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Whole effluent toxicity (WET) testing uses whole animal exposures to assess the toxicity of complex mixtures, like wastewater. These assessments typically include four apical endpoints: mortality, growth, development, and reproduction. In the last decade, there has been a shift to alternative methods that align with the 3Rs to replace, reduce, and refine the use of animals in research. In vitro bioassays can provide a cost-effective, high-throughput, ethical alternative to in vivo assays. In addition, they can potentially include additional, more sensitive, environmentally relevant endpoints than traditional toxicity tests. However, the ecological relevance of these endpoints must be established before they are adopted into regulatory frameworks. This is Part 2 of a two-part review that aims to identify in vitro bioassays that are linked to ecologically relevant endpoints that could be included in WET testing. Part 2 of this review focuses on non-apical endpoints that should be incorporated into WET testing. In addition to the four apical endpoints addressed in Part 1, this review identified seven additional toxic outcomes: endocrine disruption, xenobiotic metabolism, carcinogenicity, oxidative stress, inflammation, immunotoxicity and neurotoxicity. For each, the response at the molecular or cellular level measured in vitro was linked to the response at the organism level through a toxicity pathway. Literature from 2015 to 2020 was used to identify suitable bioassays that could be incorporated into WET testing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

"The TOXI Chemical Exposures and Impact on Health" session at the 2020 Fall ACS meeting presented analytical and biological approaches, advancing our understanding of legacy and emerging chemical pollutants and their impact on human health.

Intravital microscopy (IVM) is an essential experimental approach for evaluating, in real time, cell interactions in the blood and rheological parameters in the microcirculation of the living animals. Different tissues are surgically exposed to the visualization of the microvascular network in optical microscopies connected to video cameras and image software. By evaluating in situ microcirculatory network, IVM allows the visualization and quantification of physiological and pathological processes in the blood or in the adjacent tissues considering the whole system. Therefore, IVM has been used to evaluate the effects and mechanisms of actions in the microvascular network caused by pharmacological or toxic chemical agents. In this chapter, different experimental approaches are described to study the toxic effects and mechanisms of xenobiotics in the microcirculatory network.

Insights into the modes of action (MoAs) of xenobiotics are of utmost importance for the definition of adverse outcome pathways (AOPs), which are essential for a mechanism-based risk assessment. A well-established strategy to reveal MoAs of xenobiotics is the use of omics. However, often an even more comprehensive approach is needed, which can be achieved using multi-omics. Since the immune system plays a central role in the defense against foreign substances and pathogens, with the innate immune system building a first barrier, we systematically reviewed multi-omics studies investigating the effects of xenobiotics on macrophages. Surprisingly, only nine publications were identified, combining proteomics with transcriptomics or metabolomics. We summarized pathways and single proteins, transcripts, or metabolites, which were described to be affected upon treatment with xenobiotics in the reviewed studies, thus revealing a broad range of effects. In summary, we show that macrophages are a relevant model system to investigate the toxicological effects induced by xenobiotics. Furthermore, the multi-omics approaches led to a more comprehensive overview compared to only one omics layer with slight advantages for combinations that complement each other directly, e.g., proteome and metabolome.

Zebrafish has become a popular model organism in several lines of biological research sharing physiological, morphological and histological similarities with mammals. In fact, many human cytochrome P450 (CYP) enzymes have direct orthologs in zebrafish, suggesting that zebrafish xenobiotic metabolic profiles may be similar to those in mammals. The focus of the review is to analyse the studies that have evaluated the metabolite production in zebrafish over the years, either of the drugs themselves or xenobiotics in general (environmental pollutants, natural products, etc.), bringing a vision of how these works were performed and comparing, where possible, with human metabolism. Early studies that observed metabolic production by zebrafish focused on environmental toxicology, and in recent years the main focus has been on toxicity screening of pharmaceuticals and drug candidates. Nevertheless, there is still a lack of standardization of the model and the knowledge of the extent of similarity with human metabolism. Zebrafish screenings are performed at different life stages, typically being carried out in adult fish through in vivo assays, followed by early larval stages and embryos. Studies comparing metabolism at the different zebrafish life stages are also common. As with any non-human model, the zebrafish presents similarities and differences in relation to the profile of generated metabolites compared to that observed in humans. Although more studies are still needed to assess the degree to which zebrafish metabolism can be compared to human metabolism, the facts presented indicate that the zebrafish is an excellent potential model for assessing xenobiotic metabolism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Human lungs are susceptible to the toxicity induced by soluble xenobiotics. However, the direct cellular effects of many pulmonotoxic chemicals are not always clear, and thus, a general in vitro assay for testing pulmonotoxicity applicable to a wide variety of chemicals is not currently available. Here, we report a study that uses high-throughput imaging and artificial intelligence to build an in vitro pulmonotoxicity assay by automatically comparing and selecting human lung-cell lines and their associated quantitative phenotypic features most predictive of in vivo pulmonotoxicity. This approach is called "High-throughput In vitro Phenotypic Profiling for Toxicity Prediction" (HIPPTox). We found that the resulting assay based on two phenotypic features of a human bronchial epithelial cell line, BEAS-2B, can accurately classify 33 reference chemicals with human pulmonotoxicity information (88.8% balance accuracy, 84.6% sensitivity, and 93.0% specificity). In comparison, the predictivity of a standard cell-viability assay on the same set of chemicals is much lower (77.1% balanced accuracy, 84.6% sensitivity, and 69.5% specificity). We also used the assay to evaluate 17 additional test chemicals with unknown/unclear human pulmonotoxicity, and experimentally confirmed that many of the pulmonotoxic reference and predicted-positive test chemicals induce DNA strand breaks and/or activation of the DNA-damage response (DDR) pathway. Therefore, HIPPTox helps us to uncover these common modes-of-action of pulmonotoxic chemicals. HIPPTox may also be applied to other cell types or models, and accelerate the development of predictive in vitro assays for other cell-type- or organ-specific toxicities.

Xenobiotic safety assessment is an area that impacts a multitude of different industry sectors such as medicinal drugs, agrochemicals, industrial chemicals, cosmetics and environmental contaminants. As such there are a number of well-developed in vitro, in vivo and in silico approaches to evaluate their properties and potential impact on the environment and to humans. Additionally, there is the continual investment in multidisciplinary scientists to explore non-animal surrogate technologies to predict specific toxicological outcomes and to improve our understanding of the biological processes regarding the toxic potential of xenobiotics. Here we provide a concise, critical evaluation of a number of in vitro systems utilised to assess the hepatotoxic potential of xenobiotics., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In marine animals with external fertilization, gametes are released into seawater where fertilization and embryo development occur. Consequently, pollutants introduced into the marine environment by human activities may affect gametes and embryos. These xenobiotics can alter cell physiology with consequent reduction of fertilization success. Here the adverse effects on the reproductive processes of the marine invertebrate Ciona intestinalis (ascidian) of different xenobiotics: lead, zinc, an organic tin compound and a phenylurea herbicide were evaluated. By using the electrophysiological technique of whole-cell voltage clamping, the effects of these compounds on the mature oocyte plasma membrane electrical properties and the electrical events of fertilization were tested by calculating the concentration that induced 50% normal larval formation (EC50). The results demonstrated that sodium currents in mature oocytes were reduced in a concentration-dependent manner by all tested xenobiotics, with the lowest EC50 value for lead. In contrast, fertilization current frequencies were differently affected by zinc and organic tin compound. Toxicity tests on gametes demonstrated that sperm fertilizing capability and fertilization oocyte competence were not altered by xenobiotics, whereas fertilization was inhibited in zinc solution and underwent a reduction in organic tin compound solution (EC50 value of 1.7 µM). Furthermore, fertilized oocytes resulted in a low percentage of normal larvae with an EC50 value of 0.90 µM. This study shows that reproductive processes of ascidians are highly sensitive to xenobiotics suggesting that they may be considered a reliable biomarker and that ascidians are suitable model organisms to assess marine environmental quality.

Organ toxicity, particularly liver toxicity, remains one of the major reasons for the termination of drug candidates in the development pipeline as well as withdrawal or restrictions of marketed drugs. A screening-amenable alternative in vivo model such as zebrafish would, therefore, find immediate application in the early prediction of unacceptable organ toxicity. To identify highly upregulated genes as biomarkers of toxic responses in the zebrafish model, a set of well-characterized reference drugs that cause drug-induced liver injury (DILI) in the clinic were applied to zebrafish larvae and adults. Transcriptome microarray analysis was performed on whole larvae or dissected adult livers. Integration of data sets from different drug treatments at different stages identified common upregulated detoxification pathways. Within these were candidate biomarkers which recurred in multiple treatments. We prioritized 4 highly upregulated genes encoding enzymes acting in distinct phases of the drug metabolism pathway. Through promoter isolation and fosmid recombineering, eGFP reporter transgenic zebrafish lines were generated and evaluated for their response to DILI drugs. Three of the 4 generated reporter lines showed a dose and time-dependent induction in endodermal organs to reference drugs and an expanded drug set. In conclusion, through integrated transcriptomics and transgenic approaches, we have developed parallel independent zebrafish in vivo screening platforms able to predict organ toxicities of preclinical drugs., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

This paper describes the further development of a read-across approach applicable to the toxicological assessment of structurally-related xenobiotic metabolites. The approach, which can be applied in the absence of definitive identification of all the individual metabolites, draws on the use of chemical descriptors and multi-variate statistical analysis to define a composite "chemical space" and to classify and characterize closely-related subgroups within this. In this example, consideration of the descriptors driving grouping, combined with empirical evidence for lack of significant further biotransformation of metabolites, leads to the conclusion that, in the absence of any specific structural alerts, the relative toxicity of metabolites within a single grouping will be determined by their relative systemic exposure as described by their ADME characteristics. The in vivo testing of a smaller number of exemplars, selected to have representative ADME properties for each grouping, is sufficient, therefore, to evaluate the toxicity of the remainder. The approach is exemplified using the metabolites of the herbicide S-metolachlor, detected in the leachate of a soil lysimeter., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

We analyzed hepatotoxicity of three drugs: acetaminophen, metformin, and isoniazid. Spheroids of differentiated HepaRG cells cultured under microfluidic conditions were used as the model. Acute toxicity of substances was assessed by analyzing cell viability, while lactate concentration in the culture medium was used as the potential marker for evaluation of chronic exposure and non-lethal side effects of xenobiotics. The results were compared with mitochondrial activity and DNA fragmentation data. The efficiency and possibility of applying the integrated approach for assessment of drug hepatotoxicity are discussed.

Blood is a fluid connective tissue which plays a vital role for normal body function. It consist different type of blood cells which is continuously reproduce inside the bone marrow from hematopoietic system. Xenobiotics could be specifically toxic to the hematopoietic system and they can cause hematological disorders by disturbing the normal functions. In vitro hematopoietic colony-forming cell assays play a crucial role to evaluate potential toxic effects of new xenobiotics and also helpful in bridging the gap between preclinical toxicology studies in animal models and clinical investigations. Use of these assays in conjunction with, high-throughput screening reduces the cost and time associated with these assays. This article provides a critical view over in vitro hematopoietic colony-forming cell assays in assessment of hematotoxicity.

The erythrocytes of mammals represent a good model to evaluate the cytotoxicity of molecules, organic and inorganic, natural or synthetic, by cellular damage measure. Indeed, before any investigation on the mechanism of action of different molecules, it is important to perform a cytotoxicity assay. Among the different cytotoxicity assays that assess a possible toxicity in the red blood cells is the rate of haemolysis. This essay is based on the evaluation of the alterations of red cell membranes in the presence of an eventual xenobiotic. Red blood cells are the main cells in circulation, and they are responsible for transporting oxygen; in fact, any alterations of this process could be lethal. The plasma membrane of red blood cells is a multi-component structure such as to confer to these cells their characteristic biconcave shape, high flexibility, elasticity and deformability. However, there are clear signs of cellular suffering if there are any alterations to this structure. One method of toxicity assessment is based on measurement of the efflux of haemoglobin from suspended red blood cells. Haemolysis, and therefore the loss of haemoglobin, is the signal stability of the cell membrane of the erythrocytes. In recent years, the discovery of programmed cell death in mammalian red blood cells presented a diversification of the response to injury by these a-nucleated cells. This review shows that mammals' erythrocytes might serve well as a model cell to study on the cellular and molecular mechanisms of many treatments., (Copyright © 2015 John Wiley & Sons, Ltd.)

The need for models that recapitulate liver physiology is perceived for drug development, study of liver disease and bioartificial liver support. The bipotent cell line HepaRG constitutes an efficient surrogate of liver function, yet its differentiated status relies on high concentrations of DMSO, which may compromise the study of drug metabolism and limit the applicability of this hepatic model. Herein, we present a three-dimensional (3D) strategy for the differentiation of HepaRG based on alginate microencapsulation of cell spheroids and culture in dimethyl sulfoxide (DMSO)-free conditions. A ratio of 2.9:1 hepatocyte-like to biliary-like cells was obtained in the 3D culture, with an improvement of 35.9 % in the hepatocyte differentiation when compared with two-dimensional (2D) cultures. The expression of the hepatic identity genes HNF4α and PXR in 3D cultures was comparable to 2D differentiated cultures, while the expression of homeostatic-associated genes albumin and carbamoyl phosphate synthase 1 was higher in 3D. Moreover, the spheroids presented a polarized organization, exhibiting an interconnected bile canalicular network and excretory functionality, assessed by specific activity of MRP2. Importantly, despite variability in basal gene expression levels, the activity of the phase I enzymes cytochrome P450 family 3, subfamily A, polypeptide 4 and cytochrome P450 family 1, subfamily A, polypeptide 2 upon induction was comparable to differentiated 2D cultures and albumin production and ammonia detoxification were enhanced in 3D. The presented model is suitable for toxicological applications, as it allows high throughput analysis of multiple compounds in a DMSO-free setting. Due to the high xenobiotic metabolism and maintenance of biosynthetic functions, the applicability of this model might be broadened to understand liver physiology and for bioartificial liver applications.

Primary human hepatocytes (PHHs) are a limited resource for drug screening, their quality for in vitro use can vary considerably across different lots, and a lack of available donor diversity restricts our understanding of how human genetics affect drug-induced liver injury (DILI). Induced pluripotent stem cell-derived human hepatocyte-like cells (iPSC-HHs) could provide a complementary tool to PHHs for high-throughput drug screening, and ultimately enable personalized medicine. Here, we hypothesized that previously developed iPSC-HH-based micropatterned co-cultures (iMPCCs) with murine embryonic fibroblasts could be amenable to long-term drug toxicity assessment. iMPCCs, created in industry-standard 96-well plates, were treated for 6 days with a set of 47 drugs, and multiple functional endpoints (albumin, urea, ATP) were evaluated in dosed cultures against vehicle-only controls to enable binary toxicity decisions. We found that iMPCCs correctly classified 24 of 37 hepatotoxic drugs (65% sensitivity), while all 10 non-toxic drugs tested were classified as such in iMPCCs (100% specificity). On the other hand, conventional confluent cultures of iPSC-HHs failed to detect several liver toxins that were picked up in iMPCCs. Results for DILI detection in iMPCCs were remarkably similar to published data in PHH-MPCCs (65% versus 70% sensitivity) that were dosed with the same drugs. Furthermore, iMPCCs detected the relative hepatotoxicity of structural drug analogs and recapitulated known mechanisms of acetaminophen toxicity in vitro. In conclusion, iMPCCs could provide a robust tool to screen for DILI potential of large compound libraries in early stages of drug development using an abundant supply of commercially available iPSC-HHs., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The TTC concept uses toxicological data from animal testing to derive generic human exposure threshold values (TTC values), below which the risk of adverse effects on human health is considered to be low. It uses distributions of no-observed-adverse-effect levels (NOAELs) for substances. The 5th percentile value is divided by an uncertainty factor (100) to give a TTC value. As the toxicological data underpinning the TTC concept are from tests with oral exposure, the exposure is to be understood as an external oral exposure. For risk assessment of substances with a low absorption (by the oral route, or through skin), the internal exposure is more relevant than the external exposure. European legislation allows that tests might not be necessary for substances with negligible absorption with low internal exposure. The aim of this work is to derive internal TTC values to allow the TTC concept to be applied to situations of low internal exposure. The external NOAEL of each chemical of three databases (Munro, ELINCS, Food Contact Materials) was multiplied by the bioavailability of the individual chemical. Oral bioavailability was predicted using an in silico prediction tool (ACD Percepta). After applying a reduced uncertainty factor of 25, we derived internal TTC values. For Cramer class I, the internal TTC values are 6.9 μg/kg bw/d (90 % confidence interval: 3.8-11.5 mg/kg bw/d); for Cramer class II/III 0.1 μg/kg bw/d (90 % confidence interval: 0.08-0.14 μg/kg bw/d).

Conventional approaches for toxicity evaluation of drugs and chemicals, such as animal tests, can be impractical due to the large experimental scale and the immunological differences between species. Organ-on-a-chip models have recently been recognized as a prominent alternative to conventional toxicity tests aiming to simulate the human in vivo physiology. This review focuses on the organ-on-a-chip applications for high-throughput screening of candidate drugs against toxicity, with a particular emphasis on bone-marrow-on-a-chip. Studies in which organ-on-a-chip models have been developed and utilized to maximize the efficiency and predictability in toxicity assessment are introduced. The potential of these devices to replace tests of acute systemic toxicity in animals, and the challenges that are inherent in simulating the human immune system are also discussed. As a promising approach to overcome the limitations, we further focus on an in-depth analysis of the development of bone-marrow-on-a-chip that is capable of simulating human immune responses against external stimuli due to the key roles of marrow in immune systems with hematopoietic activities. Owing to the complex interactions between hematopoietic stem cells and marrow microenvironments, precise control of both biochemical and physical niches that are critical in maintenance of hematopoiesis remains a key challenge. Thus, recently developed bone-marrow-on-a-chip models support immunogenicity and immunotoxicity testing in long-term cultivation with repeated antigen stimulation. In this review, we provide an overview of clinical studies that have been carried out on bone marrow transplants in patients with immune-related diseases and future aspects of clinical and pharmaceutical application of bone-marrow-on-a-chip.

Evaluating immunomodulatory effects of xenobiotics is an important component of the toxicity studies. Herein we report on the establishment of a novel invitro test system for the immunotoxicity screening of xenobiotics based on human lymphoblastoid cell lines (LCLs). Four immunotoxic compounds; tributyltin chloride, cyclosporine A, benzo(a)pyrene and verapamil hydrochloride, as well as three immune-inert compounds; urethane, furosemide and mannitol were selected for characterization. The treatment of LCLs with immunosuppressive compounds resulted in reduced viability. The IC50 values determined in human LCLs were in agreement with the data obtained for human peripheral mononuclear cells. Since cytokine production reflects lymphocytes responses to external stimuli, we evaluated the functional responses of LCLs by monitoring their pro-inflammatory and immunoregulatory cytokine production. Our findings prove that LCLs allowed for reliable differentiation between immunomodulatory and immune-inert compounds. Hence, pre-treatment with immunomodulatory compounds led to a decrease in the production of pro-inflammatory TNFα, IL-6 and immunoregulatory IL-2, IL-4, IL-10 and IFNγ cytokines, when compared to untreated ionomycin/PMA stimulated cells. Moreover, testing a panel of ten LCLs derived from unrelated healthy individuals reflects inter-individual variability in response to immunomodulatory xenobiotics. In conclusion, LCLs provide a novel alternative method for the testing of the immunotoxic effects of xenobiotics., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Momentum is growing worldwide to use in vitro high-throughput screening (HTS) to evaluate human health effects of chemicals. However, the integration of dosimetry into HTS assays and incorporation of population variability will be essential before its application in a risk assessment context. Previously, we employed in vitro hepatic metabolic clearance and plasma protein binding data with in vitro in vivo extrapolation (IVIVE) modeling to estimate oral equivalent doses, or daily oral chemical doses required to achieve steady-state blood concentrations (Css) equivalent to media concentrations having a defined effect in an in vitro HTS assay. In this study, hepatic clearance rates of selected ToxCast chemicals were measured in vitro for 13 cytochrome P450 and five uridine 5'-diphospho-glucuronysyltransferase isozymes using recombinantly expressed enzymes. The isozyme-specific clearance rates were then incorporated into an IVIVE model that captures known differences in isozyme expression across several life stages and ethnic populations. Comparison of the median Css for a healthy population against the median or the upper 95th percentile for more sensitive populations revealed differences of 1.3- to 4.3-fold or 3.1- to 13.1-fold, respectively. Such values may be used to derive chemical-specific human toxicokinetic adjustment factors. The IVIVE model was also used to estimate subpopulation-specific oral equivalent doses that were directly compared with subpopulation-specific exposure estimates. This study successfully combines isozyme and physiologic differences to quantitate subpopulation pharmacokinetic variability. Incorporation of these values with dosimetry and in vitro bioactivities provides a viable approach that could be employed within a high-throughput risk assessment framework., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In fresh waters cyanobacterial blooms can produce a variety of toxins, such as microcystin variants (MCs) and anatoxin-a (ANA). ANA is a well-known neurotoxin, whereas MCs are hepatotoxic and, to a lesser degree, also neurotoxic. Neurotoxicity applies especially to invertebrates lacking livers. Current standardized neurotoxicity screening methods use rats or mice. However, in order to minimize vertebrate animal experiments as well as experimental time and effort, many investigators have proposed the nematode Caenorhabditis elegans as an appropriate invertebrate model. Therefore, four known neurotoxic compounds (positive compounds: chlorpyrifos, abamectin, atropine, and acrylamide) were chosen to verify the expected impacts on autonomic (locomotion, feeding, defecation) and sensory (thermal, chemical, and mechanical sensory perception) functions in C. elegans. This study is another step towards successfully establishing C. elegans as an alternative neurotoxicity model. By using this protocol, anatoxin-a adversely affected locomotive behavior and pharyngeal pumping frequency and, most strongly, chemotactic and thermotactic behavior, whereas MC-LR impacted locomotion, pumping, and mechanical behavior, but not chemical sensory behavior. Environmental samples can also be screened in this simple and fast way for neurotoxic characteristics. The filtrate of a Microcystis aeruginosa culture, known for its hepatotoxicity, also displayed mild neurotoxicity (modulated short-term thermotaxis). These results show the suitability of this assay for environmental cyanotoxin-containing samples.

FT-IR spectroscopy is a useful tool for determining the biomolecular profile of micro-samples of body fluids such as coelomic fluid of earthworms. The present study focuses on the usefulness of the earthworm (Perionyx sansibaricus) coelomic fluid for observing pathologically induced biochemical changes. Compared to controls, appreciable changes in expression of peaks were observed in worms exposed to seven selected xenobiotics (pesticides, heavy metals, herbicides and detergents). Observation of bands in the region 1600-1690 cm(-1) indicates the presence of amide I band in all the worms. The peak at 2364 cm(-1) present as a weak band on day 7 of treatment, is shifted to 2358/2359 cm(-1) and more pronounced in most of the treated groups on day 14. Presence of band at 1663 cm(-1) in controls is attributed to CO stretching vibration representing the amino acid, glutamic acid. Under toxicological conditions vibration in this region is absent. Presence of the amino acid arginine (1633 cm(-1)) and lysine (1629 cm(-1)) and absence of glutamic acid (1663 cm(-1)) under toxicological stress were characteristic. FT-IR spectra of the coelomic fluid were similar under the sublethal and lethal concentrations of the test chemicals. The potential use of FT-IR spectral information as baseline data for toxicological studies and for monitoring the quality of the environment is recommended., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Fighting against water pollution requires the ability to evaluate the toxicity of pollutants such as herbicides. Tetrahymena pyriformis are ubiquitous ciliated protozoans commonly used in ecotoxicological research. Microcalorimetry can be used in many biological investigations as a universal, non-destructive and highly sensitive tool that provides a continuous real-time monitoring of the metabolic activity. This technique based on the thermal power output was applied to evaluate the influence of herbicide diuron on cultures of T. pyriformis. The heat flux produced upon addition of 0, 3.5, 7.0, 14.0, 28.0, and 56.0 µg mL⁻¹ of diuron was monitored. The biomass change during the growth was also determined by flow cytometry. The results confirmed that the growth of T. pyriformis is progressively inhibited as the concentration of diuron increases and revealed that the state of the living system is severely altered at a concentration of 56.0 µg mL⁻¹. The IC₅₀ was estimated at 13.8 µg mL⁻¹ by microcalorimetry and at 18.6 µg mL⁻¹ by flow cytometry. It was shown that microcalorimetry is not only a very effective tool for the determination of the growth rate constant but that it is also a valuable probe for a rapid detection of the metabolic perturbations and, in ultimate cases, of the critical alterations of the living system under the action of a toxic agent., (© 2013 Elsevier Inc. All rights reserved.)

While exposure to chemical mixtures is an everyday reality, an understanding of their combined effects, and any potential prediction thereof, is extremely limited. Realistic exposures potentially consist of hundreds to thousands of chemicals per day, but even relatively simple binary mixture interactions can be inherently difficult to predict based upon the lack of temporal and spatial mechanisms for the individual constituents. To this end, we explore the concept of capitalizing on downstream convergence of intracellular signal transduction to experimentally simplify the means of determining xenobiotics that, when combined, could result in increased or unexpected toxicity. In a proof of principle study, we exposed HepG2 cells to deguelin, a natural isoflavonoid, alone and in combination with KCN, and determined the relative post-translational phosphorylation responses to several key proteins related to mitochondrial outer membrane permeabilization. Dose-dependent phosphorylation activity provides a clear identification of threshold response to low-level exposures, and crosstalk amongst selected proteins correctly forecasts mixtures interactions that may lead to increased toxicity. We then used Bliss Independence to determine if the experimentally measured mixture phosphorylation responses could be predicted with individual responses. Independence accurately predicted mixture interactions for deguelin and KCN (87.5%). To more fully exhaust independence as a model for determining potential pharmacodynamic interactions, we exposed HepG2 cells to deguelin and staurosporine, a broad kinase inhibitor; independence accurately predicted these mixture responses (77.5%). In this study, we demonstrate the potential of a new in vitro approach for the prediction of toxic mixtures interactions that is fundamentally driven by the interdependence of signal transduction and apoptosis., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Toxicity screening of compounds provides a means to identify compounds harmful for human health and the environment. Here, we further develop the technique of genomic phenotyping to improve throughput while maintaining specificity. We exposed cells to eight different compounds that rely on different modes of action: four genotoxic alkylating (methyl methanesulfonate (MMS), N-Methyl-N-nitrosourea (MNU), N,N'-bis(2-chloroethyl)-N-nitroso-urea (BCNU), N-ethylnitrosourea (ENU)), two oxidizing (2-methylnaphthalene-1,4-dione (menadione, MEN), benzene-1,4-diol (hydroquinone, HYQ)), and two non-genotoxic (methyl carbamate (MC) and dimethyl sulfoxide (DMSO)) compounds. A library of S. cerevisiae 4,852 deletion strains, each identifiable by a unique genetic 'barcode', were grown in competition; at different time points the ratio between the strains was assessed by quantitative high throughput 'barcode' sequencing. The method was validated by comparison to previous genomic phenotyping studies and 90% of the strains identified as MMS-sensitive here were also identified as MMS-sensitive in a much lower throughput solid agar screen. The data provide profiles of proteins and pathways needed for recovery after both genotoxic and non-genotoxic compounds. In addition, a novel role for aromatic amino acids in the recovery after treatment with oxidizing agents was suggested. The role of aromatic acids was further validated; the quinone subgroup of oxidizing agents were extremely toxic in cells where tryptophan biosynthesis was compromised.

For ethical and regulatory reasons, in vitro tests for scoring potential toxicities of cosmetics are essential. A test strategy for investigating potential skin sensitization using two human keratinocytic and two human dendritic cell lines has been developed (Mehling et al. Arch Toxicol 86:1273–1295, 2012). Since prohaptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (XME) activities in these cell lines is of high interest. In this study, XME activity assays, monitoring metabolite or cofactor, showed the following: all three passages of keratinocytic (KeratinoSens® and LuSens) and dendritic (U937 und THP-1) cells displayed N-acetyltransferase 1 (NAT1) activities (about 6–60 nmol/min/mg S9-protein for acetylation of para-aminobenzoic acid). This is relevant since reactive species of many cosmetics are metabolically controlled by cutaneous NAT1. Esterase activities of about 1–4 nmol fluorescein diacetate/min/mg S9-protein were observed in all passages of investigated keratinocytic and about 1 nmol fluorescein diacetate/min/mg S9-protein in dendritic cell lines. This is also of practical relevance since many esters and amides are detoxified and others activated by cutaneous esterases. In both keratinocytic cell lines, activities of aldehyde dehydrogenase (ALDH) were observed (5–17 nmol product/min/mg cytosolic protein). ALDH is relevant for the detoxication of reactive aldehydes. Activities of several other XME were below detection, namely the investigated cytochrome P450-dependent alkylresorufin O-dealkylases 7-ethylresorufin O-deethylase, 7-benzylresorufin O-debenzylase and 7-pentylresorufin O-depentylase (while NADPH cytochrome c reductase activities were much above the limit of quantification), the flavin-containing monooxygenase, the alcohol dehydrogenase as well as the UDP glucuronosyl transferase activities.

Several human skin models employing primary cells and immortalized cell lines used as monocultures or combined to produce reconstituted 3D skin constructs have been developed. Furthermore, these models have been included in European genotoxicity and sensitization/irritation assay validation projects. In order to help interpret data, Cosmetics Europe (formerly COLIPA) facilitated research projects that measured a variety of defined phase I and II enzyme activities and created a complete proteomic profile of xenobiotic metabolizing enzymes (XMEs) in native human skin and compared them with data obtained from a number of in vitro models of human skin. Here, we have summarized our findings on the current knowledge of the metabolic capacity of native human skin and in vitro models and made an overall assessment of the metabolic capacity from gene expression, proteomic expression, and substrate metabolism data. The known low expression and function of phase I enzymes in native whole skin were reflected in the in vitro models. Some XMEs in whole skin were not detected in in vitro models and vice versa, and some major hepatic XMEs such as cytochrome P450-monooxygenases were absent or measured only at very low levels in the skin. Conversely, despite varying mRNA and protein levels of phase II enzymes, functional activity of glutathione S-transferases, N-acetyltransferase 1, and UDP-glucuronosyltransferases were all readily measurable in whole skin and in vitro skin models at activity levels similar to those measured in the liver. These projects have enabled a better understanding of the contribution of XMEs to toxicity endpoints.

Assessing chemicals for acute oral toxicity is a standard information requirement of regulatory testing. However, animal testing is now prohibited in the cosmetics sector in Europe, and strongly discouraged for industrial chemicals. Building on the results of a previous international validation study, a follow up study was organised to assess if the 3T3 Neutral Red Uptake cytotoxicity assay could identify substances not requiring classification as acute oral toxicants under the EU regulations. Fifty-six coded industrial chemicals were tested in three laboratories, each using one of the following protocols: the previously validated protocol, an abbreviated version of the protocol and the protocol adapted for an automation platform. Predictions were very similar among the three laboratories. The assay exhibited high sensitivity (92-96%) but relatively low specificity (40-44%). Three chemicals were under predicted. Assuming that most industrial chemicals are not likely to be acutely toxic, this test method could prove a valuable component of an integrated testing strategy, a read-across argument, or weight-of-evidence approach to identify non toxic chemicals (LD50>2000 mg/kg). However, it is likely to under predict chemicals acting via specific mechanisms of action not captured by the 3T3 test system, or which first require biotransformation in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Advances in high throughput and high content (HT/HC) methods such as those used in the fields of toxicogenomics, bioinformatics, and computational toxicology have the potential to improve both the efficiency and effectiveness of toxicity evaluations and risk assessments. However, prior to use, scientific confidence in these methods should be formally established. Traditional validation approaches that define relevance, reliability, sensitivity and specificity may not be readily applicable. HT/HC methods are not exact replacements for in vivo testing, and although run individually, these assays are likely to be used as a group or battery for decision making and use robotics, which may be unique in each laboratory setting. Building on the frameworks developed in the 2010 Institute of Medicine Report on Biomarkers and the OECD 2007 Report on (Q)SAR Validation, we present constructs that can be adapted to address the validation challenges of HT/HC methods. These are flexible, transparent, and require explicit specification of context and purpose of use such that scientific confidence (validation) can be defined to meet different regulatory applications. Using these constructs, we discuss how anchoring the assays and their prediction models to Adverse Outcome Pathways (AOPs) could facilitate the interpretation of results and support scientifically defensible fit-for-purpose applications., (Copyright © 2012 Elsevier Inc. All rights reserved.)

An outcome and statistical review of male reproductive performance assessed by including a mating phase within 6-month general toxicity studies in the Han Wistar rat was undertaken. The basic study design was 16-20 animals per group dosed for approximately 9 weeks before pairing the male rats with undosed females. This design provides opportunity for remating and automatically includes general toxicity parameters. The dose levels used in the 1- and 6-month studies show that male reproduction was assessed at generally similar doses. The majority of males (compound-dosed and controls) mated within 7 days. All vehicle-dosed males mated and 98.5% of these females were pregnant. Modeling shows that a pregnancy rate of less than 14 out of 16 pregnant animals is very unlikely to occur due to biological variability. Power calculations based on vehicle control data show that group sizes of >10 males have a >80% power of detecting a decrease in median of three embryos per group compared with the control group. Even if the number of pregnancies decreased by a third, a group size of ≥12 would still detect a decrement in the median of three embryos with >80% power. Based on the statistical modeling and inherent strengths of the study design, this review indicates that decrements in male reproductive function can be successfully detected by incorporating a mating phase into a 6-month rat study and that a group size of 12-16 is generally adequate rather than the 16-20 group size indicated as a generic default within ICHS5(R2)., (© 2012 Wiley Periodicals, Inc.)

There is increased awareness of the specific position of children when it comes to hazards of xenobiotic exposures. Children are not small adults, since their exposure patterns, compound kinetics and metabolism, and sensitivity of their developing organs may differ extensively from adults. Current international hazard assessment test guidelines do not specifically address juvenile exposures and effects. In conjunction with the Annual Meeting of the European Teratology Society, a satellite meeting was organized to specifically address juvenile toxicity testing issues for chemicals. The workshop focused on developmental neurotoxicity and developmental immune toxicity testing in juvenile animals. A clear case was made for the importance of juvenile toxicity testing, showing that in animal studies developmental neurotoxicity and immunotoxicity parameters express specifically high sensitivities after exposure during the juvenile period. Additional data will be generated in the coming years, and OECD initiatives will need to further the issue at the global regulatory level., (Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

When test article-related testicular toxicity or Leydig cell tumors are identified in nonclinical studies, the measurement of circulating hormones such as luteinizing hormone, follicle-stimulating hormone, inhibin, testosterone, or prolactin is often considered in order to aid mechanistic investigations or to identify potential biomarkers in man. Although some hormone levels are relatively constant, others are subject to wide variability owing to pulsatility of secretion, diurnal rhythms, and stress. To avoid being misled, it is important that this variation is factored into any study design that includes hormone measurements. Since all these possibilities start from the pathologist's reading of the tissue sections, we begin with a review of the morphologic changes that are tied to underlying alterations in hormones. We then provide the reader with basic information and representative hormone data, including coefficients of variation, for the major male reproductive hormones in the three main nonclinical species (rats, dogs, and cynomolgus monkeys). Power and probability tables for rats and dogs allow estimates of the number of animals or samples needed to provide a given likelihood of detecting a hormonal change of a given size. More importantly, we highlight the variability of this process and the real value in readers developing this information at their own site.

Standard protocols are given for assessing metabolic stability in rainbow trout using the liver S9 fraction. These protocols describe the isolation of S9 fractions from trout livers, evaluation of metabolic stability using a substrate depletion approach, and expression of the result as in vivo intrinsic clearance. Additional guidance is provided on the care and handling of test animals, design and interpretation of preliminary studies, and development of analytical methods. Although initially developed to predict metabolism impacts on chemical accumulation by fish, these procedures can be used to support a broad range of scientific and risk assessment activities including evaluation of emerging chemical contaminants and improved interpretation of toxicity testing results. These protocols have been designed for rainbow trout and can be adapted to other species as long as species-specific considerations are modified accordingly (e.g., fish maintenance and incubation mixture temperature). Rainbow trout is a cold-water species. Protocols for other species (e.g., carp, a warm-water species) can be developed based on these procedures as long as the specific considerations are taken into account., (© 2012 by John Wiley & Sons, Inc.)

The integrated discrete multiple organ co-culture system (IdMOC) allows the co-culturing of multiple cell types as physically separated cells interconnected by a common overlying medium. We report here the application of IdMOC with two cell types: the metabolically competent primary human hepatocytes, and a metabolically incompetent cell line, mouse 3T3 fibroblasts, in the definition of the role of hepatic metabolism on the cytotoxicity of three model toxicants: cyclophosphamide (CPA), aflatoxin B1 (AFB) and tamoxifen (TMX). The presence of hepatic metabolism in IdMOC with human hepatocytes was demonstrated by the metabolism of the P450 isoform 3A4 substrate, luciferin-IPA. The three model toxicants showed three distinct patterns of cytotoxic profile: TMX was cytotoxic to 3T3 cells in the absence of hepatocytes, with slightly lower cytotoxicity towards both 3T3 cells and hepatocytes in the IdMOC. AFB was selective toxic towards the human hepatocytes and relatively noncytotoxic towards 3T3 cells both in the presence and absence of the hepatocytes. CPA cytotoxicity to the 3T3 cells was found to be significantly enhanced by the presence of the hepatocytes, with the cytotoxicity dependent of the number of hepatocytes, and with the cytotoxicity attenuated by the presence of a non-specific P450 inhibitor, 1-aminobenzotriazole. We propose here the following classification of toxicants based on the role of hepatic metabolism as defined by the human hepatocyte-3T3 cell IdMOC assay: type I: direct-acting cytotoxicants represented by TMX as indicated by cytotoxicity in 3T3 cells in the absence of hepatocytes; type II: metabolism-dependent cytotoxicity represented by AFB1 with effects localized within the site of metabolic activation (i. e. hepatocytes); and type III: metabolism-dependent cytotoxicity with metabolites that can diffuse out of the hepatocytes to cause toxicity in cells distal from the site of metabolism, as exemplified by CPA., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Hepatotoxicity is a major reason for drug nonapprovals and withdrawals. The multiparametric analysis of xenobiotic toxicity at the single cells level using flow cytometry and cellular imaging-based approaches, such as high-content screening (HCS) technology, could play a key role in the detection of toxicity and the classification of compounds based on patterns of cellular injury. This study aimed to develop and validate a practical, reproducible, in vitro multiparametric cell-based protocol to assess those drugs that are potentially hepatotoxic to humans and to suggest their mechanisms of action. The assay was applied to HepG2 human cell line cultured in 96-well plates and exposed to 78 different compounds for 3 and 24 h at a range of concentrations (1-1000μM). After treatments, cells were simultaneously loaded with five fluorescent dyes showing optical compatibility and were then analyzed with the High-Content Screening Station Scan^R (Olympus). By using the new technology of HCS cell parameters associated with nuclear morphology, plasma membrane integrity, mitochondrial function, intracellular calcium concentration, and oxidative stress, indicative of prelethal cytotoxic effects and representative of different mechanisms of toxicity, were measured at the single cells level, which allows high-throughput screening. This strategy appears to identify early and late events in the hepatotoxic process and also suggests the mechanism(s) implicated in the toxicity of compounds to thereby classify them according to their degree of injury (no injury, low, moderate, and high injury).

Caenorhabiditis elegans (C. elegans) offers an attractive experimental platform as it has a short life cycle, is inexpensive to maintain and most importantly has high degree of evolutionary conservation with higher eukaryotes. Understanding the contribution of inherent genes that regulate neurotoxicity and antioxidant stress responses in the worm provides critical insight into mechanisms of mammalian neurotoxicity. The C. elegans model readily enables multi-gene approach, allowing for combinatorial genetic variation to be studied within the context of the influence of multigenic polymorphisms in environmental risk and vulnerability. This review provides a synopsis of recent studies on metal and pesticides toxicity in C. elegans, highlighting the utility of the model system in understanding molecular mechanisms that underlie developmental, reproductive and neuronal damage. The continuation of these investigations combining basic toxicological experimentation with novel genetic and high throughput methods will continue to make C. elegans an invaluable tool for future research, providing insight into molecular and cellular mechanisms of toxicity.

New approaches to generating and evaluating toxicity data for chemicals are needed to cope with the ever-increasing demands of new programs. One such approach involves the use of an integrated testing and evaluation strategy based on the specific properties and activities of a chemical. Such an integrated strategy, whether applied to existing or future programs, can promote efficient use of resources and save animals. We demonstrate the utility of such a strategy by applying it to the current U.S. Environmental Protection Agency Endocrine Disruptor Screening Program (EDSP). Launched in October 2009, the EDSP utilizes a two-tiered approach, whereby each tier requires a battery of animal-intensive and expensive tests. Tier 1 consists of five in vitro and six in vivo assays that are intended to determine a chemical's potential to interact with the estrogen (E), androgen (A), or thyroid (T) hormone pathways. Tier 2 is proposed to consist of multigenerational reproductive and developmental toxicity tests in several species and is intended to determine whether a chemical can cause adverse effects resulting from E, A, or T modulation. In contrast to the existing EDSP structure, we show, using the pesticide atrazine as an example, that a multilevel testing framework combined with an integrated evaluation process would significantly increase efficiency by minimizing testing.

Different methods for determining the toxicity and biodegradability of hazardous compounds evaluating their susceptibility to biological treatment were studied. Several compounds including chlorophenols and herbicides have been evaluated. Toxicity was analyzed in terms of EC50 and by a simple respirometric procedure based on the OECD Method 209 and by the Microtox® bioassay. The values of EC50 obtained from respirometry were in all the cases higher than those from the Microtox® test. The respirometric inhibition values of chlorophenols were related well with the number of chlorine atoms and their position in the aromatic ring. In general, herbicides showed lower inhibition, being alachlor the less toxic from this criterion. For determination of biodegradability an easier and faster alternative to the OECD Method 301, with a higher biomass to substrate ratio is proposed. When this test was negative, the Zahn-Wellens one was performed in order to evaluate the inherent biodegradability. In the fast test of biodegradability, 4-chlorocatechol and 4-chlorophenol showed a complete biodegradation by an unacclimated sludge upon 48 h. These results together with their low respirometric inhibition, allow concluding that these compounds could be conveniently removed in a WWTP. Alachlor, 2,4-dichlorophenol, 2,4,6-trichlorophenol and MCPA showed a partial biodegradation upon 28 days by the Zahn-Wellens inherent biodegradability test.

A process for evaluating analogs for use in SAR (Structure-Activity Relationship) assessments was previously published (Wu et al. 2010). Subsequently, this process has been updated to include a decision tree for estrogen binding (from US EPA) and flags for developmental and reproductive toxicity (DART). This paper presents the results of blinded case studies designed to test this updated framework. The results of these case studies support the conclusion that the process outlined by Wu et al. (2010) can be successfully applied to develop surrogate values for risk assessment. The read across results generated by the process were shown to be protective when compared to the actual toxicity data. Successful application of the approach requires significant expertise as well as discipline to not overstep the boundaries of the defined analogs and the rating system. The end result of this rigor can be the inability to read across all endpoints for all chemicals resulting in data gaps that cannot be filled using read across, however, this reflects the current state of the science and is preferable to making non-protective decisions. Future work will be targeted towards expanding read across capabilities. Two examples of a broader category approach are also shown., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We performed an interspecies comparison for the human hepatoma cell line HepG2 and the eukaryotic single cell organism Tetrahymena pyriformis (T. pyriformis) for 17 xenobiotics with diverse structures and four metals. The cytotoxicity was assessed by four different cell viability assays (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide reduction (MTT), neutral red uptake (NRU), resazurin dye (AlamarBlue), 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM)) for the HepG2 and by cell count and MTT for T. pyriformis. For HepG2 cells, the results revealed interassay variations depending on the compound. The highest assay conformity was found for the metal Hg(2+) and the fungicide prochloraz. The AlamarBlue assay was the most sensitive assay according to low-effect concentrations. By contrast, the NRU assay was comprised of more frequent whole concentration response relationships and was more susceptible to EC(50). For T. pyriformis the EC(50) values of the two applied assays displayed a high conformity (R(2) = 0.97). Comparing the EC(50) values obtained by the MTT assay for the two cell models, a direct correlation was absent for the xenobiotics and only present for the metals (Cd(2+), Cu(2+), and Ni(2+)). Moreover, the protozoa T. pyriformis displayed a 20 times higher sensitivity than the cell line. The highest interspecies difference of three log degrees was obtained for the polycyclic aromatic hydrocarbon fluoranthene. In addition, a correlation of the EC(50) values and octanol-water partition coefficient (log K(OW)) of the xenobiotics was performed. No correlation was found for HepG2, and a weak one for T. pyriformis. Interestingly, the interspecies difference of logarithmized EC(50) correlated positive with the log K(OW) (R(2) = 0.65). In conclusion, to obtain reliable evidence for human cytotoxicity, more than one viability/cytotoxicity assay had to be applied for cell lines. Second, the human hepatoma cell line was less affected by the organic compounds than the eukaryotic single-cell organism and was also less dependent on the log K(OW) of the xenobiotic., (Copyright © 2009 Wiley Periodicals, Inc.)

This "Commentary" has examined the use of human stem cells for detection of toxicities of physical, chemical, and biological toxins/toxicants in response to the challenge posed by the NRC Report, "Toxicity Testing in the 21st Century: A vision and Strategy." Before widespread application of the use of human embryonic, pluripotent, "iPS," or adult stem cells be considered, the basic characterization of stem cell biology should be undertaken. Because no in vitro system can mimic all factors that influence cells in vivo (individual genetic, gender, developmental, immunological and diurnal states; niche conditions; complex intercellular interactions between stem, progenitor, terminal differentiated cells, and the signaling from extracellular matrices, oxygen tensions, etc.), attempts should be made to use both embryonic and adult stem cells, grown in three dimension under "niche-like" conditions. Because many toxins and toxicants work by "epigenetic" mechanisms and that epigenetic mechanisms play important roles in regulating gene expression and in the pathogenesis of many human diseases, epigenetic toxicity must be incorporated in toxicity testing. Because modulation of gap junctional intercellular communication by epigenetic agents plays a major role in homeostatic regulation of both stem and progenitor cells in normal tissues, the modulation of this biological process by both endogenous and endogenous chemicals should be incorporated as an end point to monitor for potential toxicities or chemo-preventive attributes. In addition, modulation of quantity, as well as the quality, of stem cells should be considered as potential source of a chemical's toxic potential in affecting any stem cell-based pathology, such as cancer.

The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro-cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed.

In the present study, expression levels of vitellogenin (vtg) and cytochrome P450 aromatase genes (cyp19a and cyp19b) in zebrafish larvae during the early stages of development were investigated by quantitative real time-PCR assay. The results indicated that vtg gene transcription was induced seven days after zebrafish larvae fertilization, whereas the expression of cyp19a and cyp19b genes was detected as early as 3 and 4 days post-fertilization (dpf). Investigations into the effects of 17β-estradiol (E2) exposure on the expression of these genes showed that both vtg and cyp19b were upregulated by E2 in zebrafish larvae as early as four dpf, whereas no variation was observed in cyp19a gene expression. The estrogenic potential of pharmaceutical estrogen (DES), phenol estrogen (BPA) and the brominated flame retardants, TBBPA, DE-71 and 4-BP, were evaluated by analyzing the expression of these three genes in zebrafish larvae. The results demonstrated that natural estrogen, endocrine disrupting compounds and brominated flame retardants act as endocrine disrupters through different mechanisms. We have demonstrated for the first time that the polybrominated diphenyl ether mixture, DE-71, acts as an endocrine disrupter by upregulation of cyp19b gene expression at a relatively low concentration. These results indicate that analysis of vtg and cyp19b gene expression in zebrafish during early embryogenesis and organogenesis represents the basis of a sensitive and fast bioassay for the routine assessment of xenoestrogen effects.

This session at the 2010 joint symposium of the Society of Toxicologic Pathology (STP) and the International Federation of Societies of Toxicologic Pathologists (IFSTP) explored modern neuropathology methods for assessing the neurotoxicologic potential of xenobiotics. Conventional techniques to optimally prepare and evaluate the central and peripheral neural tissues while minimizing artifact were reviewed, and optimal schemes were set forth for evaluation of the nervous system during both routine (i.e., general toxicity) studies and enhanced (i.e., specialized neurotoxicity) studies. Stereology was introduced as the most appropriate means of examining the possible impact of toxicants on neural cell numbers. A focused discussion on brain sampling took place among a panel of expert neuroscientists (anatomists and pathologists) and the audience regarding the proper balance between sufficient sampling and cost- and time-effectiveness of the analysis. No consensus was reached on section orientation (coronal sections of both sides vs. a parasagittal longitudinal section with several unilateral hemisections from the contralateral side), but most panelists favored sampling at least 8 sections (or approximately double to triple the current complement) in routine toxicity studies.

Large-scale toxicogenomic screening approaches offer great promise for generating a bias-free system-wide view of toxicological effects and modes-of-action of chemicals and ecotoxicants. However, early applications of microarray technology have identified relatively small groups of responding genes with which to define new targets for analysis by conventional means. We have trialled a more intensive approach to the design and interpretation of array experiments incorporating a balanced interwoven ANOVA design with higher levels of biological replication, a more thorough analysis of errors and false discovery rates, and an analysis of response patterns using gene network models. Zebrafish embryos were exposed from 1.5 h post-fertilization for 72 h to ecotoxicants representing different classes--2,4-dichlorophenol, 3,4-dichloroaniline, pentachlorophenol, and cadmium chloride--at low concentrations producing a developmental disturbance to 10% of embryos and half of this dose. Extracted whole embryo RNA was then analyzed on microarrays. Analysis revealed responses of 3000-5000 genes, which is 10-1000 times greater than previously reported, with significance at lower levels of fold change. Some gene responses were common to multiple toxicants, and others were restricted to just one or two toxicants. The gene expression profiles for the different toxicants were distinctive, and analysis using network-based models provided a high level of detail of affected processes, some of which were novel. This approach provides a more highly refined view of toxic effects, from which meaningful patterns of response can be discerned and related to functional deficits and from which more reliable indicators of toxicological effect can be predicted.

Hepatic drug metabolizing enzyme (DME) induction complicates the development of new drugs owing to altered efficacy of concomitant treatments, reduction in exposure resulting from autoinduction, and potential generation of toxic metabolites. Risk assessment of DME induction during clinical evaluation is confounded by several uncertainties pertaining to hazard identification and dose response analysis. Hepatic DME induction rarely leads to clinical evidence of altered metabolism and toxicity in the patient, which typically occur only if the DME induction is relatively severe. High drug doses are associated with a greater likelihood of hepatic DME induction and downstream effects; therefore, drugs of low potency requiring higher dosing tend to lead to a greater risk of drug-drug interactions. Vigilance in clinical trials for increased or diminished drug effect and, specifically, pharmacokinetic studies in the presence of other drugs and concomitant diseases are necessary for a drug risk assessment profile. Efforts to remove hepatic DME-inducing drugs from development can be facilitated with current in vitro and in vivo assessments and will improve with the development of newer technologies. A carefully tailored case-by-case approach will lead to the development of efficacious drugs with an acceptable risk/benefit profile available to patients.