A proposed in vitro cytotoxicity test battery to detect and predict hepatotoxicity via multiple mechanisms and pathways: a minireview.

The 21st-century toxicity testing program recommends the use of cytotoxicity data from human cells in culture for rapid in vitro screening focusing on biological pathways of potential toxicants to predict in vivo toxicity. Liver is the major organ for both endogenous and exogenous chemical metabolism of xenobiotics. Therefore, this review was undertaken to evaluate side by side five different currently used commercial cytotoxicity assay kits for purpose of rapid predictive screening of potential hepatotoxicants. The test compounds for this review were selected from the NIH LiverTox and FDA Liver Toxicity Knowledge Base (LTKB) databases. Human liver HepG2, HepaRG, and rat liver Clone 9 cell cultures were used as the in vitro liver models. Five commercial assay kits representing different biomarkers or pathways were selected for this review. These kits are Vita-Orange Cell Viability Assay Kit (Sigma-Aldrich), CellTiter-Glo Cell Viability Assay Kit (Promega), CytoTox-ONE Homogeneous Membrane Integrity Assay Kit (Promega), DNA Quantitation Fluorescence Assay Kit (Sigma-Aldrich), and Neutral Red Based In Vitro Toxicology Assay Kit (Sigma-Aldrich). This review found that these kits can all be used for rapid predictive cytotoxicity screening of potential hepatotoxicants in human liver HepG2 and rat liver Clone 9 cells in culture as in vitro liver models without compromising quality and accuracy of endpoint measurements as well as the length of toxicity screening time. Unraveling the structure-activity relationship of potential hepatotoxins would help to classify their hepatotoxic effects. Therefore, in addition to the current regulatory hepatotoxicity testing strategies, development and regulatory approval of hepatotoxins need to be discussed in order to identify potential gaps in the safety assessment. The overall results of our study support the hypothesis that a battery of rapid, simple, and reliable assays is an excellent tool for predicting in vivo effects of suspected liver toxins. The human liver HepaRG cells do not appear to be an ideal in vitro liver model for this purpose.

A microscale 3D organ on a chip for recapitulating reciprocal neuroendocrine crosstalk between the hypothalamus and the pituitary gland.

Conventional 2D or even recently developed 3Din vitroculture models for hypothalamus and pituitary gland cannot successfully recapitulate reciprocal neuroendocrine communications between these two pivotal neuroendocrine tissues known to play an essential role in controlling the body's endocrine system, survival, and reproduction. In addition, most currentvitroculture models for neuroendocrine tissues fail to properly reflect their complex multicellular structure. In this context, we developed a novel microscale chip platform, termed the 'hypothalamic-pituitary (HP) axis-on-a-chip,' which integrates various cellular components of the hypothalamus and pituitary gland with biomaterials such as collagen and hyaluronic acid. We used non-toxic blood coagulation factors (fibrinogen and thrombin) as natural cross-linking agents to increase the mechanical strength of biomaterials without showing residual toxicity to overcome drawbacks of conventional chemical cross-linking agents. Furthermore, we identified and verified SERPINB2 as a reliable neuroendocrine toxic marker, with its expression significantly increased in both hypothalamus and pituitary gland cells following exposure to various types of toxins. Next, we introduced SERPINB2-fluorescence reporter system into loaded hypothalamic cells and pituitary gland cells within each chamber of the HP axis on a chip, respectively. By incorporating this SERPINB2 detection system into the loaded hypothalamic and pituitary gland cells within our chip platform, Our HP axis-on-chip platform can better mimic reciprocal neuroendocrine crosstalk between the hypothalamus and the pituitary gland in the brain microenvironments with improved efficiency in evaluating neuroendocrine toxicities of certain drug candidates.

3D hepatic organoid production from human pluripotent stem cells.

Hepatic organoids might provide a golden opportunity for realizing precision medicine in various hepatic diseases. Previously described hepatic organoid protocols from pluripotent stem cells rely on complicated multiple differentiation steps consisting of both 2D and 3D differentiation procedures. Therefore, the spontaneous formation of hepatic organoids from 2D monolayer culture is associated with a low-throughput production, which might hinder the standardization of hepatic organoid production and hamper the translation of this technology to the clinical or industrial setting. Here we describe the stepwise and fully 3D production of hepatic organoids from human pluripotent stem cells. We optimized every differentiation step by screening for optimal concentrations and timing of differentiation signals in each differentiation step. Hepatic organoids are stably expandable without losing their hepatic functionality. Moreover, upon treatment of drugs with known hepatotoxicity, we found hepatic organoids are more sensitive to drug-induced hepatotoxicity compared with 2D hepatocytes differentiated from PSCs, making them highly suitable for in vitro toxicity screening of drug candidates. The standardized fully 3D protocol described in the current study for producing functional hepatic organoids might serve as a novel platform for the industrial and clinical translation of hepatic organoid technology.

Molecular design of environment-friendly chlorophenol (CP) derivatives based on 3D-QSAR assisted with a comprehensive evaluation method combining bioaccumulation and degradation.

In this study, a chlorophenol (CP) 3D-QSAR model with a double activity (bioaccumulation and degradation) combination was established. 19 CPs were divided into a training set and test set according to the ratio of 4:1. The cross-validation coefficient (q2) and non-cross-validation coefficient (R2) of the model were 0.803 (> 0.5) and 0.925 (> 0.9), respectively, indicating a good stability and predictive ability of the 3D-QSAR. 2,4,6-trichlorophenol (2,4,6-TCP) was used as a target molecule, and 46 derivatives with low comprehensive effects were designed. Out of the 46 derivatives, 11 derivatives were screened to have the good insecticidal and preservative properties. From the perspective of the toxicity of zebrafish, 4 out of the 11 derivatives were found to have lower aquatic toxicity effects. Through the food chain simulation of cyanobacteria-daphnia-swamp-mandarin fish, it was found that the bioaccumulation effect of the four derivatives was lower than that of 2,4, 6-TCP. Finally, molecular dynamics simulation was conducted using 2-CH2NH2 substituted derivatives, and it was found that the degradation effect by laccase (white rot fungi) was significantly improved in the presence of violuric acid, hydroxybenzotriazole, and syringaldehyde. This study can provide theoretical support for the development of environment-friendly technology for emerging pollutants.

Antineoplastic drugs in urban wastewater: Occurrence, nanofiltration treatment and toxicity screening.

Antineoplastic drugs are pharmaceuticals that have been raising concerns among the scientific community due to: (i) their increasing prescription in the fight against the disease of the twentieth century (cancer); (ii) their recalcitrance to conventional wastewater treatments; (iii) their poor environmental biodegradability; and (iv) their potential risk to any eukaryotic organism. This emerges the urgency in finding solutions to mitigate the entrance and accumulation of these hazardous chemicals in the environment. Advanced oxidation processes (AOPs) have been taken into consideration to improve the degradation of antineoplastic drugs in wastewater treatment plants (WWTPs), but the formation of by-products that are more toxic or exhibit a different toxicity profile than the parent drug is frequently reported. This work evaluates the performance of a nanofiltration pilot unit, equipped with a Desal 5DK membrane, in the treatment of real WWTP effluents contaminated (without spiking) with eleven pharmaceuticals, five of which were never studied before. Average removals of 68 ± 23% were achieved for the eleven compounds, with decreasing risks from feed to permeate for aquatic organisms from receiving waterbodies (with the exception of cyclophosphamide, for which a high risk was estimated in the permeate). Aditionally, no significative impact on the growth and germination of three different seeds (Lepidium sativum, Sinapis alba, and Sorghum saccharatum) were determined for permeate matrix in comparison to the control.

Identification of cardiorespiratory toxic components of Nemopilema nomurai jellyfish venom using sequential chromatography methods.

Jellyfish stings pose a significant threat to humans in coastal areas worldwide, with venomous jellyfish species stinging millions of individuals annually. Nemopilema nomurai is one of the largest jellyfish species, with numerous tentacles rich in nematocysts. N. nomurai venom (NnV) is a complex mixture of proteins, peptides, and small molecules that serve as both prey-capture and defense mechanisms. Yet, the molecular identity of its cardiorespiratory and neuronal toxic components of NnV has not been clearly identified yet. Here, we isolated a cardiotoxic fraction, NnTP (Nemopilema nomurai toxic peak), from NnV using chromatographic methods. In the zebrafish model, NnTP exhibited strong cardiorespiratory and moderate neurotoxic effects. LC-MS/MS analysis identified 23 toxin homologs, including toxic proteinases, ion channel toxins, and neurotoxins. The toxins demonstrated a synergistic effect on the zebrafish, leading to altered swimming behavior, hemorrhage in the cardiorespiratory region, and histopathological changes in organs such as the heart, gill, and brain. These findings provide valuable insights into the mechanisms underlying the cardiorespiratory and neurotoxic effects of NnV, which could be useful in developing therapeutic strategies for venomous jellyfish stings.

Role of three-dimensional cell culture in therapeutics and diagnostics: an updated review.

Drug development and testing are a tedious and expensive process with a high degree of uncertainty in the clinical success and preclinical validation of manufactured therapeutic agents. Currently, to understand the drug action, disease mechanism, and drug testing, most therapeutic drug manufacturers use 2D cell culture models to validate the drug action. However, there are many uncertainties and limitations with the conventional use of 2D (monolayer) cell culture models for drug testing that are primarily attributed due to poor mimicking of cellular mechanisms, disturbance in environmental interaction, and changes in structural morphology. To overcome such odds and difficulties in the preclinical validation of therapeutic medications, newer in vivo drug testing cell culture models with higher screening efficiencies are required. One such promising and advanced cell culture model reported recently is the "three-dimensional cell culture model." The 3D cell culture models are reported to show evident benefits over conventional 2D cell models. This review article outlines and describes the current advancement in cell culture models, their types, significance in high-throughput screening, limitations, applications in drug toxicity screening, and preclinical testing methodologies to predict in vivo efficacy.

Deep Learning-Enabled Morphometric Analysis for Toxicity Screening Using Zebrafish Larvae.

Toxicology studies heavily rely on morphometric analysis to detect abnormalities and diagnose disease processes. The emergence of ever-increasing varieties of environmental pollutants makes it difficult to perform timely assessments, especially using in vivo models. Herein, we propose a deep learning-based morphometric analysis (DLMA) to quantitatively identify eight abnormal phenotypes (head hemorrhage, jaw malformation, uninflated swim bladder, pericardial edema, yolk edema, bent spine, dead, unhatched) and eight vital organ features (eye, head, jaw, heart, yolk, swim bladder, body length, and curvature) of zebrafish larvae. A data set composed of 2532 bright-field micrographs of zebrafish larvae at 120 h post fertilization was generated from toxicity screening of three categories of chemicals, i.e., endocrine disruptors (perfluorooctanesulfonate and bisphenol A), heavy metals (CdCl2 and PbI2), and emerging organic pollutants (acetaminophen, 2,7-dibromocarbazole, 3-monobromocarbazo, 3,6-dibromocarbazole, and 1,3,6,8-tetrabromocarbazo). Two typical deep learning models, one-stage and two-stage models (TensorMask, Mask R-CNN), were trained to implement phenotypic feature classification and segmentation. The accuracy was statistically validated with a mean average precision >0.93 in unlabeled data sets and a mean accuracy >0.86 in previously published data sets. Such a method effectively enables subjective morphometric analysis of zebrafish larvae to achieve efficient hazard identification of both chemicals and environmental pollutants.

Fluorescent hiPSC-derived MYH6-mScarlet cardiomyocytes for real-time tracking, imaging, and cardiotoxicity assays.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) hold great potential in the cardiovascular field for human disease modeling, drug development, and regenerative medicine. However, multiple hurdles still exist for the effective utilization of hiPSC-CMs as a human-based experimental platform that can be an alternative to the current animal models. To further expand their potential as a research tool and bridge the translational gap, we have generated a cardiac-specific hiPSC reporter line that differentiates into fluorescent CMs using CRISPR-Cas9 genome editing technology. The CMs illuminated with the mScarlet fluorescence enable their non-invasive continuous tracking and functional cellular phenotyping, offering a real-time 2D/3D imaging platform. Utilizing the reporter CMs, we developed an imaging-based cardiotoxicity screening system that can monitor distinct drug-induced structural toxicity and CM viability in real time. The reporter fluorescence enabled visualization of sarcomeric disarray and displayed a drug dose-dependent decrease in its fluorescence. The study also has demonstrated the reporter CMs as a biomaterial cytocompatibility analysis tool that can monitor dynamic cell behavior and maturity of hiPSC-CMs cultured in various biomaterial scaffolds. This versatile cardiac imaging tool that enables real time tracking and high-resolution imaging of CMs has significant potential in disease modeling, drug screening, and toxicology testing.

Utilizing a Population-Genetic Framework to Test for Gene-Environment Interactions between Zebrafish Behavior and Chemical Exposure.

Individuals within genetically diverse populations display broad susceptibility differences upon chemical exposures. Understanding the role of gene-environment interactions (GxE) in differential susceptibility to an expanding exposome is key to protecting public health. However, a chemical's potential to elicit GxE is often not considered during risk assessment. Previously, we've leveraged high-throughput zebrafish (Danio rerio) morphology screening data to reveal patterns of potential GxE effects. Here, using a population genetics framework, we apportioned variation in larval behavior and gene expression in three different PFHxA environments via mixed-effect modeling to assess significance of GxE term. We estimated the intraclass correlation (ICC) between full siblings from different families using one-way random-effects model. We found a significant GxE effect upon PFHxA exposure in larval behavior, and the ICC of behavioral responses in the PFHxA exposed population at the lower concentration was 43.7%, while that of the control population was 14.6%. Considering global gene expression data, a total of 3746 genes showed statistically significant GxE. By showing evidence that heritable genetics are directly affecting gene expression and behavioral susceptibility of individuals to PFHxA exposure, we demonstrate how standing genetic variation in a heterogeneous population such as ours can be leveraged to test for potential GxE.

Reproductive and developmental toxicity screening of bisphenol F by oral gavage in rats.

Bisphenol F (BPF, 4,4'-methylenediphenol) has recently been selected as an alternative to bisphenol A (BPA), which is used in the manufacturing of polycarbonates and epoxy resins. This study aimed to investigate the general, and reproductive/developmental effects of BPF. Therefore, BPF at dose levels of 0, 1, 5, 20, and 100 mg/kg/day was administered daily by oral gavage to Sprague-Dawley rats during the pre-mating, mating, gestation, and early lactation periods, and reproductive and developmental toxicities including general systemic toxicities were investigated. A decrease in body weight and food consumption was observed in the female rats treated with BPF at 20 and 100 mg/kg/day during the pre-mating and gestation periods. Additionally, gamma glutamyl transpeptidase levels were increased in the female rats administered 100 mg/kg/day. At 100 mg/kg/day, ovarian weight decreased and vaginal mucification increased according to a necropsy and histopathological examination, respectively. Moreover, the number of implantation sites and litter size decreased at 100 mg/kg/day. However, no significant BPF-related changes were observed in the male rats. Based on the results of this study, the no-observed-adverse-effect levels (NOAELs) of BPF for general systemic and reproductive effects were 5 and 20 mg/kg/day, respectively.

Reproductive and developmental toxicity screening test of new TiO2 GST in Sprague-Dawley rats.

TiO2 nanoparticles are widely used in paints, plastics, cosmetics, printing ink, rubber, food products, pharmaceuticals and other products (photocatalyst, etc.). However, there is little toxicological information during reproduction and developmental period. This study was performed to obtain safety data for new TiO2 powder, GST (Green Sludge Titanium) produced through sludge recycling of the sewage treatment plant for Reproduction/developmental toxicity screening test in Sprague-Dawley (SD) rats in according to the OECD test guideline (TG 421). Based on the results of the dose-range finding study (14-day repeated toxicity), GST was orally administered to rats at doses of 0, 500, 1000, and 2000 mg/kg B.W/day. Males were dosed for 35 days beginning 14 days before mating, and females for a maximum of 53 days beginning 14 days before mating to day 13 of lactation, including throughout the mating, gestation and lactation periods. In the reproductive and developmental examinations, there were no marked toxicities in terms of general clinical signs, body weight, food consumption, organ weights, macroscopic / microscopic findings, stages of spermatogenesis in the testis, reproductive finding (estrous cycle, copulation-fertility-gestation index), developmental finding (number of corpora lutea and implantations, pups parameters including live birth and viability index). The NOAEL for reproductive/developmental screening toxicity was concluded to be 2000 mg/kg/day under the present study conditions.

Generation of self-assembling cardiac organoids using hiPSC-derived cardiomyocytes.

The combination of the human induced pluripotent stem cell (hiPSC) and organoid technology enables the generation of human 3D culture systems, providing the opportunity to model human tissue-like structures in vitro. This protocol offers the details to generate and characterize self-assembling 3D cardiac organoids in a controlled and efficient manner based on hiPSC-derived cardiomyocytes. Cardiac organoids can be used as 3D-based assay systems and offer a wide range of applications in pharmacological and toxicological research as well as an alternative to animal experiments.

Identification of novel cyanoacrylate monomers for use in nanoparticle drug delivery systems prepared by miniemulsion polymerisation - A multistep screening approach.

Poly (alkyl cyanoacrylate) (PACA) polymeric nanoparticles (NPs) are promising drug carriers in drug delivery. However, the selection of commercially available alkyl cyanoacrylate (ACA) monomers is limited, because most monomers were designed for use in medical and industrial glues and later repurposed for drug encapsulation. This study therefore aimed to seek out novel ACA materials for use in NP systems using a toxicity led screening approach. A multistep strategy, including cytotoxicity screening of alcohols as degradation products of PACA (44 alcohols), NPs (14 polymers), and a final in vivo study (2 polymers) gave poly (2-ethylhexyl cyanoacrylate) PEHCA as a promising novel PACA candidate. For the first time, this work presents cytotoxicity data on several novel ACAs, PEHCA in vivo toxicity data, and miniemulsion polymerisation-based encapsulation of the cabazitaxel and NR688 in novel PACA candidates. Furthermore, several of the ACA candidates were compatible with a wider selection of lipophilic active pharmaceutical ingredients (APIs) versus commercially available controls. Combined, this work demonstrates the potential benefits of expanding the array of available ACA materials in drug delivery. Novel ACAs have the potential to encapsulate a wider range of APIs in miniemulsion polymerisation processes and may also broaden PACA applicability in other fields.

Phytochemical and Safety Evaluations of Finger Lime, Mountain Pepper, and Tamarind in Zebrafish Embryos.

Plants play a pivotal role in drug discovery, constituting 50% of modern pharmacopeia. Many human diseases, including age-related degenerative diseases, converge onto common cellular oxidative stress pathways. This provides an opportunity to develop broad treatments to treat a wide range of diseases in the ageing population. Here, we characterize and assess the toxicological effects of finger lime (Citrus australasica), mountain pepper (Tasmannia lanceolata), and small-leaved tamarind (Diploglottis australis) extracts. The characterization demonstrates that these Australian native plants have antioxidant potential and, importantly, they have high concentrations of distinct combinations of different antioxidant classes. Using zebrafish larvae as a high-throughput pre-clinical in vivo toxicology screening model, our experiment effectively discriminates which of these extracts (and at what exposure levels) are suitable for development towards future therapies. The LC50-96h for finger lime and tamarind were >480 mg/L, and 1.70 mg/L for mountain pepper. Critically, this work shows that adverse effects are not correlated to the properties of these antioxidants, thus highlighting the need for combining characterization and in vivo screening to identify the most promising plant extracts for further development. Thus, we present a high-throughput pre-clinical screening that robustly tests natural plant products to utilize the diversity of antioxidant compounds for drug development.

Extreme Interocular Asymmetry in an Atypical Case of a Hydroxychloroquine-Related Retinopathy.

Background and Objectives: Long-term hydroxychloroquine (HCQ) therapy can lead to retinal toxicity. Typically, it is characterized by a bull's eye maculopathy. More recently, a "pericentral" form of HCQ retinopathy that predominantly affects patients of Asian descent has been described. To our knowledge, this is the first reported case where such an asymmetry between the right and the left eye in the toxicity profile is observed. Case presentation: The patient presented with a 12-year exposure to HCQ at a daily dose of 4.35 mg/kg. She presented an inferior pericentral-only phenotype of HCQ toxicity on the right eye and a perifoveal-only toxicity on the left eye. Modest progression of toxicity was observed on both eyes over the seven years of follow-up, despite drug discontinuation. Conclusions: To our knowledge, this is the first time that two different phenotypes of HCQ-related retinopathy are found in the same patient, challenging our understanding of the pathophysiology of HCQ retinal toxicity.

Bioluminescence of Aliivibrio Fischeri in Artificial Seawater and Its Application in Fungicide Sensing.

It has been well investigated that the bioluminescence (BL) intensity of marine luminous bacteria is enhanced depending on cell density. In contrast, the correlation between seawater components and BL intensity is still a challenging subject to be addressed. In addition, the marine luminous bacteria rapidly lose the BL intensity when exposed to toxic substances, but unclear to fungicides. Herein, we introduce a new approach to investigate (i) the correlation between the components of artificial seawater (ASW) and BL intensity and (ii) the corresponding protocol to determine the susceptibility of marine luminous bacteria to fungicide using A. fischeri. The examples show that (i) ionic ingredients (K+, HCO3-, and SO42-) activate the BL cell density independently and (ii) A. fischeri cultured with the ionic ingredients shows the susceptibility to fungicide (sodium ortho-phenylphenol and imazalil). These protocols provide a new insight how to investigate the correlation between inorganic salts and BL intensity in a low cell density environment such as seawater.

Microelectrode array membranes to simultaneously assess cardiac and neurological signals of xenopus laevis under chemical exposures and environmental changes.

Simultaneous monitoring of electrocardiogram (ECG) and electroencephalogram (EEG) in studied animal models requires innovative engineering techniques that can capture minute physiological changes. However, this is often administered with a bulky and/or invasive system that may cause discomfort to animals and signal distortions. Here, we develop an integrated bioelectronic sensing system to provide simultaneous recordings of ECG and EEG in real-time for Xenopus laevis. The microelectrode array (MEA) membrane and the distinct anatomy of Xenopus offer noninvasive multi-modal electrophysiological monitoring with favorable spatial resolution. The system was validated under different environmental conditions, including drug exposure and temperature changes. Under the exposure of Pentylenetetrazol (PTZ), an epilepsy-inducing drug, clear ECG and EEG alterations, including frequent ictal and interictal EEG events, 30 dB average EEG amplitude elevations, abnormal ECG morphology, and heart rate changes, were observed. Furthermore, the ECG and EEG were monitored and analyzed under different temperatures. A decrease in relative power of delta band was observed when cold environment was brought about, in contrast to an increase in relative power of other higher frequency bands while the ECG remained stable. Overall, the real-time electrophysiology monitoring system using the Xenopus model holds potential for many applications in drug screening and remote environmental monitoring.

Suitability of Human Mesenchymal Stem Cells Derived from Fetal Umbilical Cord (Wharton's Jelly) as an Alternative In Vitro Model for Acute Drug Toxicity Screening.

Preclinical toxicity screening is the first and most crucial test that assesses the safety of new candidate drugs before their consideration for further evaluation in clinical trials. In vitro drug screening using stem cells has lately arisen as a promising alternative to the "gold standard" of animal testing, but their suitability and performance characteristics in toxicological studies have so far not been comprehensively investigated. In this study, we focused on the evaluation of human mesenchymal stem cells isolated from the matrix (Wharton's jelly) of fetal umbilical cord (WJSCs), which bear enhanced in vitro applicability due to their unique biological characteristics. In order to determine their suitability for drug-related cytotoxicity assessment, we adopted a high-throughput methodology that evaluated their sensitivity to a selected panel of chemicals in different culture environments. Cytotoxicity was measured within 48 h by means of MTS and/or NRU viability assays, and was compared directly (in vitro) or indirectly (in silico) to adult human mesenchymal stem cells and to reference cell lines of human and murine origin. Our data clearly suggest that human WJSCs can serve as a robust in vitro alternative for acute drug toxicity screening by uniquely combining rapid and versatile assay setup with high-throughput analysis, good representation of human toxicology, high reproducibility, and low cost.

Assessing Fish Immunotoxicity by Means of In Vitro Assays: Are We There Yet?

There is growing awareness that a range of environmental chemicals target the immune system of fish and may compromise the resistance towards infectious pathogens. Existing concepts to assess chemical hazards to fish, however, do not consider immunotoxicity. Over recent years, the application of in vitro assays for ecotoxicological hazard assessment has gained momentum, what leads to the question whether in vitro assays using piscine immune cells might be suitable to evaluate immunotoxic potentials of environmental chemicals to fish. In vitro systems using primary immune cells or immune cells lines have been established from a wide array of fish species and basically from all immune tissues, and in principal these assays should be able to detect chemical impacts on diverse immune functions. In fact, in vitro assays were found to be a valuable tool in investigating the mechanisms and modes of action through which environmental agents interfere with immune cell functions. However, at the current state of knowledge the usefulness of these assays for immunotoxicity screening in the context of chemical hazard assessment appears questionable. This is mainly due to a lack of assay standardization, and an insufficient knowledge of assay performance with respect to false positive or false negative signals for the different toxicant groups and different immune functions. Also the predictivity of the in vitro immunotoxicity assays for the in vivo immunotoxic response of fishes is uncertain. In conclusion, the currently available database is too limited to support the routine application of piscine in vitro assays as screening tool for assessing immunotoxic potentials of environmental chemicals to fish.