Finding a Direct Method for a Dynamic Process: The DD (Direct and Dynamic) Cell-Tox Method.

The main focus of in vitro toxicity assessment methods is to assess the viability of the cells, which is usually based on metabolism changes. Yet, when exposed to toxic substances, the cell triggers multiple signals in response. With this in mind, we have developed a promising cell-based toxicity method that observes various cell responses when exposed to toxic substances (either death, division, or remain viable). Based on the collective cell response, we observed and predicted the dynamics of the cell population to determine the toxicity of the toxicant. The method was tested with two different conformations: In the first conformation, we exposed a monoculture model of blood macrophages to UV light, hydrogen peroxide, nutrient deprivation, tetrabromobisphenol A, fatty acids, and 5-fluorouracil. In the second, we exposed a coculture liver model consisting of hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells to rifampicin, ibuprofen, and 5-fluorouracil. The method showed good accuracy compared to established toxicity assessment methods. In addition, this approach provided more representative information on the toxic effects of the compounds, as it considers the different cellular responses induced by toxic agents.

In Vitro Human Liver Model for Toxicity Assessment with Clinical and Preclinical Instrumentation.

The existing in vitro toxicological models lack translational potential, which makes difficult the application of gathered information to clinical usage. To tackle this issue, we built a model with four different types of primary liver cells: hepatic sinusoidal endothelial cells, hepatic stellate cells, Kupffer cells and hepatocytes. We cultured them in different combinations of composition and volumes of cell medium, hepatocyte proportions of total cells and additions of extracellular matrixes. We added rifampicin (RIF), ibuprofen (IBU) and 5-fluorouracil (5-FU) to this model and observed the microanatomy and physiology changes for a week with preclinical and clinical instruments. Among the different model configurations, we selected the feature combination of the in vitro model that had similar biomarker values to those measured in clinical diagnostics. When we exposed the selected model configuration to RIF, IBU and 5-FU, we observed similar glucose, triglyceride and albumin dynamics as in vivo (from clinical data). Therefore, we have built an in vitro liver model that resembles the liver microenvironment, and we have analysed it with clinical instrumentation to facilitate data translation. Furthermore, during these observations, we found that Kupffer and LSEC cells are suitable candidates for the search for clinical diagnostic markers of liver function.

A Promising Method for the Determination of Cell Viability: The Membrane Potential Cell Viability Assay.

Determining the viability of cells is fraught with many uncertainties. It is often difficult to determine whether a cell is still alive, approaching the point of no return, or dead. Today, there are many methods for determining cell viability. Most rely on an indirect determination of cell death (metabolism, molecular transport, and leakage, to name a few). In contrast, we have developed a promising novel method for a "direct" determination of cell viability. The potential method assesses cell membrane integrity (which is essential for all viable cells) by measuring the electrical potential of the cell membrane. To test the assay, we chose two different cell types, blood macrophages (TLT) and breast cancer epithelial cells (MCF 7). We exposed them to seven different toxic scenarios (arsenic (V), UV light, hydrogen peroxide, nutrient starvation, Tetrabromobisphenol A, fatty acids, and 5-fluorouracil) to induce different cell death pathways. Under controlled test conditions, the assay showed good accuracy when comparing the toxicity assessment with well-established methods. Moreover, the method showed compatibility with live cell imaging. Although we know that further studies are needed to confirm the performance of the assay in other situations, the results obtained are promising for their wider application in the future.

In vitro toxicity model: Upgrades to bridge the gap between preclinical and clinical research.

The Centers for Disease Control and Prevention (CDC) provides extensive data that indicate our need for drugs to maintain human population health. Despite the substantial availability of drugs on the market, many patients lack specific drugs. New drugs are required to tackle this issue. Moreover, we need more reliable models for testing drug toxicity, as too many drug approval failures occur with the current models. This article briefly describes various approaches of the currently used models for toxicity screening, to justify the selection of in vitro cell-based models. Cell-based toxicity models have the best potential to reliably predict drug toxicity in humans, as they are developed using the cells of the target organism. However, currently, a large gap exists between in vitro cell-based approach to toxicity testing and the clinical approach, which may be contributing to drug approval failures. We propose improvements to in vitro cell-based toxicity models, which is often an insight approach, to better match this approach with the clinical homeostatic approach. This should enable a more accurate comparison of data between the preclinical as well as clinical models and provide a more comprehensive understanding of human physiology and biological effects of drugs.

Occurrence of cyclic imines in European commercial seafood and consumers risk assessment.

Cyclic imines constitute a quite recently discovered group of marine biotoxins that act on neural receptors and that bioaccumulate in seafood. They are grouped together due to the imino group functioning as their common pharmacore, responsible for acute neurotoxicity in mice. Cyclic imines (CIs) have not been linked yet to human poisoning and are not regulated in the European Union (EU), although the European Food Safety Authority (EFSA) requires more data to perform conclusive risk assessment for consumers. Several commercial samples of bivalves including raw and processed samples from eight countries (Italy, Portugal, Slovenia, Spain, Ireland, Norway, The Netherlands and Denmark) were obtained over 2 years. Emerging cyclic imine concentrations in all the samples were analysed on a LC-3200QTRAP and LC-HRMS QExactive mass spectrometer. In shellfish, two CIs, pinnatoxin G (PnTX-G) and 13-desmethylspirolide C (SPX-1) were found at low concentrations (0.1-12µg/kg PnTX-G and 26-66µg/kg SPX-1), while gymnodimines and pteriatoxins were not detected in commercial (raw and processed) samples. In summary, SPX-1 (n: 47) and PnTX-G (n: 96) were detected in 9.4% and 4.2% of the samples, respectively, at concentrations higher than the limit of quantification (LOQ), and in 7.3% and 31.2% of the samples at concentrations lower than the LOQ (25µg/kg for SPX-1 and 3µg/kg for PnTX-G), respectively. For the detected cyclic imines, the average exposure and the 95th percentile were calculated. The results obtained indicate that it is unlikely that a potential health risk exists through the seafood diet for CIs in the EU. However, further information about CIs is necessary in order to perform a conclusive risk assessment.

Toxic effects of perfluorinated compounds at human cellular level and on a model vertebrate.

This work aims at deepening the understanding of the mode of action of some of the most prominent perfluorinated compounds (PFCs) by detecting in a realistic way their effects. To this end, after adjusting the exposure media taking into account the biological model employed and the physico-chemical properties of PFCs, we evaluated the toxic effects of PFOA, PFOS and PFNA in a human macrophage cell line (TLT cells) and in zebrafish embryos. We performed such evaluation on individual compounds and mixtures. Acute toxicity was greater for PFOS in zebrafish; however, it was greater for PFNA in TLT cells. PFNA was also the compound producing the greatest levels of oxidative stress, both in zebrafish and TLT cells. Additionally, in both biological systems, it showed a much stronger effect on mixtures in comparison to the others PFCs tested in this work. Mixture studies in zebrafish showed that acute toxicity depended on the concentration and that the mixture was far more toxic than the individual compounds. This study highlights the importance of studying PFCs in realistic conditions on various biological models.