. Herein, we examined the relationship between cellular circadian rhythm disruption and cytotoxicity in neural cells. Moreover, we evaluated the potential application of an in vitro cellular circadian rhythm assay in determining circadian rhythm disruption as a sensitive and early marker of neurotoxicant-induced adverse effects. To explore these objectives, we established an in vitro cellular circadian rhythm assay using human glioblastoma (U87 MG) cells stably transfected with a circadian reporter vector (PER2-dLuc) and determined the lowest-observed-adverse-effect levels (LOAELs) of several common neurotoxicants. Additionally, we determined the LOAEL of each compound on multiple cytotoxicity endpoints (nuclear size [NC], mitochondrial membrane potential [MMP], calcium ions, or lipid peroxidation) using a multiparametric high-content screening (HCS) assay using transfected U87 MG cells treated with the same neurotoxicants for 24 and 72h. Based on our findings, the LOAEL for cellular circadian rhythm disruption for most chemicals was slightly higher than that for most cytotoxicity indicators detected using HCS, and the LOAEL for MMP in the first 24h was the closest to that for cellular circadian rhythm disruption. Dietary antioxidants (methylselenocysteine and N-acetyl-l-cysteine) prevented or restored neurotoxicant-induced cellular circadian rhythm disruption. Our results suggest that cellular circadian rhythm disruption is as sensitive as cytotoxicity indicators and occurs early as much as cytotoxic events during disease development. Moreover, the in vitro cellular circadian rhythm assay warrants further evaluation as an early screening tool for neurotoxicants.

Cytogenetic Studies on Genoprotective Effect of Rosa damascena Mill. Hydrosol in Plant and Lymphocyte Test Systems.

Bulgarian Rosa damascena Mill. is has been known since ancient times for its high-quality oil, hydrosol, and other aromatic products. Rose hydrosol has various biological activities, but no research on its anticytotoxic/antigenotoxic effects exists. This study aimed to assess its defense potential against the genotoxin N-methyl-N'-nitro-N-nitrosoguanidine and to test its cytotoxic/genotoxic activity in plant and human lymphocyte test systems. Endpoints for cytotoxicity (mitotic index and nuclear division index) and genotoxicity (chromosome aberration and micronuclei) were used. Hydrosol was applied as a single treatment in concentrations ranging from 3% to 20% (4 h) to assess its cytotoxic and genotoxic effects. Its protective potential against MNNG was tested by applying an experimental scheme involving (i) conditioning treatment with non-toxic or slightly toxic concentrations of hydrosol, followed by genotoxin challenge (50 μg/mL) with a 4 h intertreatment time and (ii) treatment with hydrosol and mutagen with no time between the treatments. Hydrosol induces low cytotoxicity and clastogenicity, demonstrating cytoprotective/genoprotective effects against the mutagen in both applied test systems. The hydrosol defense potential was expressed by a more than twofold reduction in both chromosomal aberrations and micronuclei and by enhancing the mitotic activity compared with that of the mutagen, regardless of the experimental conditions. The results are promising for further hydrosol applications in pharmaceutical and medical practice.

Machine Learning-Based Hazard-Driven Prioritization of Features in Nontarget Screening of Environmental High-Resolution Mass Spectrometry Data.

Nontarget high-resolution mass spectrometry screening (NTS HRMS/MS) can detect thousands of organic substances in environmental samples. However, new strategies are needed to focus time-intensive identification efforts on features with the highest potential to cause adverse effects instead of the most abundant ones. To address this challenge, we developed MLinvitroTox, a machine learning framework that uses molecular fingerprints derived from fragmentation spectra (MS2) for a rapid classification of thousands of unidentified HRMS/MS features as toxic/nontoxic based on nearly 400 target-specific and over 100 cytotoxic endpoints from ToxCast/Tox21. Model development results demonstrated that using customized molecular fingerprints and models, over a quarter of toxic endpoints and the majority of the associated mechanistic targets could be accurately predicted with sensitivities exceeding 0.95. Notably, SIRIUS molecular fingerprints and xboost (Extreme Gradient Boosting) models with SMOTE (Synthetic Minority Oversampling Technique) for handling data imbalance were a universally successful and robust modeling configuration. Validation of MLinvitroTox on MassBank spectra showed that toxicity could be predicted from molecular fingerprints derived from MS2 with an average balanced accuracy of 0.75. By applying MLinvitroTox to environmental HRMS/MS data, we confirmed the experimental results obtained with target analysis and narrowed the analytical focus from tens of thousands of detected signals to 783 features linked to potential toxicity, including 109 spectral matches and 30 compounds with confirmed toxic activity.

122 Elucidating the Impact of Inhaled Micro-, Nanoplastics (MNPs) from Surgical Face Masks In Vitro

Abstract Surgical face masks are routinely used in clinical settings, but also extensively by the public during the SARS-CoV-2 pandemic. These masks contain a high proportion of plastic, specifically polypropylene (PP), which could shed microscopic particles to the environment or nasal cavity. However, to-date, despite focus upon an increased environmental burden, the (human) hazard relevant to inhaled MNPs remains largely unknown. The aim of this project therefore, was to determine the toxicity of inhaled PP (commercially available powders, or extracted from surgical masks; either (i) all layers, or (ii) the innermost layer only), using an advanced in vitro approach. Type-II lung epithelial cells (NCI-H441) were cultured at the air-liquid interface and exposed to respirable PP via aerosol for 24hrs at depositions of 500, 100 and 2000 ng/cm2 (Vitrocell Cloud12). Carbon black (Printex 90) was used as a positive particle control. Respirable MNP samples required cryomilling and cryotome slicing, and were analysed using pyrolysis-GCMS. In addition to acellular deposition, as well as particle and cellular morphology (electron microscopy) assessed endpoints of cytotoxicity (trypan blue exclusion), barrier integrity (dextran blue), (pro-)inflammatory response (IL-1β/IL-6/IL-8), oxidative stress (SOD-1), and genotoxicity (mononucleate micronucleus) were conducted. So far, a concentration-dependent decrease in cell viability was observed following exposure to commercial PP and complete mask PP samples, whilst a concentration-dependent increase in all (pro-)inflammatory mediators assessed was noted. Further, initial genotoxicity assessment suggests a minor increase to micronucleus formation for both exposure samples. Preliminary data therefore suggests that aerosolised MNPs instigate a hazardous response in vitro.

55 Investigating the co-Exposure of Micro(nano)Plastics and Indoor/Outdoor Particulate Matter Using an Alveolar BarrierIn Vitro Model

Abstract The impact of micro-, nanoplastics (MNPs) and particulate matter (PM) upon human health are escalating concerns worldwide. MNPs released from surgical face masks, and PM from ambient air pollution, can both be inhaled and consequently have potentially significant negative impacts upon human health, particularly on our lung epithelial cell structure and function. Currently, the impact of the realistic co-exposure of these samples is unknown. Therefore, the purpose of this study was to determine the potential inhaled hazardous health effects of the co-exposure of MNPs and ambient (indoor and outdoor) air PM. Using an advanced in vitro testing model combined with a specific mechanistic toxicology approach, it is hypothesised that the co-exposure approach will elucidate important information regarding the differential toxicology of ambient (indoor and outdoor) air PM. The in vitro system will consist of alveolar epithelial type-II cells (-NCI-H441) combined with differentiated monocytes (dTHP-1), cultured at the relevant in vivo ratio and at the air-liquid interface. Cell cultures will be exposed for both 4 and 24hrs to MNP (commercially available polypropylene (PP) and blue face mask extracted PP), PM (NIST 2584 and NIST 1649a respectively) and then subsequent MNP+PM (both NIST 2584 and NIST 1649a respectively) via aerosol (VITROCELL Cloud12), using alternative dosimetry patterns. The biochemical endpoints of cytotoxicity, oxidative stress, (pro-)inflammatory response and genotoxicity, will then be assessed to provide the basis for future global analytical techniques to examine the currently unknown human hazard of inhaled MNPs (+/- PM).

94 Metalsafety – In Vitro, ex Vivo and in Vivo Investigations on Particulate and Nanofibrous Metal Compounds

Abstract Metals and metal compounds are essential in daily life. Besides particulate materials nanofibrous material (nanowires) gain importance in innovative processes like optoelectronics. Toxicological data to assess the hazard is often available only for granular metals while data on nanowires is sparse. The project MetalSafety funded by the German BMBF aims at the development of an in vitro model to allow the grouping of different metal compounds with different bioavailability according to their toxicological hazard. Ex vivo and in vivo investigations are used to analyse the predictivity of the in vitro results and the underlying mode-of-action and dose-response relationship. Within this contribution the study concept is introduced. Silver and copper, both in particulate and nanofibrous form, are used across all examinations in vitro, ex vivo and in vivo. To allow an interspecies comparison cell lines derived from human and rat lung are used. Exposure to the materials can be investigated either submerse or with air-liquid-interface (ALI) conditions. The correlation between both species can also be examined ex vivo using precision-cut lung slices (PCLS) derived from human and rat lung tissue. Application in vivo by intratracheal instillation in rats is conducted to enable a validation of the in vitro and ex vivo screening models in a best case scenario under physiological conditions. Comparable endpoints in vitro, ex vivo as well as in vivo are measured during the study. This includes cytotoxicity endpoints and cytokine measurements as well as gene expression analyses. The lung tissue is also subjected to a histopathological examination.

In vitro analysis of the cytotoxic effect of two different sizes ITER-like tungsten nanoparticles on human dermal fibroblasts

BackgroundBased on the current configuration of the International Thermonuclear Experimental Reactor, tungsten (W) was chosen as the armour material. Nevertheless, during operation, the expected power and temperature of plasma can trigger the formation of W dust in the plasma chamber. According to the scenario for a Loss Of Vacuum Accident (LOVA), in the case of confinement failure dust is released, which can lead to occupational or accidental exposure. MethodsFor a first evidence of potential risks, fusion devices relevant W dust has been produced on purpose, using a magnetron sputtering gas aggregation source. We aimed to assess the in vitro cytotoxicity of synthesized tungsten nanoparticles (W-NPs) with diameters of 30 and 100 nm, on human BJ fibroblasts. That was systematically investigated using different cytotoxic endpoints (metabolic activity, cellular ATP, AK release and caspase-3/7 activity) and by direct observation with optical and scanning electron microscopy. ResultsIncreasing concentrations of W-NPs of both sizes induced cell viability decrease, but the effect was significantly higher for large W-NPs, starting from 200 μg/mL. In direct correlation with the effect on the cell membrane integrity, high concentrations of large W-NPs appear to increase AK release in the first 24 h of treatment. On the other hand, activation of the cellular caspase 3/7 was found significantly increased after 16 h of treatment solely for low concentrations of small W-NPs. SEM images revealed an increased tendency of agglomeration of small W-NPs in liquid medium, but no major differences in cells development and morphology were observed after treatment. An apparent internalization of nanoparticles under the cell membrane was also identified. ConclusionThese results provide evidence for different toxicological outputs identified as mechanistic responses of BJ fibroblasts to different sizes of W-NPs, indicating also that small W-NPs (30 nm) display lower cytotoxicity compared to larger ones (100 nm).

Evaluation of the risk of human exposure to thiamethoxam by extrapolation from a toxicokinetic experiment in rats and literature data

Previous studies suggest that exposure to thiamethoxam (TMX) may cause adverse effects to human. However, the distribution of TMX in various organs of human body and the associated risk are little-known. This study aimed to explore the distribution of TMX in human organs by extrapolation from a toxicokinetic experiment in rats and to assess the associated risk based on literature data. The rat exposure experiment was performed using 6-week female SD rats. Five groups of rats were oral-exposed to 1 mg/kg TMX (water as solvent) and executed at 1 h, 2 h, 4 h, 8 h and 24 h after treatment, respectively. The concentrations of TMX and its metabolites in rat liver, kidney, blood, brain, muscle, uterus and urine were measured in different time points using LC-MS. Data on concentrations of TMX in food, human urine and blood as well as human cell-based in vitro toxicity of TMX were collected from the literature. After oral exposure, TMX and its metabolite clothianidin (CLO) were detected in all organs of the rats. The steady-state tissue-plasma partition coefficients of TMX for liver, kidney, brain, uterus and muscle were 0.96, 1.53, 0.47, 0.60 and 1.10, respectively. Based on literature analysis, the concentration of TMX in human urine and blood for general population were 0.06–0.5 ng/mL and 0.04–0.6 ng/mL, respectively. For some people, the concentration of TMX in human urine reached 222 ng/mL. By extraplation from rat experiment, the estimated concentrations of TMX in human liver, kidney, brain, uterus and muscle for general population were 0.038–0.58, 0.061–0.92, 0.019–0.28, 0.024–0.36 and 0.044–0.66 ng/g, respectively, well below the relevant concentrations for cytotoxic endpoints (HQs ≤ 0.012); however, for some people they could be up to 253.44, 403.92, 124.08, 158.40 and 290.40 ng/g, respectively, with very high developmental toxicity (HQ = 5.4). Therefore, the risk for highly exposed people should not be neglected.

Detrimental effects of individual versus combined exposure to tetrabromobisphenol A and polystyrene nanoplastics in fish cell lines

The potential interactions between the diverse pollutants that can be released into the environment and the resulting outcomes are a challenging issue that needs to be further examined. This in vitro study was aimed to assess potential toxic effects caused by combined exposure to tetrabromobisphenol A, a flame retardant widely used and frequently detected in aquatic matrices, and commercially available polystyrene nanoparticles as reference material to evaluate nanoplastics risks. Our results, using freshwater fish cell lines and a set of relevant cytotoxicity endpoints including cell viability, oxidative stress, and DNA damage, provide additional mechanistic insights that could help to fully characterize the toxicity profiles of tetrabromobisphenol A and polystyrene nanoparticles. Furthermore, we describe subtle changes in cell viability as well as the generation of oxidative DNA damage after coexposure to subcytotoxic concentrations of the tested pollutants.

Cytotoxicity Assays with Zebrafish Cell Lines.

Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute toxicity. Thus, this protocol presents cytotoxicity assays modified to evaluate cell viability in zebrafish (Danio rerio) embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates. The cytotoxicity endpoints evaluated are mitochondrial integrity (Alamar Blue [AB] and MTT assays), membrane integrity via esterase activity (CFDA-AM assay), and lysosomal membrane integrity (Neutral Red [NR] assay). After the exposure of the test substances in a 96-well plate, the cytotoxicity assays are performed; here, AB and CFDA-AM are carried out simultaneously, followed by NR on the same plate, while the MTT assay is performed on a separate plate. The readouts for these assays are taken by fluorescence for AB and CFDA-AM, and absorbance for MTT and NR. The cytotoxicity assays performed with these fish cell lines can be used to study the acute toxicity of chemical substances on fish.

Predicting the cytotoxicity of nanomaterials through explainable, extreme gradient boosting

Nanoparticles (NPs) are a wide class of materials currently used in several industrial and biomedical applications. Due to their small size (1-100 nm), NPs can easily enter the human body, inducing tissue damage. NP toxicity depends on physical and chemical NP properties (e.g., size, charge and surface area) in ways and magnitudes that are still unknown. We assess the average as well as the individual importance of NP atomic descriptors, along with chemical properties and experimental conditions, in determining cytotoxicity endpoints for several nanomaterials. We employ a multicenter cytotoxicity nanomaterial database (12 different materials with first and second dimensions ranging between 2.70 and 81.2 nm and between 4.10 and 4048 nm, respectively). We develop a regressor model based on extreme gradient boosting with hyperparameter optimization. We employ Shapley additive explanations to obtain good cytotoxicity prediction performance. Model performances are quantified as statistically significant Spearman correlations between the true and predicted values, ranging from 0.5 to 0.7. Our results show that i) size in situ and surface areas larger than 200 nm and 50 m2/g, respectively, ii) primary particles smaller than 20 nm; iii) irregular (i.e., not spherical) shapes and iv) positive Z-potentials contribute the most to the prediction of NP cytotoxicity, especially if lactate dehydrogenase (LDH) assays are employed for short experimental times. These results were moderately stable across toxicity endpoints, although some degree of variability emerged across dose quantification methods, confirming the complexity of nano-bio interactions and the need for large, systematic experimental characterization to reach a safer-by-design approach.

Study on Cytotoxic and Genotoxic Potential of Bulgarian Rosa damascena Mill. and Rosa alba L. Hydrosols—In Vivo and In Vitro

The Rosa alba L. and Rosa damascena Mill. growing in Bulgaria are known for their extremely fine essential oil and valuable hydrosols. Irrespectively of its wide use in human life, little research exists on the cytotoxic and genotoxic activity of the hydrosols. This set our goal to conduct cytogenetic analyses to study these effects. A complex of classical cytogenetic methods was applied in three types of experimental test systems—higher plant in vivo, ICR mice in vivo, and human lymphocytes in vitro. Mitotic index, PCE/(PCE + NCE) ratio, and nuclear division index were used as endpoints for cytotoxicity and for genotoxicity—induction of chromosome aberrations and micronuclei. Rose hydrosol treatments range in concentrations from 6% to 20%. It was obtained that both hydrosols did not show considerable cytotoxic and genotoxic effects. These effects depend on the type of the tested rose hydrosols, the concentrations applied in the experiments, and the sensitivity and specificity of the test systems used. Human lymphocytes in vitro were the most sensitive to hydrosols, followed by higher plant and animal cells. Chromosomal aberrations and micronucleus assays suggested that R. damascena and R. alba hydrosols at applied concentrations possess low genotoxic risk. Due to the overall low values in terms of cytotoxic and/or genotoxic effects in all test systems, hydrosols are promising for further use in various areas of human life.