Anne S. Kienhuis

Systems toxicology: applications of toxicogenomics, transcriptomics, proteomics and metabolomics in toxicology

Toxicogenomics can facilitate the identification and characterization of toxicity, as illustrated in this review. Toxicogenomics, the application of the functional genomics technologies (transcriptomics, proteomics and metabolomics) in toxicology enables the study of adverse effects of xenobiotic substances in relation to structure and activity of the genome. The advantages and limitations of the different technologies are evaluated, and the prospects for integration of the technologies into a systems biology or systems toxicology approach are discussed. Applications of toxicogenomics in various laboratories around the world show that the crucial steps and sequence of events at the molecular level can be studied to provide detailed insights into mechanisms of toxic action. Toxicogenomics allowed for more sensitive and earlier detection of adverse effects in (animal) toxicity studies. Furthermore, the effects of exposure to mixtures could be studied in more detail. This review argues that in the (near) future, human health risk assessment will truly benefit from toxicogenomics (systems toxicology).

Assessing the Metabolic Competence of Sandwich-Cultured Mouse Primary Hepatocytes

Primary human and rat hepatocyte cultures are well established in vitro systems used in toxicological studies. However, whereas transgenic mouse models provide an opportunity for studying mechanisms of toxicity, mouse primary hepatocyte cultures are less well described. The potential usefulness of a mouse hepatocyte-based in vitro model was assessed in this study by investigating time-dependent competence for xenobiotic metabolism and gene expression profiles. Primary mouse hepatocytes, isolated using two-step collagenase perfusion, were cultured in a collagen sandwich configuration. Gene expression profiles and the activities of various cytochrome P450 (P450) enzymes were determined after 0, 42, and 90 h in culture. Principal component analysis of gene expression profiles shows that replicates per time point are similar. Gene expression levels of most phase I biotransformation enzymes decrease to approximately 69 and 57% of the original levels at 42 and 90 h, respectively, whereas enzyme activities for most of the studied P450s decrease to 59 and 34%. The decrease for phase II gene expression is only to 96 and 92% of the original levels at 42 and 90 h, respectively. Pathway analysis reveals initial effects at the level of proteins, external signaling pathways, and energy production. Later effects are observed for transcription, translation, membranes, and cell cycle-related gene sets. These results indicate that the sandwich-cultured primary mouse hepatocyte system is robust and seems to maintain its metabolic competence better than that of the rat hepatocyte system.

Exploring the zebrafish embryo as an alternative model for the evaluation of liver toxicity by histopathology and expression profiling

The whole zebrafish embryo model (ZFE) has proven its applicability in developmental toxicity testing. Since functional hepatocytes are already present from 36 h post fertilization onwards, whole ZFE have been proposed as an attractive alternative to mammalian in vivo models in hepatotoxicity testing. The goal of the present study is to further underpin the applicability of whole ZFE for hepatotoxicity testing by combining histopathology and next-generation sequencing-based gene expression profiling. To this aim, whole ZFE and adult zebrafish were exposed to a set of hepatotoxic reference compounds. Histopathology revealed compound and life-stage-specific effects indicative of toxic injury in livers of whole ZFE and adult zebrafish. Next-generation sequencing (NGS) was used to compare transcript profiles in pooled individual RNA samples of whole ZFE and livers of adult zebrafish. This revealed that hepatotoxicity-associated expression can be detected beyond the overall transcription noise in the whole embryo. In situ hybridization verified liver specificity of selected highly expressed markers in whole ZFE. Finally, cyclosporine A (CsA) was used as an illustrative case to support applicability of ZFE in hepatotoxicity testing by comparing CsA-induced gene expression between ZFE, in vivo mouse liver and HepaRG cells on the levels of single genes, pathways and transcription factors. While there was no clear overlap on single gene level between the whole ZFE and in vivo mouse liver, strong similarities were observed between whole ZFE and in vivo mouse liver in regulated pathways related to hepatotoxicity, as well as in relevant overrepresented transcription factors. In conclusion, both the use of NGS of pooled RNA extracts analysis combined with histopathology and traditional microarray in single case showed the potential to detect liver-related genes and processes within the transcriptome of a whole zebrafish embryo. This supports the applicability of the whole ZFE model for compound-induced hepatotoxicity screening.

Parallelogram approach using rat-human in vitro and rat in vivo toxicogenomics predicts acetaminophen-induced hepatotoxicity in humans.

The frequent use of rodent hepatic in vitro systems in pharmacological and toxicological investigations challenges extrapolation of in vitro results to the situation in vivo and interspecies extrapolation from rodents to humans. The toxicogenomics approach may aid in evaluating relevance of these model systems for human risk assessment by direct comparison of toxicant-induced gene expression profiles and infers mechanisms between several systems. In the present study, acetaminophen (APAP) was used as a model compound to compare gene expression responses between rat and human using in vitro cellular models, hepatocytes, and between rat in vitro and in vivo. Comparison at the level of modulated biochemical pathways and biological processes rather than at that of individual genes appears preferable as it increases the overlap between various systems. Pathway analysis by T-profiler revealed similar biochemical pathways and biological processes repressed in rat and human hepatocytes in vitro, as well as in rat liver in vitro and in vivo. Repressed pathways comprised energy-consuming biochemical pathways, mitochondrial function, and oxidoreductase activity. The present study is the first that used a toxicogenomics-based parallelogram approach, extrapolating in vitro to in vivo and interspecies, to reveal relevant mechanisms indicative of APAP-induced liver toxicity in humans in vivo.

Application of toxicogenomics in hepatic systems toxicology for risk assessment: Acetaminophen as a case study

Hepatic systems toxicology is the integrative analysis of toxicogenomic technologies, e.g., transcriptomics, proteomics, and metabolomics, in combination with traditional toxicology measures to improve the understanding of mechanisms of hepatotoxic action. Hepatic toxicology studies that have employed toxicogenomic technologies to date have already provided a proof of principle for the value of hepatic systems toxicology in hazard identification. In the present review, acetaminophen is used as a model compound to discuss the application of toxicogenomics in hepatic systems toxicology for its potential role in the risk assessment process, to progress from hazard identification towards hazard characterization. The toxicogenomics-based parallelogram is used to identify current achievements and limitations of acetaminophen toxicogenomic in vivo and in vitro studies for in vitro-to-in vivo and interspecies comparisons, with the ultimate aim to extrapolate animal studies to humans in vivo. This article provides a model for comparison of more species and more in vitro models enhancing the robustness of common toxicogenomic responses and their relevance to human risk assessment. To progress to quantitative dose-response analysis needed for hazard characterization, in hepatic systems toxicology studies, generation of toxicogenomic data of multiple doses/concentrations and time points is required. Newly developed bioinformatics tools for quantitative analysis of toxicogenomic data can aid in the elucidation of dose-responsive effects. The challenge herein is to assess which toxicogenomic responses are relevant for induction of the apical effect and whether perturbations are sufficient for the induction of downstream events, eventually causing toxicity.

A toxicogenomics-based parallelogram approach to evaluate the relevance of coumarin-induced responses in primary human hepatocytes in vitro for humans in vivo.

A compound for which marked species differences have been reported in laboratory animals and humans is coumarin. In rats, metabolites of coumarin are highly toxic, whereas in humans, the compound is mainly metabolized to non-toxic metabolites. In the present study, a toxicogenomics-based parallelogram approach was used to compare effects of coumarin on gene expression in human hepatocytes relevant for the situation in vivo. To this purpose, gene expression profiling was performed on human hepatocytes treated with coumarin in a pharmacological relevant and proposed toxic concentration and results were compared to a previously performed coumarin in vivo and in vitro rat toxicogenomics study. No cytotoxicity was observed in human hepatocytes at both concentrations, whereas rats showed clear toxic effects in vitro as well as in vivo. In all three systems, coumarin affected genes involved in the blood coagulation pathway; this indicates relevant responses in cases of human exposure. However, no pathways and processes related to hepatotoxicity in rats were observed in human hepatocytes. Still, repression of energy-consuming biochemical pathways and impairment of mitochondrial function were observed in human hepatocytes treated with the highest concentration of coumarin, possibly indicating toxicity. In conclusion, although species differences in response to coumarin are evident in the present results, the toxicogenomics-based parallelogram approach enables clear discrimination between pharmacological responses at pharmacological doses and proposed toxic responses at high (toxic) doses relevant for humans in vivo.

Cyclosporine A treated in vitro models induce cholestasis response through comparison of phenotype-directed gene expression analysis of in vivo Cyclosporine A-induced cholestasis.

In vitro models for hepatotoxicity testing are a necessity for advancement of toxicological research. Assessing the in vitro response requires in vivo validated gene sets reflective of the hepatotoxic phenotype. Cholestasis, the impairment of bile flow, is induced in C57BL/6J mice treated with cyclosporine A (CsA) to identify phenotype reflective gene sets. CsA treatment through oral gavage for 25 days induced cholestasis, as confirmed by histopathology and serum chemistry. Over 1, 4, and 11 days of CsA exposure gradual increases in serum markers were correlated to gene expression. This phenotype-directed analysis identified gene sets specific to the onset and progression of cholestasis, such as PPAR related processes and drug metabolism, by circumventing other effects of CsA, such as immunosuppression, found in dose*time group analysis. In vivo gene sets are enriched in publicly available data sets of CsA-treated HepaRG and primary mouse hepatocytes. However, genes identified within these gene sets did not overlap between in vivo and in vitro. In vitro regulated genes represent the initial response to cholestasis, whereas in vivo genes represent the later adaptive response. We conclude that the applicability of in vitro models for hepatotoxicity testing fully depends on a solid in vivo phenotype anchored analysis.

Fish embryo toxicity test, threshold approach, and moribund as approaches to implement 3R principles to the acute fish toxicity test

The acute fish toxicity test (AFT) is requested by EU legal frameworks for hazard classification and risk assessment. AFT is one of the few regulatory required tests using death as an endpoint. This paper reviews efforts made to reduce, refine and replace (3Rs) AFT. We make an inventory of information requirements for AFT, summarize studies on 3Rs of AFT and give recommendations. The fish embryo toxicity test (FET) is proposed as a replacement of AFT and analyses have focused on two aspects: assessing the capacity of FET in predicting AFT and defining the applicability domain of FET. Six comparison studies have consistently shown a strong correlation of FET and AFT. In contrast, the applicability domain of FET has not yet been fully defined. FET has not yet been accepted as a replacement of AFT by any EU legal frameworks to fulfill information requirements because FET is insensitive to some chemicals. It is recommended that the outlier chemicals that do not correlate between FET and AFT should be further investigated. When necessary, additional FET data should be generated. Another effort to reduce and refine AFT is incorporation of FET into the threshold approach. Furthermore, moribund as an endpoint of fish death has been introduced in revising AFT guideline to reduce the duration of suffering for refinement. This endpoint, however, needs further work on the link of moribund and death. Global regulatory acceptance of the moribund endpoint would be critical for this development.

Chapter 7:Dissecting Modes of Action of Non-genotoxic Carcinogens

In safety assessments of chemicals, genotoxic and carcinogenic potential is considered one of the basic requirements. Overall, regulatory guidelines for carcinogenicity testing focus on genotoxic potential, because the majority of carcinogens induce tumors by inflicting irreversible DNA damage in critical genes. However, there is a group of carcinogens that induce cancer via non-genotoxic mechanisms. Apart from the carcinogenicity bioassay, suitable assays to detect these chemicals hardly exist. This is mainly due to the diversity in mode of action of non-genotoxic carcinogens. We employed toxicogenomics in primary mouse hepatocytes to categorize non-genotoxic carcinogens according to their overlap in transcriptional profile. This approach, based on a limited set of significantly regulated genes, may be further improved by using a concentration range instead of a single concentration per chemical. We explored this by performing a case study using cyclosporine A and tacrolimus. Testing multiple concentrations strongly enhanced our approach to detect modes of actions of non-genotoxic carcinogens. We therefore propose to include a concentration range when using in vitro toxicogenomics approaches to detect non-genotoxic carcinogens. This approach is a promising tool for future safety assessments, since its applicability is not necessarily limited to carcinogens, but may comprise environmental and pharmaceutical chemicals in general.

Workshop on acceleration of the validation and regulatory acceptance of alternative methods and implementation of testing strategies

This report describes the proceedings of the BfR-RIVM workshop on validation of alternative methods which was held 23 and 24 March 2017 in Berlin, Germany. Stakeholders from governmental agencies, regulatory authorities, universities, industry and the OECD were invited to discuss current problems concerning the regulatory acceptance and implementation of alternative test methods and testing strategies, with the aim to develop feasible solutions. Classical validation of alternative methods usually involves one to one comparison with the gold standard animal study. This approach suffers from the reductionist nature of an alternative test as compared to the animal study as well as from the animal study being considered as the gold standard. Modern approaches combine individual alternatives into testing strategies, for which integrated and defined approaches are emerging at OECD. Furthermore, progress in mechanistic toxicology, e.g. through the adverse outcome pathway approach, and in computational systems toxicology allows integration of alternative test battery results into toxicity predictions that are more fine-tuned to the human situation. The road towards transition to a mechanistically-based human-focused hazard and risk assessment of chemicals requires an open mind towards stepping away from the animal study as the gold standard and defining human biologically based regulatory requirements for human hazard and risk assessment.