Long-term behavioral impairment following acute embryonic ethanol exposure in zebrafish.

BACKGROUND: Developmental exposure to ethanol has long been known to cause persisting neurobehavioral impairment. However, the neural and behavioral mechanisms underlying these deficits and the importance of exposure timing are not well-characterized. Given the importance of timing and sequence in neurodevelopment it would be expected that alcohol intoxication at different developmental periods would result in distinct neurobehavioral consequences. METHODS: Zebrafish embryos were exposed to ethanol (0%, 1%, 3%) at either 8-10 or 24-27 h post-fertilization (hpf) then reared to adolescence and evaluated on several behavioral endpoints. Habituation to a repeated environmental stimulus and overall sensorimotor function were assessed using a tap startle test; measurements of anxiety and exploration behavior were made following introduction to a novel tank; and spatial discrimination learning was assessed using aversive control in a three-chambered apparatus. Overt signs of dysmorphogenesis were also scored (i.e. craniofacial malformations, including eye diameter and midbrain-hindbrain boundary morphology). RESULTS: Ethanol treated fish were more active both at baseline and following a tap stimulus compared to the control fish and were hyperactive when placed in a novel tank. These effects were more prominent following exposure at 24-27 hpf than with the earlier exposure window, for both dose groups. Increases in physical malformation were only present in the 3% ethanol group; all malformed fish were excluded from behavioral testing. DISCUSSION: These results suggest specific domains of behavior are affected following ethanol exposure, with some but not all of the tests revealing significant impairment. The behavioral phenotypes following distinct exposure windows described here can be used to help link cellular and molecular mechanisms of developmental ethanol exposure to functional neurobehavioral effects.

Cadmium uptake kinetics in rat hepatocytes: correction for albumin binding.

The relationship between cytotoxicity and kinetics of cadmium uptake was investigated in primary rat hepatocyte cultures. Primary rat hepatocytes were exposed to cadmium concentrations ranging from 1.0 to 80 micro M in albumin-free buffer or 32 to 8,000 microM in buffer containing physiological concentrations of bovine serum albumin (600 micro M) for 1 h, and cellular toxicity was observed at 23 h postexposure. Hepatocytes exposed to cadmium in the presence of albumin appeared less sensitive to cadmium toxicity when compared to cells exposed in the absence of albumin. The experimentally derived 23-h postexposure EC(50)s for hepatocytes exposed to cadmium in both presence and absence of albumin was 65.5 +/- 2.4 and 14.3 +/- 3.9 microM, respectively. A Scatchard plot of cadmium binding to albumin suggested two high-affinity binding sites. The observed uptake of cadmium by hepatocytes in the absence and presence of albumin consisted of a composite fast uptake rate and cell membrane association (Component I), and a slow, sustained uptake rate (Component II). Cadmium uptake rates in hepatocytes, based on total medium cadmium concentrations, indicated that Component II uptake rates were four times faster under albumin-free exposure conditions. However, when uptake rates were evaluated, based on the calculated equilibrium concentration of free cadmium in the exposure buffer, uptake rates in hepatocytes exposed in the presence of albumin were two times as fast. This faster cadmium uptake in the presence of albumin may result from diffusion-limited, nonequilibrium conditions occurring at the cell surface.

Simulation of trichloroacetic acid kinetics in the isolated perfused rat liver using a biologically based kinetic model.

Trichloroacetic acid (TCA) is a contaminant of drinking water. It induces peroxisome proliferation in livers of rats and mice and is hepatocarcinogenic in the latter species. Previous experimental studies of the kinetics of TCA in the isolated perfused rat liver (IPRL) at two doses have been reported. To gain more insight into the mechanistic processes controlling TCA kinetics in the liver a biologically based kinetic (BBK) model for the IPRL was used to analyze the experimental data. The IPRL was exposed to 25, 250, or 1000 microM TCA for 2 h in a recirculating perfusion system. These doses were not cytotoxic. The BBK model simulated the TCA concentration in perfusion medium and liver, and the biliary excretion of TCA. Separate protein binding studies showed that over 90% of TCA was bound to albumin in the perfusion medium whereas binding in liver homogenate was much lower. Integrating the information on protein binding into the BBK model, the hepatic uptake of TCA and its biliary excretion could be fitted assuming asymmetrical saturable transport at the sinusoidal membrane and linear transport at the bile canalicular membrane. To validate the BBK model, additional washout experiments were conducted in which the perfusion medium was replaced with TCA-free medium after 30 min of exposure of the liver to 1000 microM TCA. This approach illustrates the usefulness of BBK modeling for analyzing experimental kinetic data and gaining insight in kinetic mechanisms controlling the behavior of a chemical in the liver.

QSAR modeling of oxidative stress in vitro following hepatocyte exposures to halogenated methanes.

Volatile halogenated aliphatic compounds are among those chemicals that can cause oxidative stress in vitro and in vivo. Relationships can be identified between the potential of these chemicals to elicit certain biological responses and their specific chemical descriptors, such as molecular orbital energies (LUMO) or partition coefficients (logP). A quantitative structure-activity relationship (QSAR) model has not been reported previously for the potential of a series of brominated and chlorinated methanes to induce oxidative stress in primary rat hepatocytes. By utilizing a novel in vitro methodology to expose cultures of rat primary hepatocytes to volatile chemicals, biological responses were assessed from exposures of hepatocytes to individual halogenated methanes. Indicators of lipid peroxidation, reactive oxygen species and cytotoxicity were measured. For the 10 brominated and chlorinated methanes tested, semi-empirical molecular orbital methods were used to calculate the physical/chemical descriptors used in the QSAR models. These models were used to explain the relative potential for a given halogenated methane to induce markers of oxidative stress or related damage in vitro. The results showed that certain descriptors, such as the molecular orbital energies, bond lengths, and lipophilicity are quantitatively correlated with induction of indicators for oxidative stress and cytotoxicity by halogenated methanes in primary rat hepatocytes.

In vitro toxicity assessment of a new series of high energy compounds.

Hydrazine is an aircraft fuel and propellant used by the US Air Force. Due to its toxicity the Propulsion Directorate of the Air Force Research Laboratory (AFRL/PR) has investigated alternative chemicals to replace hydrazine. AFRL/PR has synthesized a series of high energy chemicals (HECs), primarily hydrazine derivatives and amino containing compounds such as hydrazinium nitrate (HZN), 2-hydroxyethyl-hydrazine nitrate (HEHN), diethyl hydrazine nitrate (DEHN), ethanolamine nitrate (EAN), histamine dinitrate (HDN) and methoxylamine nitrate (MAN) to study as alternative chemical candidates. Although HECs are reliable constituents of powered propellant systems, they constitute an important class of toxic agents to which military and civilian personnel can be exposed. The current study was undertaken to examine the toxicity of HECs in primary hepatocytes in vitro. The effects of short-term exposure (4 h) of hepatocytes to HECs were investigated with reference to viability, mitochondrial function and oxidative stress markers. The results showed a decrease in mitochondrial activity, increase in lactate dehydrogenase (LDH) leakage and depletion of reduced glutathione (GSH) levels. The levels of reactive oxygen species (ROS) increased dose dependently in HZN, MAN and HDN exposed cells. However, there was no induction of ROS generation in EAN, DEHN and HEHN exposed cells. Depletion of GSH in hepatocytes by buthionine sulfoximine (BSO) prior to exposure to HZN increased its toxicity. The results suggest that at least one mechanism of HEC toxicity is mediated through oxidative stress.

In vitro toxicities of experimental jet fuel system ice-inhibiting agents.

One research emphasis within the Department of Defense has been to seek the replacement of operational compounds with alternatives that pose less potential risk to human and ecological systems. Alternatives to glycol ethers, such as diethylene glycol monomethyl ether (M-DE), were investigated for use as jet fuel system ice-inhibiting agents (FSIIs). This group of chemicals includes three derivatives of 1,3-dioxolane-4-methanol (M-1, M-2, and M-3) and a 1,3-dioxane (M-27). In addition, M-DE was evaluated as a reference compound. Our approach was to implement an in vitro test battery based on primary rat hepatocyte cultures to perform initial toxicity evaluations. Hepatocytes were exposed to experimental chemicals (0, 0.001, 0.01, 0.1, 1, 10 mM dosages) for periods up to 24 h. Samples were assayed for lactate dehydrogenase (LDH) release, MTT dye reduction activity, glutathione level, and rate of protein synthesis as indicators of toxicity. Of the compounds tested, M-1, especially at the 10-mM dose, appeared to be more potent than the other chemicals, as measured by these toxicity assays. M-DE, the current FSII, elicited little response in the toxicity assays. Although some variations in toxicity were observed at the 10-mM dose, the in vitro toxicities of the chemicals tested (except for M-1) were not considerably greater than that of M-DE.

In vivo kinetics of trichloroacetate in male Fischer 344 rats.

Trichloroacetate (TCA) is a toxicologically important metabolite of the industrial solvents trichloroethylene and tetrachloroethylene, and a by-product of the chlorination of drinking water. Tissue disposition and elimination of 14C-TCA were investigated in male Fischer 344 rats injected iv with 6.1, 61, or 306 micromol TCA/kg body weight. Blood and tissues were collected at various time points up to 24 h. No metabolites were observed in plasma, urine, or tissue extracts. Overall TCA kinetics in tissues were similar at all doses. Based on similar terminal elimination rate constants, tissues could be divided into three classes: plasma, RBC, muscle, and fat; kidney and skin; and liver, small intestine, and large intestine. Nonextractable radiolabel, assumed to be biologically incorporated metabolites in both liver and plasma, increased with time, peaking at 6-9 h postinjection. The fraction of the initial dose excreted in the urine at 24 h increased from 67% to 84% as the dose increased, whereas fecal excretion decreased from 7% to 4%. The cumulative elimination of TCA as CO2 at 24 h decreased from 12% to 8% of the total dose. Two important kinetic processes were identified: a) hepatic intracellular concentrations of TCA were significantly greater than free plasma concentrations, indicating concentrative transport at the hepatic sinusoidal plasma membrane, and b) TCA appears to be reabsorbed from urine postfiltration at the glomerulus, either in the renal tubules or in the bladder. These processes have an impact on the effective tissue dosimetry in liver and kidney and may play an important role in TCA toxicity.

Kinetic modeling of slow dissociation of bromosulphophthalein from albumin in perfused rat liver: toxicological implications.

Due to strong binding between organic anions and albumin, the kinetics of the binding process must be carefully considered in biologically-based models used for predictive toxicology applications. Specifically, the slow dissociation rate of an organic anion from the protein may lead to reduced availability of free anion in its flow through the capillaries of an organ. In this work, the effect of the dissociation rate of the anion bromosulphophthalein (BSP) from albumin was studied in isolated, perfused rat livers in the presence of albumin concentrations of 0.25, 1, and 4% (w/v) and an initial BSP concentration of 20 microM. The uptake of BSP from the perfusion medium was modeled using a biologically-based kinetic model of the sinusoidal and intracellular liver compartments. The best fit of the model to data resulted in the prediction of a slow dissociation rate constant for the BSP-albumin of between 0.097 and 0.133 s(-1). Assuming BSP and albumin to be in binding equilibrium in the sinusoidal space, with rapid binding-rate constants, as is often done, produced an unacceptable fit. These results indicate that the strong binding interaction between BSP and albumin, beyond keeping the concentration of free chemical low due to a small equilibrium dissociation constant, can also reduce uptake by an organ due to the slow release of BSP from the protein during passage through the capillaries. The implication of this dissociation-limited condition, when extrapolating to other doses and in-vivo situations, is discussed.

Kinetics of trichloroacetic acid and dichloroacetic acid in the isolated perfused rat liver.

Trichloroacetic acid (TCA) and dichloroacetic acid (DCA) are environmental contaminants that are suspected human carcinogens. To obtain more detail on the role of the liver in the kinetics of TCA and DCA, experimental studies in the isolated perfused rat liver (IPRL) system were conducted. The IPRL system was dosed with either 5 or 50 micromol of either TCA or DCA (25 or 250 microM initial concentration, respectively). TCA and DCA concentrations were followed in perfusion medium and bile for 2 h. The chemical concentration in liver was determined at the end of exposure. Liver viability was monitored by measuring leakage of lactate dehydrogenase (LDH) into perfusion medium and the rate of bile production. Studies performed with TCA showed that the total TCA concentration in perfusion medium decreased slightly during the first 30 min of exposure and remained constant thereafter. Most TCA, greater than 90% of total, was bound to albumin in the perfusion medium. A low, linear excretion rate of TCA in bile was obtained. The calculated free TCA concentration in the liver intracellular water space was higher than the unbound TCA concentration in the perfusion medium. Parallel studies with DCA showed that the DCA concentration in perfusion medium decreased rapidly. Of the total DCA in the perfusion medium, 60% was bound to albumin. The concentration of DCA in bile decreased over time. There was no DCA detectable in the liver after 2 h of exposure at both DCA concentrations. Enzyme leakage and bile production did not change in the presence of TCA or DCA, indicating that these concentrations were not acutely cytotoxic to the liver.

Workshop overview: scientific and regulatory challenges for the reduction, refinement, and replacement of animals in toxicity testing.

Public concern for animal welfare has been expressed through legislative control of animal use for experimental purposes since the first legislation was introduced in 1876 in the United Kingdom. Legislative control of animal use has been introduced in virtually every developed country, with major initiatives in Europe (1986) and the United States (1966 and 1985). Advances in scientific thinking resulted in the development of the concept of the three Rs--refinement, reduction, and replacement--by Russell and Burch in 1959. The field has expanded substantially since, with specialist scientific journals dedicated to alternatives, World Congresses organized to discuss the scientific and philosophical issues, and European and U.S. validation organizations being launched. Current scientific attention is focused on validation of alternative methods. The underlying scientific principles of chemical toxicity are complicated and insufficiently understood for alternative methods for all toxicity endpoints of importance in protecting human health to be available. Important lessons have been learned about how to validate methods, including the need to have prediction models available before the validation is undertaken, the need to understand the variability of the animal-based data which is to be used as the validation standard, and the need to have well-managed validation programs. Future progress will depend on the development of novel methods, which can now be validated through international collaborative efforts.

Predictive toxicodynamics: Empirical/mechanistic approaches.

A major objective of the toxicological sciences is to predict the in vivo toxicological consequences of human exposure to pure chemicals, complex mixtures and commercial formulations. Historically, the experimental approach to this goal has been to investigate toxicological processes in whole animal models and extrapolate the results obtained to predict human risk using various extrapolation procedures (high-dose/low-dose extrapolation, interspecies extrapolation and route-to-route extrapolation). Can in vitro methods be more widely employed in quantitative risk assessment? One major limitation to the broader application of in vitro toxicity testing methods is the lack of validated techniques for the extrapolation of in vitro-derived toxicodynamic data to the in vivo situation. The objective of this paper is to describe some approaches to the development of techniques to extrapolate in vitro toxicity testing data to predict in vivo toxicological responses. An empirical approach within the context of a mechanistic framework is explored. The basic hypothesis is that the in vivo response can be constructed from a cellular toxicity factor that accounts for the cellular response and a toxicodynamic factor that relates toxicological events at the cellular level to the observable in vivo responses. A predictive paradigm to describe the in vivo acute target organ toxicity (hepatotoxicity) of a model chemical (cadmium) is discussed. The cellular toxicity factor is derived from in vitro toxicity testing studies using isolated rat hepatocytes. The toxicodynamic factor is derived through Biologically-Based Response (BBR) modelling techniques to predict target organ toxicity markers (i.e. plasma hepatic enzyme levels as markers for acute hepatotoxicity). The ultimate goal is to develop validated extrapolation procedures that can be applied to predicting target organ toxicity quantitatively in human populations based on in vitro toxicity studies using human cellular models.

Application of in vitro systems to the prediction of in vivo biokinetics.

The kinetics of xenobiotics in biological systems are a critical factor in determining the site and degree of toxicological responses observed. Historically, whole animal kinetic studies coupled with classical compartmental analysis have been used to describe the movement of xenobiotics in biological systems. Often, this traditional approach has not been adequate to meet the needs of toxicologists. In the last few years, biologically based kinetic (BBK) modelling has made a significant contribution to solving this problem. The issue arises as to how in vitro approaches can contribute to this effort. In the past, in vitro models have been used mainly for metabolism studies. Generally, these applications have been qualitative studies to: (1) identify metabolites; (2) investigate metabolic pathways; or (3) assist in interspecies extrapolation issues. The quantitative application of in vitro data has been restricted by limitations of experimental models and the lack of a theoretical framework for the incorporation of these data into predictive models. The current status of BBK modelling and the potential use of in vitro data is discussed with examples of current approaches from the areas of determination of surrogate dose, membrane transport and protein binding.

Interdisciplinary approach to toxicity test development and validation.

Toxicological processes are complex interactions of biological systems at various levels of organization. These interactions evolve in time in response to the perturbation resulting from the interaction of the toxicant, or its metabolite, with molecular targets in the organism. The successful development and validation of new in vitro toxicity tests for the toxicological evaluation of chemicals and commercial products require the concerted effort of an interdisciplinary research team. Such a team should consist of the following types of experts: (1) cell physiologist/cell culturist-someone who not only can grow cells of various origin but also can investigate the normal state of cells and how in vitro culture conditions affect this state; (2) molecular toxicologist-someone who understands the molecular mechanisms of toxicological responses (mechanisms of action) and can experimentally investigate their nature; (3) measurement technologist-an expert in instrumental technology and the application of these technologies to the measurement of cellular function and responses; (4) theoretical toxicologist/modeller-the integrator for the team who can pull the various aspects of the problem together into a unified picture connecting in vitro and in vivo; (5) chemist/structure-activity expert-an expert in chemical structure and its relationship to biological activity to guide the selection of chemicals for investigation; (6) in vivo toxicologist/pathologist-the individual who provides contact with reality; (7) kineticist-an expert in the kinetics/metabolism of chemicals in biological systems who can experimentally investigate this aspect both in vitro and in vivo; (8) statistician-an expert in experimental design and data analysis with the ability to develop new analytical tools to compare in vitro and in vivo data. Most research teams consist of a small group comprising a subset of these areas of expertise and therefore struggle with various aspects of the problem, depending on the pieces missing. It is hoped that the resources of the European Centre for the Validation of Alternative Methods (ECVAM) will be adequate to pull together such an interdisciplinary team to make rapid progress in the development and validation of new testing methodologies.

The role of mechanistic toxicology in test method validation.

There are two approaches to in vitro toxicity test validation, phenomenological and mechanistic. The phenomenological approach uses correlative mathematical techniques, with no regard to the identification of mechanistic relationships, to relate in vitro measurements of toxicity to in vivo toxicological responses in order to establish the validity of the methods under consideration. This approach has three major limitations: (1) success or failure of a particular test will depend critically on the selection of test chemicals; (2) the reason why a chemical fails in a particular test is unknown; (3) without additional information there is no rational basis for extrapolation to new cases lying outside the domain of validation. The mechanistic approach addresses all of these issues: (1) mechanistic considerations are included in the selection of chemicals for validation; (2) the failure of a particular test to identify a given toxin means that the toxin does not act through the mechanism evaluated by the test, which is useful toxicological information; (3) any chemical that acts by the mechanism evaluated by the test will be identified. The major limitation of the mechanistic approach is our lack of knowledge concerning in vivo mechanistic toxicology.

Framework for validation and implementation of in vitro toxicity tests.

The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community--academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.

In vitro models for toxicological research and testing.

The objective of this report is to discuss some of the issues involved in utilizing in vitro methods in toxicological research and testing. The subject is not new, in vitro methods have been used for many years in this context. However, there has been a significant increase in interest in the topic within the scientific community recently as witnessed by the increase in scientific journals dedicated to the topic, symposia held by scientific societies, and commitment of resources to in vitro toxicological research activities. Toxicologists should be aware of these developments as the future directions of the science will be influenced significantly by in vitro methodology.

Report of the Validation and Technology Transfer Committee of the Johns Hopkins Center for Alternatives to Animal Testing. Framework for validation and implementation of in vitro toxicity tests.

The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technological developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial and regulatory communities, is recommended. Test validation acceptance is contingent upon broad buy-in by disparate groups in the scientific community-academics, industry and government. This is best achieved by early and frequent communication among parties and agreement upon common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction and refinement alternatives in toxicity testing.