Different Classes Of Toxicants Research Articles

Specificity of time- and dose-dependent morphological endpoints in the fish embryo acute toxicity (FET) test for substances with diverse modes of action: the search for a \u201cfingerprint\u201d

The fish embryo acute toxicity (FET) test with the zebrafish (Danio rerio) embryo according to OECD TG 236 was originally developed as an alternative test method for acute fish toxicity testing according to, e.g., OECD TG 203. Given the versatility of the protocol, however, the FET test has found application beyond acute toxicity testing as a common tool in environmental hazard and risk assessment. Whereas the standard OECD guideline is restricted to four core endpoints (coagulation as well as lack of somite formation, heartbeat, and tail detachment) for simple, rapid assessment of acute toxicity, further endpoints can easily be integrated into the FET test protocol. This has led to the hypothesis that an extended FET test might allow for the identification of different classes of toxicants via a “fingerprint” of morphological observations. To test this hypothesis, the present study investigated a set of 18 compounds with highly diverse modes of action with respect to acute and sublethal endpoints. Especially at higher concentrations, most observations proved toxicant-unspecific. With decreasing concentrations, however, observations declined in number, but gained in specificity. Specific observations may at best be made at test concentrations ≤ EC10. The existence of a “fingerprint” based on morphological observations in the FET is, therefore, highly unlikely in the range of acute toxicity, but cannot be excluded for experiments at sublethal concentrations.

High-content analysis of larval phenotypes for the screening of xenobiotic toxicity using Phallusia mammillata embryos

In recent years, pollution of surface waters with xenobiotic compounds became an issue of concern in society and has been the object of numerous studies. Most of these xenobiotic compounds are man-made molecules and some of them are qualified as endocrine disrupting chemicals (EDCs) when they interfere with hormones actions. Several studies have investigated the teratogenic impacts of EDCs in vertebrates (including marine vertebrates). However, the impact of such EDCs on marine invertebrates is much debated and still largely obscure. In addition, DNA-altering genotoxicants can induce embryonic malformations. The goal of this study is to develop a reliable and effective test for assessing toxicity of chemicals using embryos of the ascidian (Phallusia mammillata) in order to find phenotypic signatures associated with xenobiotics. We evaluated embryonic malformations with high-content analysis of larval phenotypes by scoring several quantitative and qualitative morphometric endpoints on a single image of Phallusia tadpole larvae with semi-automated image analysis. Using this approach we screened different classes of toxicants including genotoxicants, known or suspected EDCs and nuclear receptors (NRs) ligands. The screen presented here reveals a specific phenotypic signature for ligands of retinoic acid receptor/retinoid X receptor. Analysis of larval morphology combined with DNA staining revealed that embryos with DNA aberrations displayed severe malformations affecting multiple aspects of embryonic development. In contrast EDCs exposure induced no or little DNA aberrations and affected mainly neural development. Therefore the ascidian embryo/larval assay presented here can allow to distinguish the type of teratogenicity induced by different classes of toxicants.

New Insights into Mechanisms of Endocrine-Disrupting Chemicals in Thyroid Diseases: The Epigenetic Way.

In recent years, the presence in the environment of chemical compounds with thyroid-disrupting effects is progressively increased. This phenomenon has risen concern for human health as the preservation of thyroid system homeostasis is essential for fetal development and for maintaining psychological and physiological wellbeing. An increasing number of studies explored the role of different classes of toxicants in the occurrence and severity of thyroid diseases, but large epidemiological studies are limited and only a few animal or in vitro studies have attempted to identify the mechanisms of chemical action. Recently, epigenetic changes such as alteration of methylation status or modification of non-coding RNAs have been suggested as correlated to possible deleterious effects leading to different thyroid disorders in susceptible individuals. This review aims to analyze the epigenetic alterations putatively induced by chemical exposures and involved in the onset of frequent thyroid diseases such as thyroid cancer, autoimmune thyroiditis and disruption of fetal thyroid homeostasis.

Human Induced Pluripotent Stem Cell-Derived 3D-Neurospheres Are Suitable for Neurotoxicity Screening

We present a hiPSC-based 3D in vitro system suitable to test neurotoxicity (NT). Human iPSCs-derived 3D neurospheres grown in 96-well plate format were characterized timewise for 6-weeks. Changes in complexity and homogeneity were followed by immunocytochemistry and transmission electron microscopy. Transcriptional activity of major developmental, structural, and cell-type-specific markers was investigated at weekly intervals to present the differentiation of neurons, astrocytes, and oligodendrocytes. Neurospheres were exposed to different well-known toxicants with or without neurotoxic effect (e.g., paraquat, acrylamide, or ibuprofen) and examined at various stages of the differentiation with an ATP-based cell viability assay optimized for 3D-tissues. Concentration responses were investigated after acute (72 h) exposure. Moreover, the compound-specific effect of rotenone was investigated by a panel of ER-stress assay, TUNEL assay, immunocytochemistry, electron microscopy, and in 3D-spheroid based neurite outgrowth assay. The acute exposure to different classes of toxicants revealed distinct susceptibility profiles in a differentiation stage-dependent manner, indicating that hiPSC-based 3D in vitro neurosphere models could be used effectively to evaluate NT, and can be developed further to detect developmental neurotoxicity (DNT) and thus replace or complement the use of animal models in various basic research and pharmaceutical applications.

Set of enzymatic bioassays for assessment of soil pollution

A concept of the comprehensive assessment of soil contamination is proposed. In this concept, the conclusion regarding the presence of toxic substances in a sample, which is being analyzed, is connected with the inhibition of enzymatic reactions that are responsible for various functions of a live organism, such as luminescence, respiration, etc., these functions are taken as test functions in classical bioassays, which are using live objects (luminous bacteria, daphnia, algae, etc.). The regularities of the impact of different classes of toxicants on the activity of individual enzymes or coupled oligo-enzyme chains have been established. These enzyme reactions are selected as potential test objects - markers of pollution. The set of enzymatic bioassays consists of three enzyme systems with maximum sensitivity to different classes of toxicants: butyrylcholinesterase, NAD(P)H:FMN‑oxidoreductase + luciferase and lactate dehydrogenase + NAD(P)H:FMN‑oxidoreductase + luciferase. The possibility of using enzymes instead of living organisms in the bioassay of natural complex systems is shown.

Set of Enzymatic Bioassays for Assessment of Soil Contamination.

A concept of the comprehensive assessment of soil contamination is proposed. According to it, the conclusion regarding the presence of toxic substances in the analyzed sample is based on the inhibition of enzymatic reactions responsible for various functions of a living organism, such as luminescence, respiration, etc. These functions are taken as test functions in classical bioassays with the use of living objects (luminous bacteria, daphnia, algae, and others). The regularities of the impact of different classes of toxicants on the activity of particular enzymes or coupled oligo-enzyme chains have been established. These enzyme reactions are selected as potential test objects: markers of contamination. Three enzyme systems with the maximal sensitivity to different classes of toxicants have been chosen for the set of enzymatic bioassays: butyrylcholinesterase, NAD(P)H:FMN-oxidoreductase + luciferase, and lactate dehydrogenase + NAD(P)H:FMN-oxidoreductase + luciferase. The possibility to use enzymes instead of living organisms in the bioassay of natural complex systems has been shown.

Proteomics in mechanistic toxicology: history, concepts, achievements, caveats, and potential.

Toxicoproteomics can be defined as the application of proteomic approaches to the understanding of toxicology problems, and this review deals with the various types of applications that have been described in the literature. Toxicoproteomics has been applied to very different classes of toxicants, from drugs and natural products to metals, or from industrial chemicals to nanoparticles and nanofibers. It has also been applied to address questions at different levels, from the search of the primary molecular targets of toxicants to the deciphering of the molecular responses of cells and tissues to toxicants. Although restricted to mammalian cells and tissues, this paper reviews these two levels of investigation and the different application areas of toxicoproteomics, leading to the discussion of the advantages and drawbacks of the most popular proteomic platforms. Some of the pending questions in toxicoproteomics are also critically addressed, such as the specificity, validation, and result hierarchization issues. The question of shared mechanisms, which are encountered in many toxicoproteomic papers dealing with different toxicants, is also discussed. Finally, the future of toxicoproteomics is briefly outlined.

Protein kinase C is a target for diverse developmental neurotoxicants: Transcriptional responses to chlorpyrifos, diazinon, dieldrin and divalent nickel in PC12 cells

Unrelated developmental neurotoxicants can elicit similar functional outcomes, whereas agents in the same class may differ. We compared two organophosphate insecticides (chlorpyrifos, diazinon) with an organochlorine (dieldrin) and a metal (Ni 2+) for similarities and differences in their effects on gene expression encoding subtypes of protein kinase C and their modulators, a cell signaling cascade that integrates the actions of neurotrophic factors involved in brain development. We conducted evaluations in PC12 cells, a model for neuronal development, with each agent introduced at 30 μM for 24 or 72 h, treatments devoid of cytotoxicity. Chlorpyrifos evoked by far the largest effect, with widespread upregulation of multiple genes; the effects were greater during neurodifferentiation than when cells were exposed prior to differentiation. Diazinon had smaller and less widespread effects, consistent with its lesser long-term impact on synaptic function and behavior noted for in vivo exposures in developing rats. Surprisingly, the effects of diazinon, dieldrin and Ni 2+ showed basic similarities despite the fact that all three come from different classes of toxicants. Our findings provide some of the first evidence for a specific mechanistic cascade contributing to the cholinesterase-independent developmental neurotoxicant actions of chlorpyrifos and its differences from diazinon, while at the same time identifying mechanistic convergence between otherwise unrelated toxicants that provides predictions about common neurodevelopmental outcomes. These results further show how combined use of cell cultures and microarray technology can guide future in vivo work on diverse developmental neurotoxicants.

Interaction of mitochondrial respiratory inhibitors and excitotoxins potentiates cell death in hippocampal slice cultures

The broad-spectrum insecticide rotenone, an inhibitor of complex I of the mitochondrial electron transport chain (ETC), gives rise to oxidative stress and bioenergetic failure. Pesticides including rotenone have been implicated in human neurodegenerative diseases, including Parkinson's disease. Another intensively investigated hypothesis of neurodegenerative disease involves the toxic action of the excitatory neurotransmitter glutamate. In the present study, we determined whether concomitant exposure of rotenone plus tetraethylammonium chloride (TEA) or the specific glutamate receptor agonists N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) would cause greater cell death in organotypic hippocampal slice cultures than when given separately. Low, sublethal rotenone (100 nM), TEA (0.5-2.0 mM), NMDA (1.0-10 microM), and AMPA (1.0-10 microM) alone resulted in little cell death as determined by propidium iodide fluorescence. However, cell death was significantly to dramatically potentiated when the hippocampal slices were coincubated with comparable concentrations of rotenone plus TEA, NMDA, or AMPA. Similarly, in the presence of 10 microM NMDA, ETC inhibitors blocking other mitochondrial complexes also potentiated cell death. Immunohistochemical analysis using glial fibrillary acidic protein antibody determined that the cell death was preferentially neuronal. These results demonstrate that two different classes of toxicants can interact, resulting in potentiation of neurotoxicity, and further suggest that a combinatorial therapeutic approach may be required to ameliorate the potentiated cell death.

Construction of a nrdA::luxCDABE Fusion and Its Use in Escherichia coli as a DNA Damage Biosensor.

The promoter of nrdA gene which is related with DNA synthesis was used to construct a DNA damage sensitive biosensor. A recombinant bioluminescent E. coli strain, BBTNrdA, harboring a plasmid with the nrdA promoter fused to the luxCDABE operon, was successfully constructed. Its response to various chemicals including genotoxic chemicals substantiates it as a DNA damage biosensor. In characterization, three different classes of toxicants were used: DNA damaging chemicals, oxidative stress chemicals, and phenolics. BBTNrdA only responded strongly to DNA damaging chemicals, such as nalidixic acid (NDA), mitomycin C (MMC), 1-methyl-1-nitroso-N-methylguanidine (MNNG), and 4-nitroquinoline N-oxide (4-NQO). In contrast, there were no responses from the oxidative stress chemicals and phenolics, except from hydrogen peroxide (H2O2) which is known to cause DNA damage indirectly. Therefore, the results of the study demonstrate that BBTNrdA can be used as a DNA damage biosensor.

Characterization ofgltA: luxCDABE fusion inEscherichia coli as a toxicity biosensor

The use ofgltA gene, as a new biomarker for environmental stress biomonitoring, was investigated because of its key position as the first enzyme of the tricarboxylic acid (TCA) cycle. A recombinant bioluminescentEscherichia coli strain, EBJM2, was constructed using a plasmid carrying the citrate synthase (gltA) promoter transcribing thePhotorhabdus luminescens luxCDABE genes (gltA::luxCDABE). The responses from this strain were studied with five different classes of toxicants: DNA damage chemicals, phenolics, oxidative-stress chemicals, PAHs, and organic solvents. EBJM2 responded strongly to DNA damage chemicals, such as mitomycin C (MMC) and methyl-nitro-nitrosoguanidine (MNNG), and nalidixic acid with the strongest responses. In contrast, tests with several compounds from the other four classes of toxicants gave no significant response. Therefore, EBJM2 was found to be sensitive to DNA damage chemicals.

Supervised Sorting: Training Computers to Classify Toxicants

Supervised Sorting: Training Computers to Classify Toxicants

Discriminating Different Classes of Toxicants by Transcript Profiling

Male rats were treated with various model compounds or the appropriate vehicle controls. Most substances were either well-known hepatotoxicants or showed hepatotoxicity during preclinical testing. The aim of the present study was to determine if biological samples from rats treated with various compounds can be classified based on gene expression profiles. In addition to gene expression analysis using microarrays, a complete serum chemistry profile and liver and kidney histopathology were performed. We analyzed hepatic gene expression profiles using a supervised learning method (support vector machines; SVMs) to generate classification rules and combined this with recursive feature elimination to improve classification performance and to identify a compact subset of probe sets with potential use as biomarkers. Two different SVM algorithms were tested, and the models obtained were validated with a compound-based external cross-validation approach. Our predictive models were able to discriminate between hepatotoxic and nonhepatotoxic compounds. Furthermore, they predicted the correct class of hepatotoxicant in most cases. We provide an example showing that a predictive model built on transcript profiles from one rat strain can successfully classify profiles from another rat strain. In addition, we demonstrate that the predictive models identify nonresponders and are able to discriminate between gene changes related to pharmacology and toxicity. This work confirms the hypothesis that compound classification based on gene expression data is feasible.

Discriminating different classes of toxicants by transcript profiling.

Male rats were treated with various model compounds or the appropriate vehicle controls. Most substances were either well-known hepatotoxicants or showed hepatotoxicity during preclinical testing. The aim of the present study was to determine if biological samples from rats treated with various compounds can be classified based on gene expression profiles. In addition to gene expression analysis using microarrays, a complete serum chemistry profile and liver and kidney histopathology were performed. We analyzed hepatic gene expression profiles using a supervised learning method (support vector machines; SVMs) to generate classification rules and combined this with recursive feature elimination to improve classification performance and to identify a compact subset of probe sets with potential use as biomarkers. Two different SVM algorithms were tested, and the models obtained were validated with a compound-based external cross-validation approach. Our predictive models were able to discriminate between hepatotoxic and nonhepatotoxic compounds. Furthermore, they predicted the correct class of hepatotoxicant in most cases. We provide an example showing that a predictive model built on transcript profiles from one rat strain can successfully classify profiles from another rat strain. In addition, we demonstrate that the predictive models identify nonresponders and are able to discriminate between gene changes related to pharmacology and toxicity. This work confirms the hypothesis that compound classification based on gene expression data is feasible.

Haber's Rule: The Search for Quantitative Relationships in Toxicology

Dose (or concentration) and time of exposure are both important in determining the intensity of response to a toxic agent. For a given response intensity, Haber's Rule (c×t=K) has been proposed as a law of toxicology, but this rule is just one special case of a more general relationship c×t m=K, where the exponent m is quite variable. For inhaled toxicants m generally has a value between 0 and 1, whereas for carcinogens, m is usually between 1 and 5. The absence of a universal value for m, or one that is generally applicable to different classes of toxicants, makes it not yet possible to develop a Haber-type rule with which to extrapolate successfully between exposure scenarios

Haber's Rule: The Search for Quantitative Relationships in Toxicology

Dose (or concentration) and time of exposure are both important in determining the intensity of response to a toxic agent. For a given response intensity, Haber's Rule (c×t=K) has been proposed as a law of toxicology, but this rule is just one special case of a more general relationship c×t m =K, where the exponent m is quite variable. For inhaled toxicants m generally has a value between 0 and 1, whereas for carcinogens m is usually between 1 and 5. The absence of a universal value for m, or one that is generally applicable to different classes of toxicants, makes it not yet possible to develop a Haber-type rule with which to extrapolate successfully between exposure scenarios.

Applications of Gene Arrays in Environmental Toxicology: Fingerprints of Gene Regulation Associated with Cadmium Chloride, Benzo(a)pyrene, and Trichloroethylene

Toxicity testing of unknown chemicals currently uses a number of short-term bioassays. These tests are costly and time consuming, require large numbers of animals, and generally focus on a single end point. The recent development of DNA arrays provides a potential mechanism for increasing the efficiency of standard toxicity testing through genome-wide assessments of gene regulation. In this study, we used DNA arrays containing 148 genes for xenobiotic metabolizing enzymes, DNA repair enzymes, heat shock proteins, cytokines, and housekeeping genes to examine gene expression patterns in the liver in response to cadmium chloride, benzo(a)pyrene (BaP), and trichloroethylene (TCE). Dose-response studies were carried out in mice for each chemical; each produced a unique pattern of gene induction. As expected, CdCl2 markedly up-regulated metallothionine I and II (5- to 10,000-fold at the highest doses) and several of the heat shock/stress response proteins and early response genes. In contrast, administration of BaP up-regulated only Cyp1a1 and Cyp1a2 genes and produced no significant increases in any of the stress response genes or any of the DNA repair genes present on the array. Likewise, TCE-induced gene induction was highly selective; only Hsp 25 and 86 and Cyp2a were up-regulated at the highest dose tested. Microarray analysis with a highly focused set of genes is capable of discriminating between different classes of toxicants and has potential for differentiating highly noxious versus more subtle toxic agents. These data suggest that use of microarrays to evaluate the potential hazards of unknown chemicals or chemical mixtures must include multiple doses and time points to provide effective assessments of potential toxicity of these substances.

Applications of gene arrays in environmental toxicology: fingerprints of gene regulation associated with cadmium chloride, benzo(a)pyrene, and trichloroethylene.

Toxicity testing of unknown chemicals currently uses a number of short-term bioassays. These tests are costly and time consuming, require large numbers of animals, and generally focus on a single end point. The recent development of DNA arrays provides a potential mechanism for increasing the efficiency of standard toxicity testing through genome-wide assessments of gene regulation. In this study, we used DNA arrays containing 148 genes for xenobiotic metabolizing enzymes, DNA repair enzymes, heat shock proteins, cytokines, and housekeeping genes to examine gene expression patterns in the liver in response to cadmium chloride, benzo(a)pyrene (BaP), and trichloroethylene (TCE). Dose-response studies were carried out in mice for each chemical; each produced a unique pattern of gene induction. As expected, CdCl2 markedly up-regulated metallothionine I and II (5- to 10,000-fold at the highest doses) and several of the heat shock/stress response proteins and early response genes. In contrast, administration of BaP up-regulated only Cyp1a1 and Cyp1a2 genes and produced no significant increases in any of the stress response genes or any of the DNA repair genes present on the array. Likewise, TCE-induced gene induction was highly selective; only Hsp 25 and 86 and Cyp2a were up-regulated at the highest dose tested. Microarray analysis with a highly focused set of genes is capable of discriminating between different classes of toxicants and has potential for differentiating highly noxious versus more subtle toxic agents. These data suggest that use of microarrays to evaluate the potential hazards of unknown chemicals or chemical mixtures must include multiple doses and time points to provide effective assessments of potential toxicity of these substances.

High Performance Liquid Chromatography in Veterinary Toxicology

Abstract Since the late 1960's, high performance liquid chromatography (HPLC) has performed an increasingly important role as a separative analytical technique. Its range of application is far wider than that of gas chromatography which makes it attractive to those disciplines that require the capability to analyze a diversity of compound in a variety of matrices. The field of veterinary toxicology is such an area. Veterinary toxicology laboratories must have the capability to identify and quantify compounds from many different classes of toxicants. This demand requires a wide range of analytical capabilities so that inorganic, volatile organic, and complex high molecular weight toxicantscan a11 be quickly determined. The matrices from which the analytes must be separated are frequently complex; typically feeds, biological fluids, and tissues. Often clues as to the nature of the analyte are lacking in the sample history requiring methods that can screen for many classes of compounds. This review attempts ...

Field and Laboratory Protocols for Evaluating the Effects of Chemical Substances on Aquatic Life

AbstractOne of the difficulties of evaluating the hazard associated with the release of a chemical substance into the environment is to determine how much testing is necessary to delimit its effects adequately. Since different classes of toxicants have different biological effects, it would be wasteful of both time and money to carry out extremely detailed and comprehensive tests for even the most innocuous materials. It would be equally inappropriate to carry out limited tests for very dangerous materials—as painful experience has already shown. Protocols to sort out which materials require extensive testing and which do not, as well as which decision criteria should be used at each stage in a test series, are essential to the establishment of reasonable water quality standards. This is true not only for laboratory studies but also for assessments of effects in the receiving system itself. Two illustrative protocols, one for the laboratory studies of aquatic organisms and the other for field studies in freshwater receiving systems, are described.