Toxicological Applications Research Articles

Mass Spectrometry Imaging and Its Application in Forensic Toxicology.

Mass spectrometry imaging （MSI） is a new imaging technology that can simultaneously detect and record the spatial distribution information of multiple molecules on the sample surface without labeling. The main principle of MSI is to combine mass spectrometry with imaging technology and irradiate the sample slice with ion beam or laser to ionize the molecules on its surface, obtain the mass spectrometry signal through the detector, convert the obtained data into pixel points by the imaging software, and then construct the spatial distribution image of the target compound on the tissue surface. The sample preparation for MSI include： sample collection and storage, tissue section, tissue pretreatment, selection and application of matrix. At present, this technology has been widely used in the fields of biomedicine, new drug development and proteomics, and its application in the field of forensic toxicology has also gradually attracted attention. This article reviews the principles and sample preparation process of MSI, describes the application of MSI in abused substances and metabolites of various material matrices, herbal mixtures, latent fingerprints, hair and animal and plant tissues, and discusses the prospects of the application of this technology in forensic toxicology, in order to provide ideas and references for the application of MSI technology in forensic toxicology.

An Alternative to the Beta-Binomial Distribution with Application in Developmental Toxicology

An Alternative to the Beta-Binomial Distribution with Application in Developmental Toxicology

3D human liver spheroids for translational pharmacology and toxicology.

Drug development is a failure-prone endeavour, and more than 85% of drugs fail during clinical development, showcasing that current preclinical systems for compound selection are clearly inadequate. Liver toxicity remains a major reason for safety failures. Furthermore, all efforts to develop pharmacological therapies for a variety of chronic liver diseases, such as non-alcoholic steatohepatitis (NASH) and fibrosis, remain unsuccessful. Considering the time and expense of clinical trials, as well as the substantial burden on patients, new strategies are thus of paramount importance to increase clinical success rates. To this end, human liver spheroids are becoming increasingly utilized as they allow to preserve patient-specific phenotypes and functions for multiple weeks in culture. We here review the recent application of such systems for i) predictive and mechanistic analyses of drug hepatotoxicity, ii) the evaluation of hepatic disposition and metabolite formation of low clearance drugs and iii) the development of drugs for metabolic and infectious liver diseases, including NASH, fibrosis, malaria and viral hepatitis. We envision that with increasing dissemination, liver spheroids might become the new gold standard for such applications in translational pharmacology and toxicology.

Microfluidic Model to Mimic Initial Event of Neovascularization.

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Oxygenation as a driving factor in epithelial differentiation at the air-liquid interface.

Culture at the air-liquid interface is broadly accepted as necessary for differentiation of cultured epithelial cells towards an in vivo-like phenotype. However, air-liquid interface cultures are expensive, laborious and challenging to scale for increased throughput applications. Deconstructing the microenvironmental parameters that drive these differentiation processes could circumvent these limitations, and here we hypothesize that reduced oxygenation due to diffusion limitations in liquid media limits differentiation in submerged cultures; and that this phenotype can be rescued by recreating normoxic conditions at the epithelial monolayer, even under submerged conditions. Guided by computational models, hyperoxygenation of atmospheric conditions was applied to manipulate oxygenation at the monolayer surface. The impact of this rescue condition was confirmed by assessing protein expression of hypoxia-sensitive markers. Differentiation of primary human bronchial epithelial cells isolated from healthy patients was then assessed in air-liquid interface, submerged and hyperoxygenated submerged culture conditions. Markers of differentiation, including epithelial layer thickness, tight junction formation, ciliated surface area and functional capacity for mucociliary clearance, were assessed and found to improve significantly in hyperoxygenated submerged cultures, beyond standard air-liquid interface or submerged culture conditions. These results demonstrate that an air-liquid interface is not necessary to produce highly differentiated epithelial structures, and that increased availability of oxygen and nutrient media can be leveraged as important strategies to improve epithelial differentiation for applications in respiratory toxicology and therapeutic development.

Using Monochlorobimane to Visualize Glutathione Utilization in the Developing Zebrafish (Danio rerio) Embryo.

Glutathione (GSH) plays fundamental roles in cellular redox buffering and is a common detoxification pathway for excretion of xenobiotics. This is especially crucial during vertebrate embryogenesis, when an organism is at one of its most vulnerable life stages. Importantly, GSH content and redox potential can dictate cell fate decisions, which can have profound consequences if altered by early life xenobiotic exposures. Owing to technical limitations, the best available method to detect and quantify changes in GSH has been high-pressure liquid chromatography, a terminal method that prevents suborganism-level resolution of these changes in developing embryos. Here, we describe a protocol that leverages the transparent nature of zebrafish embryos and the compatibility of monochlorobimane with the zebrafish GSH-S-transferase enzymes, to allow for the visualization of changes in GSH via S-glutathionylation in a live, developing embryo. This method can find broad application in developmental biology and toxicology. © 2021 Wiley Periodicals LLC.

Validation of a method for ethanol analysis in biological and non-biological samples and its toxicological application

A simple, cost-effective and fast headspace gas chromatography method coupled with flame ionization detection (HS-GC/FID) for determination of ethanol was developed and validated for clinical and forensic toxicology purposes. HS-GC/FID is often used for alcohol determination in different biological and non-biological samples. The calibration dependence of the method was linear in the range from 0.15 to 4.00 g dm-3 (r2=0.999) with adequate accuracy (99?106 %) and precision. The limit of detection (LOD) was 0.006 g dm-3. The method was quantitative (LOQ) above 0.020 g dm-3. The new method was successfully used for determination of ethanol in biological samples of intoxicated patients, car accidents participants, participants in criminal acts, and postmortem samples, non-biological samples such as alcoholic beverages, alcohol-based herbal preparations, cosmetic preparations, etc. This method is easy to perform, making it suitable not only for the routine applications in clinical biochemistry and forensic laboratories, but also in different fields of industry (e.g. for pharmaceutical preparations, cosmetics, dietary supplements, etc.). Some of the applications for ethanol determination in different samples related to various clinical-forensic cases are presented.

Approaches in metabolomics for regulatory toxicology applications.

Innovative methodological approaches are needed to conduct human health and environmental risk assessments on a growing number of marketed chemicals. Metabolomics is progressively proving its value as an efficient strategy to perform toxicological evaluations of new and existing substances, and it will likely become a key tool to accelerate chemical risk assessments. However, additional guidance with widely accepted and harmonized procedures is needed before metabolomics can be routinely incorporated in decision-making for regulatory purposes. The aim of this review is to provide an overview of metabolomic strategies that have been successfully employed in toxicity assessment as well as the most promising workflows in a regulatory context. First, we provide a general view of the different steps of regulatory toxicology-oriented metabolomics. Emphasis is put on three key elements: robustness of experimental design, choice of analytical platform, and use of adapted data treatment tools. Then, examples in which metabolomics supported regulatory toxicology outputs in different scenarios are reviewed, including chemical grouping, elucidation of mechanisms of toxicity, and determination of points of departure. The overall intention is to provide insights into why and how to plan and conduct metabolomic studies for regulatory toxicology purposes.

Development of sol-gel phenyl/methyl/poly (dimethylsiloxane) sorbent coating for fabric phase sorptive extraction and its application in monitoring human exposure to selected polycyclic aromatic hydrocarbons using high performance liquid chromatography-fluorescence detection

Following the convenient, yet very powerful pathway to create designer extraction sorbent using sol-gel chemistry, a novel sol-gel phenyl/methyl/poly(dimethylsiloxane) sorbent coating was created on polyester fabric substrate for fabric phase sorptive extraction (FPSE) and was subsequently applied to monitor human exposure to selected polycyclic aromatic hydrocarbons (PAHs) including pyrene, chrysene, and benzo[a]pyrene in plasma samples obtained from tobacco smoker volunteers using high performance liquid chromatography-fluorescence detector (HPLC-FLD). A rapid FPSE-HPLC-FLD method was developed that adequately resolved the PAHs chromatographically, after their successful extraction from human plasma using fabric phase absorption extraction (FPSE) and subsequently analysed in the liquid chromatographic system by means of an analytical column (InterSustain C-18 column 150 × 4.6 mm, 5 μm) using acetonitrile (ACN) and water as mobile phases in gradient elution mode. With the optimized conditions, the retention times were found to be 6.168, 7.214, and 10.404 min for pyrene, chrysene, and benzo[a]pyrene, respectively. The total chromatographic runtime was limited to 12.5 min.The method, validated through the calculation of all the analytical parameters according to the International Guidelines, was applied to the analysis of real samples collected from informed volunteers. The proposed approach which included the use of sol-gel phenyl/methyl/poly(dimethylsiloxane) immobilized on hydrophobic polyester substrate and C18 stationary phase used in HPLC, has shown a high potential as a rapid tool for future clinical, forensic and toxicological applications, also in the light of the LOD and LOQ values comparable to those normally obtainable with more sophisticated, and expensive instruments that often require highly trained personnel. The results reported here further consolidate the application of FPSE in the analysis of biological samples for both diagnostic and analytical-clinical purposes.

Machine Learning in Predictive Toxicology: Recent Applications and Future Directions for Classification Models.

In recent times, machine learning has become increasingly prominent in predictive toxicology as it has shifted from in vivo studies toward in silico studies. Currently, in vitro methods together with other computational methods such as quantitative structure-activity relationship modeling and absorption, distribution, metabolism, and excretion calculations are being used. An overview of machine learning and its applications in predictive toxicology is presented here, including support vector machines (SVMs), random forest (RF) and decision trees (DTs), neural networks, regression models, naïve Bayes, k-nearest neighbors, and ensemble learning. The recent successes of these machine learning methods in predictive toxicology are summarized, and a comparison of some models used in predictive toxicology is presented. In predictive toxicology, SVMs, RF, and DTs are the dominant machine learning methods due to the characteristics of the data available. Lastly, this review describes the current challenges facing the use of machine learning in predictive toxicology and offers insights into the possible areas of improvement in the field.

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.

Absolutely continuous copulas with prescribed support constructed by differential equations, with an application in toxicology

A new method for constructing absolutely continuous two-dimensional copulas by differential equations is presented. The copulas are symmetric with respect to reflection in the opposite diagonal. The support of the copula density may be prescribed to arbitrary opposite symmetric hypographs of invertible functions, containing the diagonal. The method is applied to toxicological probit modeling, where new compatibility conditions for the probit parameters are derived.

Application of microextraction techniques in alternative biological matrices with focus on forensic toxicology: a review.

The interest in alternative biological matrices (e.g., hair and saliva) for forensic toxicology analysis has increased, and recent developments in sample preparation have targeted rapid, cheap, efficient and eco-friendly methods, including microextraction techniques. For this review, we have gathered information about these two hot topics. We discuss the composition, incorporation of analytes and advantages and disadvantages of different biological matrices, and also present the operation principles of the most reported microextraction procedures and their application in forensic toxicology. The outcome of this review may encourage future forensic researches into alternative samples and microextraction techniques.

Widespread fluorescence in terrestrial and marine samples investigated from India

The discovery of green fluorescent protein (GFP) from jellyfish Aequorea victoria has provoked numerous studies to unveil the myriads of biomedical applications. Consequently, several studies also revealed the prevalence of fluorescence in different marine and terrestrial organisms. However, since GFP's discovery or the Nobel prize award on GFP, the fluorescence has not been explored so far from India. The current study presents the widespread fluorescent organisms resources available in India for biomedical and toxicological applications. Fluorescence emission from different plant and animal components were examined by direct observations using UV torchlight. Investigation revealed that blue light excited fluorescence in several organisms. For the first time, this study observed GFP like fluorescence in many terretrial and marine organisms of India. Observations are indicating that fluorescent proteins have essential ecological functions that are yet to be determined. The examined plant and animal components were not bioluminescent. In comparison, the potential untouched research areas on fluorescence aspects are detailed. A thorough review of fluorescent organisms reported hitherto is also provided to spread the current knowledge on fluorescence.

Establishment and characterization of a gill cell line from pearl gentian grouper (Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀) and its application in cadmium toxicology

A novel gill cell line from pearl gentian grouper (Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀, PGGG cell line) was established, its application in cadmium (Cd) toxicology was demonstrated in this study. Primary cultures and PGGG subcultures were carried out at 25 °C in Dulbecco’s Modified Eagle medium/F12 medium (1:1; pH 7.2) supplemented with 15% fetal bovine serum (FBS). Primary PGGG cells were spindle-shaped, proliferated into a confluent monolayer within two weeks and were continuously subcultured over passage 60. The growth of cells at passages 20, 40, and 60 was examined. Chromosome analysis revealed that the chromosomal number of normal PGGG cells was 48, but the number of cells with the normal chromosomes number decreased during the passaging process. Cadmium is one of the most toxic metals in aquatic systems and has been associated with multiple animal and human health problems. To interpret the cytotoxicity and related mechanisms of cadmium, PGGG cells were used as an in vitro model. After treatment with cadmium at concentrations ranging from 1 µM to 500 µM, PGGG cells demonstrated dose- and time-dependent cytotoxicity, manifested as morphological abnormalities and a viability decline. Further, it was found that the reactive oxygen species (ROS) and malondialdehyde (MDA) levels were elevated following cadmium exposure, and related genes involved in the antioxidant system, including those encoding catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and Kelch-like- ECH-associated protein 1 (Keap1), were regulated differently. In addition, PGGG cells treated with cadmium had the typical features associated with apoptosis, including phosphatidylserine (PS) externalization; upregulated expression of caspase-3, −8, and −9; and apoptotic body formation. In general, the PGGG cell line may serve as a useful tool for studying the toxic mechanisms of cadmium or other toxicants or for toxicity testing and environment monitoring.

Development and characterization of rat duodenal organoids for ADME and toxicology applications

Many in vitro gastrointestinal models have been developed with the hope that they will continue to improve in their similarity to the organs from which they were isolated. Intestinal organoids isolated from various species are now being used to investigate physiology and pathophysiology. In this study, intestinal stem cells were isolated from adult rat duodenum and culture conditions were optimized to promote the growth, differentiation and development of 3D organoids. We optimized and characterized rat duodenal organoids with light and electron microscopy, immunofluorescence and notably, global mRNA expression. The metabolic capacity of these cultures was investigated using probe substrates for multiple phase I and phase II drug metabolizing enzymes and found to be in line with previous results from intestinal primary cultures and a significant improvement over immortalized cell lines. Over the course of differentiation, the gene expression profiles of the rat duodenal organoids were consistent with expected trends in differentiation to various cell lineages reflecting the duodenum in vivo. Further, incubations of these cultures with naproxen and celecoxib resulted in cytotoxicity consistent with the direct cytotoxic effects of these drugs to duodenum in vivo. Based on these characteristics, the rat duodenal organoids described herein will provide a novel platform for investigating a wide variety of mechanistic questions.

Human hair tests to document drug environmental contamination: Application in a family law case involving N,N‐dimethyltryptamine

For 40 years, hair tests have been presented as the best approach to document long-term consumption of a drug. This unique property has found numerous applications in clinical, forensic, and occupational toxicology. However, since the beginning of its implementation in biology, external contamination, with an associated risk of false positive result, has been presented as the key in the final interpretation. Evidence of environmental contamination and subsequent health issues can be the task of any toxicologist. Because of recent progress of analytical equipment, it is now possible to quantify drugs in hair with high level of accuracy and specificity at the pg/mg range. Therefore, segmental hair tests can be used to document environmental contamination and are the objective of this publication. In a family law case, N,N-dimethyltryptamine (DMT), a powerful hallucinogen, has been found in the hair of the partner of a repetitive DMT smoker at 4 to 13 pg/mg in 6 × 1 cm segments, with a regular increase of concentrations from the proximal to the distal hair end. The low measured concentrations and the particular pattern of DMT distribution along the hair shaft seem to be typical of environmental contamination, the older hair (those of the distal part) being for a longer time in contact with the drug. Despite strong decontamination, drugs from the environment can remain bound to the hair matrix and therefore be able to be used to document environmental contamination.

Evaluation of Forensic Data Using Logistic Regression-Based Classification Methods and an R Shiny Implementation.

We demonstrate the use of classification methods that are well-suited for forensic toxicology applications. The methods are based on penalized logistic regression, can be employed when separation occurs in a two-class classification setting, and allow for the calculation of likelihood ratios. A case study of this framework is demonstrated on alcohol biomarker data for classifying chronic alcohol drinkers. The approach can be extended to applications in the fields of analytical and forensic chemistry, where it is a common feature to have a large number of biomarkers, and allows for flexibility in model assumptions such as multivariate normality. While some penalized regression methods have been introduced previously in forensic applications, our study is meant to encourage practitioners to use these powerful methods more widely. As such, based upon our proof-of-concept studies, we also introduce an R Shiny online tool with an intuitive interface able to perform several classification methods. We anticipate that this open-source and free-of-charge application will provide a powerful and dynamic tool to infer the LR value in case of classification tasks.

NURA: A curated dataset of nuclear receptor modulators

Nuclear receptors (NRs) are key regulators of human health and constitute a relevant target for medicinal chemistry applications as well as for toxicological risk assessment. Several open databases dedicated to small molecules that modulate NRs exist; however, depending on their final aim (i.e., adverse effect assessment or drug design), these databases contain a different amount and type of annotated molecules, along with a different distribution of experimental bioactivity values. Stemming from these considerations, in this work we aim to provide a unified dataset, NURA (NUclear Receptor Activity) dataset, collecting curated information on small molecules that modulate NRs, to be intended for both pharmacological and toxicological applications. NURA contains bioactivity annotations for 15,247 molecules and 11 selected NRs, and it was obtained by integrating and curating data from toxicological and pharmacological databases (i.e., Tox21, ChEMBL, NR-DBIND and BindingDB). Our results show that NURA dataset is a useful tool to bridge the gap between toxicology- and medicinal-chemistry-related databases, as it is enriched in terms of number of molecules, structural diversity and covered atomic scaffolds compared to the single sources. To the best of our knowledge, NURA dataset is the most exhaustive collection of small molecules annotated for their modulation of the chosen nuclear receptors. NURA dataset is intended to support decision-making in pharmacology and toxicology, as well as to contribute to data-driven applications, such as machine learning. The dataset and the data curation pipeline can be downloaded free of charge on Zenodo at the following DOI: https://doi.org/10.5281/zenodo.3991561.

Hepatocyte-like cells derived from human induced pluripotent stem cells using small molecules: implications of a transcriptomic study

BackgroundHepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSCs) hold great promise in toxicological applications as well as in regenerative medicine. Previous efforts on hepatocyte differentiation have mostly relied on the use of growth factors (GFs) to recapitulate developmental signals under in vitro conditions. Recently, the use of small molecules (SMs) has emerged as an attractive tool to induce cell fate transition due to its superiority in terms of both quality and cost. However, HLCs derived using SMs have not been well characterized, especially on the transcriptome level.MethodsHLCs were differentiated from human iPSCs using a protocol that only involves SMs and characterized by transcriptomic analysis using whole genome microarrays.ResultsHLCs derived using the SM protocol (HLC_SM) displayed specific hepatic marker expression and demonstrated key hepatic functions. Transcriptomic analysis of the SM-driven differentiation defined a hepatocyte differentiation track and characterized the expression of some key marker genes in major stages of hepatocyte differentiation. In addition, HLC_SM were scored with CellNet, a bioinformatics tool quantifying how closely engineered cell populations resemble their target cell type, and compared to primary human hepatocytes (PHHs), adult liver tissue, fetal liver tissue, HLCs differentiated using GFs (HLC_GF), and commercially available HLCs. Similar to HLC_GF, HLC_SM displayed a mixed phenotype of fetal and adult hepatocytes and had relatively low expression of metabolic enzymes, transporters, and nuclear receptors compared to PHHs. Finally, the differentially expressed genes in HLC_SM compared to HLC_GF and to PHHs were analyzed to identify pathways and upstream transcription regulators which could potentially be manipulated to improve the differentiation of HLCs.ConclusionsOverall, the present study demonstrated the usefulness of the SM-based hepatocyte differentiation method, offered new insights into the molecular basis of hepatogenesis and associated gene regulation, and suggested ways for further improvements in hepatocyte differentiation in order to obtain more mature HLCs that could be used in toxicological studies.