Perspectives of future lung toxicology studies using human pluripotent stem cells.

The absence of in vitro platforms for human pulmonary toxicology studies is becoming an increasingly serious concern. The respiratory system has a dynamic mechanical structure that extends from the airways to the alveolar region. In addition, the epithelial, endothelial, stromal, and immune cells are highly organized in each region and interact with each other to function synergistically. These cells of varied lineage, particularly epithelial cells, have been difficult to use for long-term culture in vitro, thus limiting the development of useful experimental tools. This limitation has set a large distance between the bench and the bedside for analyzing the pathogenic mechanisms, the efficacy of candidate therapeutic agents, and the toxicity of compounds. Several researchers have proposed solutions to these problems by reporting on methods for generating human lung epithelial cells derived from pluripotent stem cells (PSCs). Moreover, the use of organoid culture, organ-on-a-chip, and material-based techniques have enabled the maintenance of functional PSC-derived lung epithelial cells as well as primary cells. The aforementioned technological advances have facilitated the in vitro recapitulation of genetic lung diseases and the detection of ameliorating or worsening effects of genetic and chemical interventions, thus indicating the future possibility of more sophisticated preclinical compound assessments in vitro. In this review, we will update the recent advances in lung cell culture methods, principally focusing on human PSC-derived lung epithelial organoid culture systems with the hope of their future application in toxicology studies.

Vision of a near future: Bridging the human health-environment divide. Toward an integrated strategy to understand mechanisms across species for chemical safety assessment.

There is a growing recognition that application of mechanistic approaches to understand cross-species shared molecular targets and pathway conservation in the context of hazard characterization, provide significant opportunities in risk assessment (RA) for both human health and environmental safety. Specifically, it has been recognized that a more comprehensive and reliable understanding of similarities and differences in biological pathways across a variety of species will better enable cross-species extrapolation of potential adverse toxicological effects. Ultimately, this would also advance the generation and use of mechanistic data for both human health and environmental RA. A workshop brought together representatives from industry, academia and government to discuss how to improve the use of existing data, and to generate new NAMs data to derive better mechanistic understanding between humans and environmentally-relevant species, ultimately resulting in holistic chemical safety decisions. Thanks to a thorough dialogue among all participants, key challenges, current gaps and research needs were identified, and potential solutions proposed. This discussion highlighted the common objective to progress toward more predictive, mechanistically based, data-driven and animal-free chemical safety assessments. Overall, the participants recognized that there is no single approach which would provide all the answers for bridging the gap between mechanism-based human health and environmental RA, but acknowledged we now have the incentive, tools and data availability to address this concept, maximizing the potential for improvements in both human health and environmental RA.

Advancing internal exposure and physiologically-based toxicokinetic modeling for 21st-century risk assessments.

Scientifically sound, risk-informed evaluation of chemicals is essential to protecting public health. Systematically leveraging information from exposure, toxicology, and epidemiology studies can provide a holistic understanding of how real-world exposure to chemicals may impact the health of populations, including sensitive and vulnerable individuals and life-stages. Increasingly, public health policy makers are employing toxicokinetic (TK) modeling tools to integrate these data streams and predict potential human health impact. Development of a suite of tools for predicting internal exposure, including physiologically-based toxicokinetic (PBTK) models, is being driven by needs to address large numbers of data-poor chemicals efficiently, translate bioactivity, and mechanistic information from new in vitro test systems, and integrate multiple lines of evidence to enable scientifically sound, risk-informed decisions. New modeling approaches are being designed "fit for purpose" to inform specific decision contexts, with applications ranging from rapid screening of hundreds of chemicals, to improved prediction of risks during sensitive stages of development. New data are being generated experimentally and computationally to support these models. Progress to meet the demand for internal exposure and PBTK modeling tools will require transparent publication of models and data to build credibility in results, as well as opportunities to partner with decision makers to evaluate and build confidence in use of these for improved decisions that promote safe use of chemicals.

Optimizing drug discovery by Investigative Toxicology: Current and future trends.

Investigative Toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, Investigative Toxicology has contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adhering to the 3R (Replacement, Reduction and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, Investigative Toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of Investigative Toxicology is improving preclinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded due to the use of inappropriate animal models. Progress in Investigative Toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an "Investigative Toxicology Think-Tank", which aimed to enhance the interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights Investigative Toxicology's position by identifying key challenges and perspectives.

SEURAT-1 liver gold reference compounds: a mechanism-based review.

There is an urgent need for the development of alternative methods to replace animal testing for the prediction of repeat dose chemical toxicity. To address this need, the European Commission and Cosmetics Europe have jointly funded a research program for 'Safety Evaluation Ultimately Replacing Animal Testing.' The goal of this program was the development of in vitro cellular systems and associated computational capabilities for the prediction of hepatic, cardiac, renal, neuronal, muscle, and skin toxicities. An essential component of this effort is the choice of appropriate reference compounds that can be used in the development and validation of assays. In this review, we focus on the selection of reference compounds for liver pathologies in the broad categories of cytotoxicity and lipid disorders. Mitochondrial impairment, oxidative stress, and apoptosis are considered under the category of cytotoxicity, while steatosis, cholestasis, and phospholipidosis are considered under the category of lipid dysregulation. We focused on four compound classes capable of initiating such events, i.e., chemically reactive compounds, compounds with specific cellular targets, compounds that modulate lipid regulatory networks, and compounds that disrupt the plasma membrane. We describe the molecular mechanisms of these compounds and the cellular response networks which they elicit. This information will be helpful to both improve our understanding of mode of action and help in the selection of appropriate mechanistic biomarkers, allowing us to progress the development of animal-free models with improved predictivity to the human situation.

Innovative non-animal testing strategies for reproductive toxicology: the contribution of Italian partners within the EU project ReProTect.

Reproductive toxicity, with its many targets and mechanisms, is a complex area of toxicology; thus, the screening and identification of reproductive toxicants is a main scientific challenge for the safety assessment of chemicals, including the European Regulation on Chemicals (REACH). Regulatory agencies recommend the implementation of the 3Rs principle (refinement, reduction, replacement) as well as of intelligent testing strategies, through the development of in vitro methods and the use of mechanistic information in the hazard identification and characterization steps of the risk assessment process. The EU Integrated Project ReProTect (6th Framework Programme) implemented an array of in vitro tests to study different building blocks of the mammalian reproductive cycle: methodological developments and results on male and female germ cells, prostate and placenta are presented.

The role of the toxicologic pathologist in the biopharmaceutical industry.

Toxicologic pathologists contribute significantly to the development of new biopharmaceuticals, yet there is often a lack of awareness of this specialized role. As the members of multidisciplinary teams, toxicologic pathologists participate in all aspects of the drug development process. This review is part of an initiative by the Society of Toxicologic Pathology to educate scientists about toxicologic pathology and to attract junior scientists, veterinary students, and veterinarians into the field. We describe the role of toxicologic pathologists in identifying candidate agents, elucidating bioactive pathways, and evaluating efficacy and toxicity in preclinical animal models. Educational and specialized training requirements and the challenges of working in a global environment are discussed. The biopharmaceutical industry provides diverse, challenging, and rewarding career opportunities in toxicologic pathology. We hope that this review promotes understanding of the important role the toxicologic pathologist plays in drug development and encourages exploration of an important career option.

State-of-the-art genomics approaches in toxicology.

Genomics may be an effective tool in decreasing the lengthy drug development process and reducing compound attrition. It can generate specific gene expression profiles induced by chemicals that can be linked to dose and response. Toxicogenomics can identify sensitive biomarkers of early deleterious effects, distinguish genotoxic from non-genotoxic carcinogens and can provide information on the mechanism of action. It can help bridge in vitro to in vivo findings and provide context for preclinical data and thus address human health risks. Issues and shortcomings that still need to be resolved or improved for efficient incorporation of genomics in drug development and environmental toxicology research include data analysis, data interpretation tools and accessible data repositories. In addition, implementation of toxicogenomics in early screening or drug discovery phases and effective use of this information by project teams remains a challenge.

Characterizing and predicting carcinogenicity and mode of action using conventional and toxicogenomics methods.

The results of predictive toxicogenomics investigations over the past 6 years reviewed in this report have shed new light on the potential of molecular expression analysis to more properly classify both genotoxic and nongenotoxic carcinogens and to predict the carcinogenicity of untested chemicals. Predictive toxicogenomics uses global molecular expression data resulting from genomic perturbation (e.g., transcription or gene expression profiles) to predict a toxicological outcome, such as carcinogenicity. The classification of carcinogens has become an essential and highly debatable component of cancer risk assessment largely because of the default assumptions that drive regulatory decision-making regarding the presumed linearity of the dose-response curve for genotoxic carcinogens. Nongenotoxic mechanisms of carcinogenesis complicate the well-established relationship between genotoxicity and carcinogenicity and challenge the interpretation of the results of rodent carcinogenicity studies in terms of their relevance to humans. Although the number of presumed nongenotoxic rodent carcinogens has dramatically increased over the past two decades, the fact remains that more than 90% of the known human carcinogens are detected in conventional short-term tests for genotoxicity and induce tumors at multiple sites in rodents. In toxicogenomics studies, a strong DNA damage response at the gene expression level suggests direct DNA modification whereas increased expression of genes involved in cell cycle progression is more characteristic of the indirect-acting agents such as those that induce oxidative stress. Metabolism genes are prominently represented among gene expression profiles that discriminate nongenotoxic modes of action (e.g., cytotoxicity and regenerative proliferation, xenobiotic receptor agonists, peroxisome proliferator-activated receptors, or hormonal-mediated processes). The evidence accumulated to date suggests that gene expression profiles reflect underlying modes or mechanisms of action, such that they will be useful in the prediction of chemical carcinogenicity, especially in conjunction with conventional short-term tests for gene mutation, chromosomal aberration and aneuploidy.

Necesidades docentes en Evaluación y Prevención del Riesgo Tóxico: de la Toxicología Experimental a la Reguladora / Educational needs in Toxic Prevention and Risk Assessment: From experimental to Regulatory Toxicology

Se revisan las necesidades formativas para una serie de raas aplicadas de la toxicología derivadas de las áreas fundamentales mecanística y de evaluación toxicológica, que comprenden fundamentalmente la toxicología de sistemas, la evaluación del riesgo y la toxicología reguladora. Son materias necesarias para el desempeño profesional en los organismos nacionales, autonómicos y locales responsables de la regulación e inspección de la comercialización y uso de compuestos industriales, medicamentos, productos cosméticos, alimentarios o fitosanitarios, o de contaminantes ambientales o laborales, así como en industrias y consultorías. Se han identificado más de 25 actividades científicas de gran relevancia realizadas en España desde 1995, con una media de 1,6 actividades por año. Los principales grados de las ciencias de la salud incluyen una asignatura troncal de Toxicología, que puede aparecer como optativa en otros, generalmente de 6 créditos ECTS, con un programa muy uniforme, que incluye entre sus descriptores la estimación del riesgo, la evaluación toxicológica, el empleo de métodos alternativos y las bases de la toxicología reguladora. En relación con los estudios de postgrado, se identifican másteres con una gran variedad de contenidos y enfoques, que en algunos casos cubren un abanico muy amplio de disciplinas, incluyendo las toxicológicas, así como otros que cubren las áreas específicas de la toxicología. De la revisión transversal de los programas docentes de pregrado, postgrado y doctorado en España, se deduce la paulatina adaptación al EEES y la adecuada inclusión en los mismos de materias sobre evaluación experimental de la toxicidad, prevención del riesgo tóxico y toxicología reguladora (AU)

The training needs for a range of applied toxicology branches arising from the fundamental areas of mechanistic and toxicological evaluation, which mainly comprise of systems toxicology , risk assessment and regulatory toxicology , are reviewed. Thet are necessary for professional performance in national, regional and local authorities responsible for regulation and inspection of the marketing and use of industrial chemicals, pharmaceuticals, cosmetics, food or plant protection products or environmental or occupational pollutants, as well as industries and consultancies. There are more than 25 highly relevant scientific activities carried out in Spain since 1995, with an average of 1.6 activities per year . The main bachelor's degrees of the health sciences include a very uniform obligatory subject of T oxicology , which may appear as an option in others, usually with 6 ECTS credits, which includes among its descriptors risk e stimation assessment toxicology, the use of alternative methods and the basis for regulatory toxicology. In relation to postgraduate studies, masters were identified with a variety of topics and approaches, which in some cases cover a very wide range of disciplines, including toxicology and other covering in depth the specific toxicologic areas. From the transversal review of the teaching programs at under graduate, graduate and doctoral degrees in Spain, it was deduced a gradual adaptation to the EHEA and the appropriate inclusion of subjetcs on experimental evaluation of toxicity , toxic hazard prevention and regulatory toxicology (AU)

Biomarkers in translation; past, present and future.

The search for biomarkers and their evaluation by scientists and clinicians is of paramount importance if we are going to improve health care. In this paper we discuss the history of the discovery of biomarkers for renal and cardiac injury. We also summarize the use of biomarkers in preclinical evaluation in experimental animals and in patients to help diagnose or monitor a disease, predict outcome or to evaluate a therapeutic intervention. The need for validated biomarkers of tissue injury or disease that must be easy to analyse rapidly, preferably at the bedside, is essential if clinical decision making is to be optimally affected by their use.

Potential applications of human embryonic stem cells.

Advances in reproductive technologies provided opportunity for scientists to be able to grow human embryos in vitro for more than two decades. Skills and knowledge derived from in vitro fertilization and in vitro culture of mammalian embryos opened the chance for scientists to develop the strategies to derive embryonic stem cell lines from mammalian and human embryos. This achievement has initiated a new era in the fields of biotechnology, pharmacology, basic scientific research, and cell-based medicine. To date, scientists have made some progress in optimizing regimens in deriving ES cell lines from human embryos but much more research and development are still required especially in the aspect of directing stem cells into the specific cells of potential clinical use. Collaboration among clinicians and scientists from diverse fields, together with the public awareness of how useful this technology could be to modern medicine, will result in the accumulation of knowledge in this field and, in the near future, a progress in cell-based therapy.

Estimation of selenium concentration in shallow groundwater in alluvial fan area in Tsukui, Central Japan.

Total selenium (Se) and water-soluble Se in soil, and Se in a shallow groundwater were hydrogeochemically researched in an alluvial fan area in Tsukui, Central Japan. The water-soluble Se was estimated at average level of 2.6 +/- 1.2 microg Se kg(-1) dry soil (+/- SD, n = 25), showing less than 1% of the total Se (349-508 microg Se kg(-1) dry soil) in soil. The monthly Se concentration in groundwater was average 2.2 microg,L(-1), ranging 1.6-2.4 microg,L(-1) during 2001-2003. The Se in groundwater significantly decreased with increasing groundwater level after rainfall. This result indicated that Se-bearing water percolated with relatively low Se concentration through the soil layer. According to our prediction model of linear regression curve on the observation data, Se concentration in the groundwater was estimated to be increasing with the very low rate of 4.35 x 10(-3) microg Se L(-1),yr(-1). The hydrogeochemical research and the result of the prediction model showed that any explosive increase of Se will hardly occur in this groundwater without an anthropogenic Se contamination.

Future of toxicology--mechanisms of toxicity and drug safety: where do we go from here?

Recent high-profile drug withdrawals increase the pressure on regulators and the pharmaceutical industry to improve preclinical safety testing. Understanding mechanisms of drug toxicity is an essential step toward improving drug safety testing by providing the basis for mechanism-based risk assessments. Nonetheless, despite several decades of research on mechanisms of drug-induced toxicity and the application of various new technologies to preclinical safety assessment, the overall impact on preclinical safety testing has been modest. Assessing the risk of exposing humans to new drug candidates still depends on preclinical testing in animals, which in many, but not all cases, predicts outcomes in humans accurately. The following offers a perspective on the challenges and opportunities facing efforts to improve preclinical safety testing and outlines gaps and needs that must be addressed. A case is built for focusing solutions on defined problems within the current safety testing paradigm rather than imposing wholesale change. Targets for application of new technologies, including in silico screening, biomarkers, surrogate assays and 'omic technologies, are outlined. Improving drug safety testing will depend on improving the application of mechanism-based risk assessment but will also require improving public and private collaborations in order to focus research regarding the mechanism of drug-induced toxicity on the most important problems.

How omics technologies can contribute to the '3R' principles by introducing new strategies in animal testing.

In Europe, in light of ethical, political and commercial pressure, every effort should be made to replace animals with alternatives (e.g. in vitro models), to reduce the number of animals used in experiments to a minimum and to refine current testing strategies in a way that ensures animals undergo minimum pain and distress. Methods currently used in toxicology for mandatory safety tests rely heavily on the dosing of animals, followed by the detection and pathological evaluation of manifested toxic lesions. Through the integration of so-called 'omics' technologies, a global analysis of treatment-related changes on the molecular level becomes feasible and therefore might provide a means for predicting toxicity before classical toxicological endpoints. This Opinion article summarizes the key features of pushing the '3R' principles in animal testing, discusses the possible impact on safety testing in toxicology and describes the potential of using omics technologies for improved toxicity prediction to meet ethical, political and commercial expectations.

Informed toxicity assessment in drug discovery: systems-based toxicology.

Technological advances in the biological, chemical and in silico sciences have transformed many scientific disciplines, including toxicology. A vast new palate of toxicity testing tools is now available to investigators, enabling the generation of enormous amounts of data using only small amounts of test sample and at relatively low cost. In addition to these tools, the pharmaceutical industry has an urgent need for toxicity testing earlier in the process, based on the recognition that safety issues are the single largest cause of drug candidate attrition from development portfolios and the marketplace. However, along with the opportunity provided by new testing tools comes the dilemma of deciding which tools to use and, equally as important, when and why to use them. It may well be unwise to apply a new toxicity test or screening system simply because one can, as both false positive and false negative outcomes can quickly negate the value of a toxicity test system and may even have a net negative impact on drug discovery productivity. This can be true even of test systems that are considered to be 'validated' in the traditional sense. How then is an investigator or drug discovery organization to decide which of the new tools to use, and when to use them? Proposed herein is a strategy for identifying high-value toxicity testing systems and strategies based on program knowledge and informed decision-making. The decision to apply a certain toxicity testing system in this strategy is informed by knowledge of the pharmacological target, the chemical features of molecules active at the pharmacological target, and existing public domain or institutional learning. This 'fit-for-purpose' approach limits non-targeted or 'uninformed' toxicity screening to only those few test systems with high specificity, strong outcome concordance and molecular relevance to frequently encountered toxicity risks (eg, genotoxicity). Additional toxicity testing and screening is then conducted to address specific known or potential toxicity risks, based on existing knowledge of the target pharmacology and secondary pharmacology or chemical attributes with known or suspect risk, and by active 'interrogation' of both the target and active chemical moieties during the drug discovery process. This model for toxicity testing decision-making is illustrated by two case studies from recent experience.

Evaluation of cognitive function in weanling rats: a review of methods suitable for chemical screening.

Current developmental neurotoxicity (DNT) tests that are used for environmental agents require cognitive testing around the age of weaning as well as adulthood. There are challenges associated with testing weanling rodents that are not present with testing older subjects, including rapid brain development, and the impact of food or water restriction necessary for appetitive paradigms. This review provides an overview of cognitive tests that can be used for laboratory rodents in the context of such DNT studies; as such, those requiring surgery or food/water deprivation are excluded. Potential test methods described herein include spontaneous, avoidance, conditioned, spatial, and sequential behavioral assays; although, some procedures meet scientific and regulatory requirements better than others. Scientific judgment should be exercised in the choice of cognitive measures for weanling rodents in DNT studies, and should include an assessment of the sensitivity and efficiency of the procedure, an understanding of the literature and the neuronal substrates involved, and evaluation of available information on the mode(s) of action of the test chemical.