Systems Toxicology Approaches Research Articles

Antagonistic interaction between caffeine and ketamine in zebrafish: Implications for aquatic toxicity

The coexistence of caffeine (CF) and ketamine (KET) in surface waters across Asia has been widely reported. Previous studies have implied that CF and KET may share a mechanism of action. However, the combined toxicity of these two chemicals on aquatic organisms remains unclear at environmental levels, and the underlying mechanisms are not well understood. Here we demonstrate that KET antagonizes the adverse effects of CF on zebrafish larvae by modulating the gamma-aminobutyric acid (GABA)ergic synapse pathway. Specifically, KET (10–250 ng L−1) ameliorates the locomotor hyperactivity and impaired circadian rhythms in zebrafish larvae induced by 2 mg L−1 of CF, showing a dose-dependent relationship. Additionally, the developmental abnormalities in zebrafish larvae exposed to CF are mitigated by KET, with an incidence rate reduced from 26.7% to 6.7%. The competition between CF and KET for binding sites on the GABA-A receptor (in situ and in silico) elucidates the antagonistic interactions between the two chemicals. Following a seven-day recovery period, the adverse outcomes of CF exposure persist in the fish, whereas the changes observed in the CF + KET groups are significantly alleviated, especially with KET at 10 ng L−1. Based on these results, it is imperative to further assess the environmental risks associated with CF and KET co-pollution. This pilot study underscores the utility of systems toxicology approaches in estimating the combined toxicity of environmental chemicals on aquatic organisms. Moreover, the nighttime behavioral functions of fish could serve as a sensitive biomarker for evaluating the toxicity of psychoactive substances.

Classifying polycyclic aromatic hydrocarbons by carcinogenic potency using in vitro biosignatures

One of the most difficult challenges for risk assessment is evaluation of chemicals that predominately co-occur in mixtures like polycyclic aromatic hydrocarbons (PAHs). We previously developed a classification model in which systems biology data collected from mice short-term after chemical exposure accurately predict tumor outcome. The present study demonstrates translation of this approach into a human in vitro model in which chemical-specific bioactivity profiles from 3D human bronchial epithelial cells (HBEC) classify PAHs by carcinogenic potency. Gene expression profiles were analyzed from HBEC exposed to carcinogenic and non-carcinogenic PAHs and classification accuracies were identified for individual pathway-based gene sets. Posterior probabilities of best performing gene sets were combined via Bayesian integration resulting in a classifier with four gene sets, including aryl hydrocarbon receptor signaling, regulation of epithelial mesenchymal transition, regulation of angiogenesis, and cell cycle G2-M. In addition, transcriptional benchmark dose modeling of benzo[a]pyrene (BAP) showed that the most sensitive gene sets to BAP regulation were largely dissimilar from those that best classified PAH carcinogenicity challenging current assumptions that BAP carcinogenicity (and subsequent mode of action) is reflective of overall PAH carcinogenicity. These results illustrate utility of using systems toxicology approaches to analyze global gene expression towards carcinogenic hazard assessment.

Systems Toxicology Approaches Reveal the Mechanisms of Hepatotoxicity Induced by Diosbulbin B in Male Mice.

Diosbulbin B (DIOB) is an effective component of air potato yam with antitumor and anti-inflammatory activities, and it is the main toxic component leading to hepatotoxicity. However, the mechanism of its hepatotoxicity remains unclear. In this study, we aimed to systematically elucidate the molecular action of DIOB on liver metabolic function through systems toxicology approaches. C57BL/6 mice were orally treated with DIOB (10, 30, 60 mg/kg) for 28 days, and the liver metabonomics and histopathology, molecular docking, mRNA expression levels, and activities of enzymes were analyzed. The results illustrated that DIOB could affect fatty acid and glucose metabolism, block the TCA cycle, and DIOB also could disorder bile acid synthesis and transport and promote the occurrence of hyperbilirubinemia. In addition, DIOB increased Cyp3a11 expression in a dose-dependent manner. Thus, these results provide new insights into the mechanism of hepatotoxicity caused by DIOB.

Multi-omics systems toxicology study of mouse lung assessing the effects of aerosols from two heat-not-burn tobacco products and cigarette smoke

Cigarette smoke (CS) causes adverse health effects and, for smoker who do not quit, modified risk tobacco products (MRTPs) can be an alternative to reduce the risk of developing smoking-related diseases. Standard toxicological endpoints can lack sensitivity, with systems toxicology approaches yielding broader insights into toxicological mechanisms. In a 6-month systems toxicology study on ApoE−/− mice, we conducted an integrative multi-omics analysis to assess the effects of aerosols from the Carbon Heated Tobacco Product (CHTP) 1.2 and Tobacco Heating System (THS) 2.2—a potential and a candidate MRTP based on the heat-not-burn (HnB) principle—compared with CS at matched nicotine concentrations. Molecular exposure effects in the lungs were measured by mRNA/microRNA transcriptomics, proteomics, metabolomics, and lipidomics. Integrative data analysis included Multi-Omics Factor Analysis and multi-modality functional network interpretation. Across all five data modalities, CS exposure was associated with an increased inflammatory and oxidative stress response, and lipid/surfactant alterations. Upon HnB aerosol exposure these effects were much more limited or absent, with reversal of CS-induced effects upon cessation and switching to CHTP 1.2. Functional network analysis revealed CS-induced complex immunoregulatory interactions across the investigated molecular layers (e.g., itaconate, quinolinate, and miR-146) and highlighted the engagement of the heme–Hmox–bilirubin oxidative stress axis by CS. This work exemplifies how multi-omics approaches can be leveraged within systems toxicology studies and the generated multi-omics data set can facilitate the development of analysis methods and can yield further insights into the effects of toxicological exposures on the lung of mice.

Deciphering Adverse Outcome Pathway Network Linked to Bisphenol F Using Text Mining and Systems Toxicology Approaches.

Bisphenol F (BPF) is one of several Bisphenol A (BPA) substituents that is increasingly used in manufacturing industry leading to detectable human exposure. Whereas a large number of studies have been devoted to decipher BPA effects, much less is known about its substituents. To support decision making on BPF’s safety, we have developed a new computational approach to rapidly explore the available data on its toxicological effects, combining text mining and integrative systems biology, and aiming at connecting BPF to adverse outcome pathways (AOPs). We first extracted from different databases BPF-protein associations that were expanded to protein complexes using protein-protein interaction datasets. Over-representation analysis of the protein complexes allowed to identify the most relevant biological pathways putatively targeted by BPF. Then, automatic screening of scientific abstracts from literature using the text mining tool, AOP-helpFinder, combined with data integration from various sources (AOP-wiki, CompTox, etc.) and manual curation allowed us to link BPF to AOP events. Finally, we combined all the information gathered through those analyses and built a comprehensive complex framework linking BPF to an AOP network including, as adverse outcomes, various types of cancers such as breast and thyroid malignancies. These results which integrate different types of data can support regulatory assessment of the BPA substituent, BPF, and trigger new epidemiological and experimental studies.

Toxicogenomics and systems toxicology approaches to understanding adverse outcomes

Toxicogenomics and systems toxicology approaches to understanding adverse outcomes

Cardiac safety evaluation in zebrafish and in silico ADME prediction of cephalosporins with an aminothiazoyl ring at the C-7 position

Systems toxicology approaches have been used as important tools in the drug discovery and medicine quality control processes. The aim of this study was to assess the pharmacokinetic and toxicity properties of cephalosporins with an aminothiazoyl ring at the C-7 position (CATRs). Cardiac toxicity of the compounds was assessed in zebrafish embryos, and it was determined that CATRs disturbed the formation and development of the heart in a dose-dependent manner. Differentially expressed genes (DEGs) related to the heart were also identified by transcriptome analysis, and co-DEGs were obtained in the protein-protein interaction (PPI) network. Several Gene Ontology (GO) terms and pathways that were enriched by DEGs were identified, and the most significantly enriched pathways were adrenergic signaling in cardiomyocytes, cardiac muscle contraction, and vascular smooth muscle contraction. Combined molecular docking results elucidated that cardiac toxicity mainly depends on the mother nucleus structure 7-aminocephalosporanic acid (7-ACA). The predicted absorption, distribution, metabolism and excretion (ADME) profile suggests that there is a modification at the C-3 side chain of 7-ACA that could change the compound distribution in vivo. The 7-ACA mother nucleus is responsible for the CATRs induced cardiac toxicity, and the three DEGs (nppa, adra2c, and tnni1c) may potentially be utilized as novel biomarkers for CATRs. Our results show that zebrafish embryos may be used to reveal the pathways of cardiac toxicity and they play a vital role in drug safety assessments.

3S - Systematic, systemic, and systems biology and toxicology.

SummaryA biological system is more than the sum of its parts – it accomplishes many functions via synergy. Deconstructing the system down to the molecular mechanism level necessitates the complement of reconstructing functions on all levels, i.e., in our conceptualization of biology and its perturbations, our experimental models and computer modelling. Toxicology contains the somewhat arbitrary subclass “systemic toxicities”; however, there is no relevant toxic insult or general disease that is not systemic. At least inflammation and repair are involved that require coordinated signaling mechanisms across the organism. However, the more body components involved, the greater the challenge to recapitulate such toxicities using non-animal models. Here, the shortcomings of current systemic testing and the development of alternative approaches are summarized.We argue that we need a systematic approach to integrating existing knowledge as exemplified by systematic reviews and other evidence-based approaches. Such knowledge can guide us in modelling these systems using bioengineering and virtual computer models, i.e., via systems biology or systems toxicology approaches. Experimental multi-organon-chip and microphysiological systems (MPS) provide a more physiological view of the organism, facilitating more comprehensive coverage of systemic toxicities, i.e., the perturbation on organism level, without using substitute organisms (animals). The next challenge is to establish disease models, i.e., micropathophysiological systems (MPPS), to expand their utility to encompass biomedicine. Combining computational and experimental systems approaches and the challenges of validating them are discussed. The suggested 3S approach promises to leverage 21st century technology and systematic thinking to achieve a paradigm change in studying systemic effects.

Evaluation of the Tobacco Heating System 2.2. Part 7: Systems toxicological assessment of a mentholated version revealed reduced cellular and molecular exposure effects compared with mentholated and non-mentholated cigarette smoke

Modified risk tobacco products (MRTPs) are being developed with the aim of reducing smoking-related health risks. The Tobacco Heating System 2.2 (THS2.2) is a candidate MRTP that uses the heat-not-burn principle. Here, systems toxicology approaches were engaged to assess the respiratory effects of mentholated THS2.2 (THS2.2M) in a 90-day rat inhalation study (OECD test guideline 413). The standard endpoints were complemented by transcriptomics and quantitative proteomics analyses of respiratory nasal epithelium and lung tissue and by lipidomics analysis of lung tissue. The adaptive response of the respiratory nasal epithelium to conventional cigarette smoke (CS) included squamous cell metaplasia and an inflammatory response, with high correspondence between the molecular and histopathological results. In contrast to CS exposure, the adaptive tissue and molecular changes to THS2.2M aerosol exposure were much weaker and were limited mostly to the highest THS2.2M concentration in female rats. In the lung, CS exposure induced an inflammatory response, triggered cellular stress responses, and affected sphingolipid metabolism. These responses were not observed or were much lower after THS2.2M aerosol exposure. Overall, this system toxicology analysis complements and reconfirms the results from classical toxicological endpoints and further suggests potentially reduced health risks of THS2.2M.

Evaluation of the Tobacco Heating System 2.2 (THS2.2). Part 5: microRNA expression from a 90-day rat inhalation study indicates that exposure to THS2.2 aerosol causes reduced effects on lung tissue compared with cigarette smoke

Modified-risk tobacco products (MRTP) are designed to reduce the individual risk of tobacco-related disease as well as population harm compared to smoking cigarettes. Experimental proof of their benefit needs to be provided at multiple levels in research fields. Here, we examined microRNA (miRNA) levels in the lungs of rats exposed to a candidate modified-risk tobacco product, the Tobacco Heating System 2.2 (THS2.2) in a 90-day OECD TG-413 inhalation study. Our aim was to assess the miRNA response to THS2.2 aerosol compared with the response to combustible cigarettes (CC) smoke from the reference cigarette 3R4F. CC smoke exposure, but not THS2.2 aerosol exposure, caused global miRNA downregulation, which may be explained by the interference of CC smoke constituents with the miRNA processing machinery. Upregulation of specific miRNA species, such as miR-146a/b and miR-182, indicated that they are causal elements in the inflammatory response in CC-exposed lungs, but they were reduced after THS2.2 aerosol exposure. Transforming transcriptomic data into protein activity based on corresponding downstream gene expression, we identified potential mechanisms for miR-146a/b and miR-182 that were activated by CC smoke but not by THS2.2 aerosol and possibly involved in the regulation of those miRNAs. The inclusion of miRNA profiling in systems toxicology approaches increases the mechanistic understanding of the complex exposure responses.

Evaluation of the Tobacco Heating System 2.2. Part 4: 90-day OECD 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects compared with cigarette smoke

The objective of the study was to characterize the toxicity from sub-chronic inhalation of test atmospheres from the candidate modified risk tobacco product (MRTP), Tobacco Heating System version 2.2 (THS2.2), and to compare it with that of the 3R4F reference cigarette. A 90-day nose-only inhalation study on Sprague-Dawley rats was performed, combining classical and systems toxicology approaches. Reduction in respiratory minute volume, degree of lung inflammation, and histopathological findings in the respiratory tract organs were significantly less pronounced in THS2.2-exposed groups compared with 3R4F-exposed groups. Transcriptomics data obtained from nasal epithelium and lung parenchyma showed concentration-dependent differential gene expression following 3R4F exposure that was less pronounced in the THS2.2-exposed groups. Molecular network analysis showed that inflammatory processes were the most affected by 3R4F, while the extent of THS2.2 impact was much lower. Most other toxicological endpoints evaluated did not show exposure-related effects. Where findings were observed, the effects were similar in 3R4F- and THS2.2-exposed animals. In summary, toxicological changes observed in the respiratory tract organs of THS2.2 aerosol-exposed rats were much less pronounced than in 3R4F-exposed rats while other toxicological endpoints either showed no exposure-related effects or were comparable to what was observed in the 3R4F-exposed rats.

Evidence-based absorption, distribution, metabolism, excretion (ADME) and its interplay with alternative toxicity methods.

ADME (absorption, distribution, metabolism, elimination) has rapidly evolved over the past two decades, creating a unique interdisciplinary interface between medicinal chemists, biologists, formulators, toxicologists, clinicians, and regulators across industries, but has advanced most rapidly in the pharmaceutical industry. The implementation of ADME profiling of drug candidates, in conjunction with biological efficacy and safety optimization, has dramatically reduced pharmacokinetic drug failures in clinical trials and has become a lingua franca between disciplines that are involved in drug development. This article briefly reviews the basics and current state-of-the-art of ADME and the major lessons from the pharmaceutical industry on its efficient use, points out the importance of defining ADME properties leading to toxicity across industries for safety and toxicity prediction of chemicals, and raises the issues of quality, reliability, and reproducibility of tests and inclusion of ADME under the umbrella of evidence-based toxicology. Increasingly, in vitro results are used to inform ADME assessments and computer modeling. The aspects of kinetics of substances in cellular models themselves, however, are still too often neglected. ADME information will play a critical role in establishing quantitative in vitro to in vivo extrapolations (QIVIVE), integrated testing strategies, and systems toxicology approaches.

Systems Biology in Nanosafety Research

In his famous essay, Lazebnik [1] discussed whether a biologist can fix a broken radio and suggested that applying the ‘biologist approach’ to a broken radio will likely not lead to a successful end, while any engineer or even a trained repairman evidently could fix the radio. In biology, the study of molecules one by one is fine for gathering information on how molecules work, but deciphering biological mechanisms, or the function of biological systems, is a different story. One difference between an engineer and a biologist, according to Lazebnik, lies in the language: in biology, language is oftentimes vague and nonquantitative which limits the possibility of making predictions [1]. In systems biology, a quantitative and predictive language is adopted to describe biological knowledge in order to understand how molecules act together within the network of interactions that makes up a living system. Systems biology, in other words, is the holistic analysis of complex systems. Indeed, systems biology can be defined as the computational and mathematical modeling of complex biological systems, in contrast to the traditional, reductionist approach to biology. Common technology platforms that are deployed to obtain complex datasets are transcriptomics, proteomics, metabolomics and epigenomics. However, systems biology should not be seen merely as the generation of lists of genes, proteins or metabolites using such omics platforms; the objective is to exploit these data and to develop quantitative models that describe the biological system and its response to individual perturbations (see [2] for an excellent introduction to this subject). Furthermore, systems biology can be seen as an iterative interplay between discoveryand hypothesis-driven science: “global observations (discoveries) are matched against model predictions (hypotheses) in an iterative manner, leading to the formation of new models, new predictions, and new experiments to test them” [2]. Systems toxicology, in turn, has been described as “the integration of classical toxicology with quantitative analysis of large networks of molecular and functional changes occurring across multiple levels of biological organization”, and it has been suggested that its application could be part of a new and improved risk assessment [3]. This is nothing less than a paradigm shift in the way that toxicology is conducted. Importantly, systems toxicology – and systems biology in general – relies heavily on computational approaches to manage, analyze and interpret the data generated by large-scale experiments, and computer databases are thus an integral feature of this approach [2]. To this end, commonly accepted repositories and software environments are crucially important [4]. This does not mean that the introduction of systems toxicology approaches is breaking the rice bowl of the classically trained toxicologist. Instead, the ‘new’ toxicology is Systems biology in nanosafety research

Arsenic induces diabetic effects through beta-cell dysfunction and increased gluconeogenesis in mice.

Arsenic as a potential risk factor for type 2 diabetes has been received attention recently. However, the roles of arsenic on development of diabetes are unclear. In this study, we compared the influences of inorganic arsenic (iAs) on normal and diabetic mice by systems toxicology approaches. Although iAs exposure did not change glucose tolerance in normal mice, it caused the pancreatic β-cell dysfunction and increased gluconeogenesis and oxidative damages in liver. However, iAs exposure worsened the glucose tolerance in diabetic mice, which might be due to increased gluconeogenesis and impairment of pancreatic β-cell function. It is interesting that iAs exposure could improve the insulin sensitivity based on the insulin tolerance testing by the activation of glucose uptake-related genes and enzymes in normal and diabetic individuals. Our data suggested that iAs exposure could cause pre-diabetic effects by altering the lipid metabolism, gluconeogenesis and insulin secretion in normal individual, and worsen diabetic effects in diabetes individual by these processes. Insulin resistance might be not the reason of diabetic effects caused by iAs, indicating that mechanism of the diabetogenic effects of iAs exposure is different from the mechanism associated with traditional risk factors (such as obesity)-reduced type 2 diabetes.

Systems toxicology approaches enable mechanistic comparison of spontaneous and cigarette smoke-related lung tumor development in the A/J mouse model.

The A/J mouse is highly susceptible to lung tumor induction and has been widely used as a screening model in carcinogenicity testing and chemoprevention studies. However, the A/J mouse model has several disadvantages. Most notably, it develops lung tumors spontaneously. Moreover, there is a considerable gap in our understanding of the underlying mechanisms of pulmonary chemical carcinogenesis in the A/J mouse. Therefore, we examined the differences between spontaneous and cigarette smoke-related lung tumors in the A/J mouse model using mRNA and microRNA (miRNA) profiling. Male A/J mice were exposed whole-body to mainstream cigarette smoke (MS) for 18 months. Gene expression interaction term analysis of lung tumors and surrounding non-tumorous parenchyma samples from animals that were exposed to either 300 mg/m3 MS or sham-exposed to fresh air indicated significant differential expression of 296 genes. Ingenuity Pathway Analysis® (IPA®) indicated an overall suppression of the humoral immune response, which was accompanied by a disruption of sphingolipid and glycosaminoglycan metabolism and a deregulation of potentially oncogenic miRNA in tumors of MS-exposed A/J mice. Thus, we propose that MS exposure leads to severe perturbations in pathways essential for tumor recognition by the immune system, thereby potentiating the ability of tumor cells to escape from immune surveillance. Further, exposure to MS appeared to affect expression of miRNA, which have previously been implicated in carcinogenesis and are thought to contribute to tumor progression. Finally, we identified a 50-gene expression signature and show its utility in distinguishing between cigarette smoke-related and spontaneous lung tumors.

Systems Toxicology: From Basic Research to Risk Assessment

Systems Toxicology is the integration of classical toxicology with quantitative analysis of large networks of molecular and functional changes occurring across multiple levels of biological organization. Society demands increasingly close scrutiny of the potential health risks associated with exposure to chemicals present in our everyday life, leading to an increasing need for more predictive and accurate risk-assessment approaches. Developing such approaches requires a detailed mechanistic understanding of the ways in which xenobiotic substances perturb biological systems and lead to adverse outcomes. Thus, Systems Toxicology approaches offer modern strategies for gaining such mechanistic knowledge by combining advanced analytical and computational tools. Furthermore, Systems Toxicology is a means for the identification and application of biomarkers for improved safety assessments. In Systems Toxicology, quantitative systems-wide molecular changes in the context of an exposure are measured, and a causal chain of molecular events linking exposures with adverse outcomes (i.e., functional and apical end points) is deciphered. Mathematical models are then built to describe these processes in a quantitative manner. The integrated data analysis leads to the identification of how biological networks are perturbed by the exposure and enables the development of predictive mathematical models of toxicological processes. This perspective integrates current knowledge regarding bioanalytical approaches, computational analysis, and the potential for improved risk assessment.

Integrated systems toxicology approaches identified the possible involvement of ABC transporters pathway in erythromycin estolate-induced liver injury in rat

Erythromycin estolate (EE), a macrolide antibiotic, has caused hepatotoxicity both in human and experimental animals. The objective of this study was to integrate general toxicology, transcriptomics, and metabonomics approaches to determine the mechanisms of EE-induced liver injury. Histopathological examinations unveiled dose-dependent hydropicdegenerationof hepatocytes after EE administration. Further biochemical analysis of treated rats confirmed that cholestasis and oxidative stress were induced by EE treatments. Microarray analysis of the livers from EE-treated rats showed that differentially expressed genes were enriched in the ABC transporters, cell cycle, and p53 signaling pathways. Metabonomics analysis revealed that EE exposure could lead to disturbances in energy metabolism, amino acid metabolism, lipid metabolism, and nucleotide metabolism, which may be attributable to EE toxicological effects on the liver through oxidative stress. 5-Oxoproline may be used as a biomarker of EE-induced liver injury. More importantly, the integrated analysis of transcriptomics and metabonomics datasets demonstrated that the induction of ABC transporters pathway severed as an anti-cholestatic adaptive mechanism in EE-induced cholestasis. In addition, EE-induced liver injury was also related to alteration in glycogen and sucrose metabolism, arachidonic acid metabolism, and linoleic acid metabolism pathways.

Systems toxicology approaches for understanding the joint effects of environmental chemical mixtures

Environmental mixtures of chemicals constitute a prevalent issue in ecotoxicology and the development of new methods to reduce the uncertainties associated with their ecological risk assessment is a critical research need. Historically, a number of models have been explored to predict the potential combined effects of chemicals on species. These models, especially concentration addition and the independent action, have been applied to a number of mixtures. While often providing a good prediction of joint effect, there are cases where these models can have limitations: notably in cases where there are interactions for which they fail to adequately predict joint effects. To support the better mechanistic understanding of interactions in mixture toxicology a framework to support experimental studies to investigate the basis of observed interactions is proposed. The conceptual framework is derived from the extension of a three stage scheme which has previously been applied to understand chemical bioavailability. The framework considers that interactions in mixtures result from processes related to 1) the speciation, binding and transport of chemicals in the exposure medium (external exposure); 2) the adsorption, distribution, metabolism and excretion of chemicals within the organisms (toxicokinetics); 3) associations governing the binding and toxicity of the chemical(s) at the target site (toxicodynamics). The current state of the art in (eco)toxicology in relation to investigation of the mechanisms of interactions between chemicals is discussed with particular emphasis towards the multi-disciplinary tools and techniques within environmental chemistry; toxicology; biochemistry and systems biology that can be used to address such effects.