An in vitro approach to the study of target organ toxicity of drugs and chemicals

Summary A major goal of our laboratory has been the development of primary culture systems that retain differentiated fucntions and responses characteristic of intact tissues in vivo. Specifically, we have developed cellular models of primary cultures of rat heart, liver, and kidney cells to explore the mechanisms by which drugs or chemicals may be toxic to key organs of the body and to develop new techniques by which xenobiotics may be evaluated or identified as potential toxicants to living systems. The purpose of this paper is to describe our rationale and approach to the study of target organ toxicology with in vitro cellular systems.     

Submitochondrial particles asin vitro biosensors of heavy metal toxicity

The effects on mitochondrial respiratory parameters of heavy metals, such as Cu, Ni, Pb, Cd, Zn, Ag, Hg, were recorded by using thein vitro response of submitochondrial particles (SMP) from beef heart mitochondria.The toxicity of these elements was estimated by determining their effects on the energy-coupled reverse electron transfer (RET), which is induced by ATP and succinate at first site level of the respiratory chain in SMP.The RET rate was easily monitored by recording spectrophotometrically at 340 nm the production of NADH, arising from the reduction of exogenous NAD⁺ by RET.The toxicity values were expressed as the toxicant molar concentration which decreases the rate of reduction of NAD⁺ to an extent of 50 percent (EC50). The toxicity increased in the following order: Ni²⁺2+2+< Cd²⁺2+2++.The SMP data were compared with the toxicity values obtained from a variety of biological systems currently used for toxicity testing. The results obtained demonstrate that the SMP test generally provides a good estimate of metal toxicity for several fish and invertebrate species. This is demonstrated by the statistical parameters obtained in the regression analysis. The broadened 95% confidence intervals and, in particular, the poor correlations obtained for some aquatic organisms can be ascribed to the more complex metabolic interactions and competing toxic pathways in aquatic organisms, when compared to SMP.     

Acute toxicity tests on three species of the genus Lecane (Rotifera: Monogononta)

Three rotifer species, Lecane hamataL. luna, and L. quadridentata, were submitted to acute toxicity tests to compare their susceptibility to 11 toxicants. In acute tests with 48-h exposure of neonates of less than 24 h old, copper was most toxic with LC₅₀ values in the range of 0.06–0.33 mg l^–1, while acetone was the least toxic with LC₅₀ values in the range of 5000–7000 mg l^–1. Differences in LC₅₀ value of up to 22-fold were found in the susceptibility to lead between the three species. These data indicate large differences in toxicity among members of the same genus, and point out that it is necessary to submit several species to toxicity tests in order to assess the potential effects of toxicants to rotifers. The commonly used Brachionus calyciflorus cannot be considered representative of all freshwater rotifers in this respect.     

Rhodamine 123 as a probe of in vitro toxicity in MDCK cells

The effect of mercuric chloride on Madin-Darby Canine Kidney (MDCK) cells grown in culture was assayed by the mitochondrial-specific fluorescent probe, rhodamine 123. Treatment of cells with mercuric chloride resulted in a dissipation of rhodamine fluorescence from the mitochondria into the cytoplasm, followed by a release into the medium bathing the cells. Toxicity was assayed either by determining the proportion of cells with delocalized rhodamine fluorescence, or by measuring the rhodamine released from or retained in the cells. Quantifying the release or retention of rhodamine 123 is semi-automated and represents a highly sensitive method of using a vital fluorescent dye for in vitro toxicity analysis.     

Developing a statistical support system for environmental hazard evaluation

Estimating the hazard or risk to both human health and the environment has been based almost exclusively on single species toxicity tests low in environmental realism and without validation of their accuracy in more complex systems. While this may be quite appropriate for humans in a large variety of circumstances, there is no substantive body of direct experimental evidence indicating that precise predictions of harm from hazardous materials can be extrapolated from single species laboratory tests (or even multispecies laboratory tests) to the more complex highly variable natural systems. Now added to the hazardous chemical assessment problem is the accidental or deliberate release of genetically engineered microorganisms into the environment that have the additional capability of multiplying and expanding their numbers and also transferring genetic information to other organisms. This paper focuses entirely on hazard evaluation for organisms other than humans, namely predicting the potential risk or probability of harm to natural systems based on laboratory toxicity testing using single species. Not only will the basic risk assessment strategy itself be examined but also the question of determining the statistical reliability of various extrapolations from one level of biological organization to another. ‘For every complex problem, there is a simple, direct solution ... and it is invariably wrong!’ H. L. Mencken     

Characterization of a gastrointestinal tract microscale cell culture analog used to predict drug toxicity

The lining of the gastrointestinal (GI) tract is the largest surface exposed to the external environment in the human body. One of the main functions of the small intestine is absorption, and intestinal absorption is a route used by essential nutrients, chemicals, and pharmaceuticals to enter the systemic circulation. Understanding the effects of digestion on a drug or chemical, how compounds interact with and are absorbed through the small intestinal epithelium, and how these compounds affect the rest of the body is critical for toxicological evaluation. Our goal is to create physiologically realistic in vitro models of the human GI tract that provide rapid, inexpensive, and accurate predictions of the body's response to orally delivered drugs and chemicals. Our group has developed an in vitro microscale cell culture analog (µCCA) of the GI tract that includes digestion, a mucus layer, and physiologically realistic cell populations. The GI tract µCCA, coupled with a multi‐chamber silicon µCCA representing the systemic circulation, is described and challenged with acetaminophen. Proof of concept experiments showed that acetaminophen passes through and is metabolized by the in vitro intestinal epithelium and is further metabolized by liver cells, resulting in liver cell toxicity in a dose‐dependent manner. The µCCA response is also consistent with in vivo measurements in mice. The system should be broadly useful for studies on orally delivered drugs or ingestion of chemicals with potential toxicity. Biotechnol. Bioeng. 2009; 104: 193–205 © 2009 Wiley Periodicals, Inc.     

Development of a new in vitro system for continuous in vitro exposure of lung tissue to complex atmospheres: Application to diesel exhaust toxicology

The purpose of this study was the development of a new incubation system that can allow continuous exposure of lung tissue to complex atmospheres as a tool for the assessment of aerial environmental lung toxicology. To assess the pertinence of this new exposure system, we studied the impact of diesel engine exhausts as a complex atmosphere containing both gaseous and particulate fractions and have been able to discriminate between the toxicological impacts of the gaseous phase and particulate matter from diesel exhausts. Continuous flow-through rotating chambers with controlled pO₂, pCO₂, and hygrometry have been designed in which lung slices are positioned in rolling inserts that allow free access of atmosphere to the exposed lung tissue. Under control conditions, cell viability was preserved for at least 48 h as assessed by intracellular ATP, GSH, and K⁺ levels and slice O₂ consumption levels. Short-term exposure (1 h) to diesel whole exhausts did not affect intracellular potassium or slice O₂ consumption, while intracellular ATP and GSH levels were markedly decreased. Exposure to filtered exhausts showed less marked effects on both ATP and GSH levels. Superoxide dismutase activity was decreased in a similar way by both total and filtered exhausts while Se⁺-dependent glutathione peroxidase activity was induced by filtered exhausts to a larger extent than after total exhaust exposure, showing different response patterns of lung tissue after exposure to whole or filtered exhausts. In conclusion, this newly designed model opens a promising area in in vitro environmental lung toxicology testing.     

Establishing and maintaining laboratory-based microcosms of riffle insect communities: their potential for multispecies toxicity tests

The purpose of this study was to determine which of the following artificial stream designs would be most logistically simple yet effective in maintaining riffle insects during a 30 d bioassay: 1) static and no current (S-NC); 2) flow-through and no current (FT-NC); 3) static with current (S-C); or 4) flow-through with current (FT-C). Flow-through and current, when provided, were 12 ml min^–1 and 30 cm sec^–1, respectively. Streams were covered by emergence traps, and daylight equivalent lights provided a natural photoperiod. The four stream designs were evaluated in triplicate based on changes in insect species-abundances after 30 d. Test organisms were transferred to the artificial streams in rock-filled containers previously colonized for 30 d in a third-order mountain stream riffle. Additional colonized substrates were sampled immediately to provide an estimate of initial densities placed in the artificial streams. Hess samples were taken directly from the source riffle to evaluate how well the artificial substrates reflected species-abundances on the natural substrate. Adults were collected from the artificial streams every 48–72 h to determine the percentage of initial densities that emerged. After 30 d (7 Aug–6 Sept, 1986), all organisms remaining in the streams were censused. Designs were evaluated using combined densities of adults and young.Relative to benthic samples taken directly from the source riffle, the artificial substrates selected for collector-filterers and against collector-gatherers. The FT-C and S-C stream designs maintained most taxa at or above initial densities, and even in the FT-NC and S-NC streams densities of some taxa were not significantly different (P 0.05) from initial densities. Emergent adults comprised a large proportion of mayfly and chironomid densities and must be monitored during bioassays with aquatic insects. These results indicate that microcosms of riffle insect communities can be maintained for at least 30 d with moderate current and minimal flow-through.     

Cultivation of the heart urchin Echinocardium cordatum and validation of its use in marine toxicity testing for environmental risk assessment

To study environmental risk assessment, echinoderms provide a useful model for ecotoxicological testing. However, limited knowledge of the life history of field collected heart urchins is a problem and the use of cultured urchins has been investigated here. The present study describes a culture method for the heart urchin Echinocardium cordatum under controlled laboratory conditions, providing organisms with a low biological variation. Based on our optimized growth protocol both larvae and juveniles have a growth rate comparable to E. cordatum in the wild. The toxicological response of cultured and field-collected E. cordatum was compared in standard saltwater toxicity bioassays. Using ammonium chloride as a water-soluble reference toxicant, mean 96 h LC₅₀ values for cultured heart urchins versus field collected animals were 37.4 ± 7.6 mg NH₄+/l (n = 5) versus 22.5 ± 4.9 mg NH₄+/l (n = 19), respectively. Additional toxicity experiments with tributyl tin (TBT) spiked sediments revealed 14d LC₅₀ values of 1,242 (95% confidence interval 986–1,564) and 964 (95% confidence interval 843–1,102) µg Sn/kg dw respectively in cultured and field collected E. cordatum. From this it was concluded that cultured heart urchins are less sensitive to TBT than field collected E. cordatum. Furthermore in whole sediment toxicity tests, survival of cultured sea urchins was higher or at least similar to that of field collected E. cordatum. The increased sensitivity of field urchins compared to cultured urchins in various toxicity tests may be due to multiple environmental stressors reducing their overall performance. Overall it was demonstrated that the use of cultured E. cordatum provides a significant advance for urchin-based bioassays for marine environmental toxicity testing, resulting in a more homogeneous, vital population with experimental data displaying reduced variability.     

The integration of investigative toxicology in the drug discovery process

Summary— Integrating toxicology early in the drug discovery process adds value by providing the earliest possible identification of a compound's potential for toxicological and pathological effects relevant to intended clinical use. With this approach true ‘lead’ candidates, with a high probability of clinical success, are identified and advanced while reducing effort and resources expended on compounds without the requisite therapeutic index. Resources are focussed on the speed of getting a discovery ‘lead’ into early clinical development, defining the mechanisms of observed preclinical toxicity and their relevance to human use, and developing early safety data with in vitro test systems ahead of in vivo systems where possible, thus reducing animal use.     

Problems associated with selecting the most sensitive species for toxicity testing

When the field of aquatic toxicity testing began its first major expansion about 40 years ago, it was uncommon to use more than one test species (usually a fish). Later, it became customary to use individual microorganisms (usually algae) and macroinvertebrates as well. Most attention was then given to the response of the most sensitive species in that test series when calculating the biologically safe concentration acceptable for use in natural systems. However, in recent years, there has been an attempt to equate the most sensitive species in a laboratory test series to the most sensitive species in natural systems. Since laboratory test species represent only a tiny part of natural systems and since response variability is well established, that can be a dangerous assumption. The purpose of this discussion is to re-examine the scientific support for this practice.     

In situ toxicity tests of fishes in acid waters

Toxicity of waters within the North Branch of the Moose River to various life stages of lake trout (Salvelinus namaycush), brook trout (Salvelinus fontinalis), creek chub (Semotilus atromaculatus), and blacknose dace (Rhinichthys atratulus) were examined in situ. Study sites were selected that were expected to range from toxic to favourable water quality. For example, pH varied from 4.25 to 7.17, inorganic monomeric Al ranged from ND (< 0.01 mg/l) to 0.40 mg/l, and Ca, from 0.41 to 4.27 mg/l.Toxicity tests were conducted at times when the life stages would naturally occur in these waters and were continued until a range of mortality was observed at the various sites. These experiments suggested that the extent of the drainage system that is toxic to fish increases during snowmelt and major runoff events. Summer base flow water quality was generally the least toxic.Critical life stages were upon hatching and as early feeding fry. In general, young of the year fish were the most tolerant life stage tested. Yearling and adult fish, however, were very sensitive. Blacknose dace were the most sensitive fish of the four species tested, and brook trout were the most tolerant.Hydrogen ion (H⁺) concentration was the most toxic variable in the majority of tests. Inorganic monomeric Al was the most toxic in several, and the combination of H⁺ and Al seemed to cause increased toxicity in many instances. A three-variable model employing hours of exposure, H⁺ concentration, and inorganic monomeric Al predicted mortality quite well. A simple two-variable model using H⁺ and color was nearly as good (R² from 0.49 to 0.94).Documented differences in toxicity among sites and species agreed with observed patterns of fish distribution. These in situ results indicated that laboratory estimates of safe levels of pH and concentrations of Al can result in mortality of fishes in surface waters subject to acidification.     

Use of skin cell cultures for in vitro assessment of corrosion and cutaneous irritancy

Skin cell culture is one of the most promising tools for in vitro evaluation of both cutaneous irritancy and corrosion. New culture methodologies, including three-dimensional reconstruction of skin, allow the evaluation of a wide range of compounds and complex formulations. A number of tests have already been developed for the evaluation of cytotoxicity and many end-points are now currently used, including cell viability, alteration of cell growth or cell function. In recent years parameters more closely related to in vivo irritancy effects such as synthesis of inflammatory mediators and/or their release by keratinocytes after exposure to potential skin irritants have been evaluated. This paper reviews technological aspects and results of validation using skin cell culture for in vitro assessment of corrosion and skin irritancy. Advantages and limits of skin cell cultures are also presented. Current questions about the validation process of cutaneous irritation and corrosion are also considered.     

Effects of spring acid episodes on macroinvertebrates revealed by population data and in situ toxicity tests

1. While streams in Europe and North America are now recovering chemically from chronic acidification, severe episodic acidification continues to threaten sensitive biota. To appraise further the biological importance of episodic acidification, we surveyed the distribution of the mayfly Baetis alpinus in streams in the Southern Alps (Canton Ticino, Switzerland) in relation to runoff acidity during spring floods. Moreover, to improve mechanistic understanding, in situ toxicity assays were carried out on nymphal B. alpinus during low flows and spring floods, both in streams prone to acid episodes and in well‐buffered controls. 2. Streams surveyed for invertebrates represented three groups which contrasted in susceptibility to episodic acidity. Group one included streams that were acid (alkalinity <0) in spring; group two streams were susceptible to acid episodes because of low base‐flow alkalinity (<200 μeq L^?1); and group three streams were well‐buffered and unlikely ever to be acid. The abundance of B. alpinus was similar among groups during stable flows in winter and summer, but was significantly lower in groups one and two following spring snowmelt. 3. During the bioassays, control streams remained circumneutral to alkaline (pH >6.4). By contrast, episodic streams were circumneutral at low flows, but became acid (pH 4.5–5.6 and total dissolved aluminium to 276 μg L^?1) during intense spring snowmelt. After 15‐day exposures, nymphal B. alpinus survival in the circumneutral control streams exceeded 92% irrespective of flow. In the episodic streams, survival matched the controls during low flows, but declined significantly to 10–20% during acid episodes in spring. Shorter exposure (2–4 days) to pH 5.5–5.7 did not cause significant mortality either during the exposure or over the following 7 days, indicating that B. alpinus might recover from short acid episodes. 4. Our data suggest that the spring distribution of B. alpinus in acid sensitive parts of the Alps directly reflects the toxicity of acid runoff during snowmelt. Our study illustrates that even mild episodic acidification can have significant consequences in Alpine streams for one of the most important invertebrate indicators of acidity.     

Evaluation and application of aquatic toxicity tests: use of the Microtox test for the prediction of toxicity based upon concentrations of contaminants in soil

The quality control for the reuse of cleaned soil from a contaminated site consisted in the determination of the main contaminants by analytical chemical methods such as GC and HPLC. Since it is not possible to analyze for all contaminants a toxicity test should be used to detect large concentrations of not routinely analyzed chemicals. The aim of the study was to develop a system for toxicity testing, which should be able to predict the toxicity of soil samples based on the concentration of chemicals in the soil and to detect toxic chemicals not analyzed by the routinely conducted soil analysis.Based upon the relative sensitivity to various contaminants as well as practical aspects such as test duration and costs the Microtox® test was favoured over the bioassays with Daphnia magna and Scenedesmus subspicatus. The Microtox® test was used to measure the toxicity of various pesticides and their major metabolites. The toxicity data of the pure compounds were used to predict the toxicity (EC₅₀ and % inhibition of the bioluminescence reaction) of defined mixtures of chemicals in water by applying two different mathematical appriaches which are based on the additivity of the effects of the single chemicals. The predicted values were compared with the experimental data and showed good agreement.In order to be able to predict the toxicity of soil samples using the Microtox® test the soil/water partition coefficient (K_d) was measured for the main contaminants. The toxicity of soil samples was predicted by calculating the concentration of the contaminants in the leachate by using the corresponding concentration in the soil and applying the K_d values determined. From the calculated composition of the leachate the expected toxicity was estimated. This value was compared with the toxicity experimentally determined in the Microtox® test.     

In vitro toxicity of various classes of test agents using the neutral red assay on a human three-dimensional physiologic skin model

Summary A new three-dimensional human skin model consisting of several layers of actively dividing and metabolically active human neonatal foreskin-derived fibroblasts and epidermal keratinocytes grown on nylon mesh has been used to assess the in vitro toxicity of test agents from various classes. Utilizing a slight modification of the published neutral red viability assay for endpoint determination, we have assayed and obtained dose-dependent toxicity curves for test agents from the following classes: detergents (n=15), alcohols (n=5), metal chlorides (n=10), perfumes and colognes (n=5), shampoos (n=4), conditioners (n=3), moisturizers (n=3), pesticides (n=3), and antimicrobial preservatives (n=4). Limited comparisons to in vivo ocular irritancy data with alcohols and detergents are encouraging. We have demonstrated the utility of this metabolically active dermal substrate containing naturally secreted collagen and other extracellular matrix proteins along with the neutral red viability assay for assessing the toxicity of a number of test agents from a variety of different classes with broad industrial applications.     

Mutants of luminous bacteria selected for bioluminescent toxicity tests

Mutants of the luminescent bacterial strain NRRL B-11177 were isolated with pleiotropic hypersensitivity towards hydrophobic antimicrobial agents. SDS-PAGE analyses of outer membrane proteins and lipopolysaccharides revealed that the outer membrane structure of the ahs-mutants was altered. QSAR analysis showed that the inhibitory effect of chloro-substituted phenols on bioluminescence of the ahs-mutants depended on their hydrophobicity. The effect of chlorinated phenols and detergents on bioluminescence was increased in the ahs-mutants. The potential use of these mutants in bioluminescent toxicity tests was discussed.     

Invited contribution: Acute toxicity testing,public responsibility and scientific challenges

Knowledge of the acute toxic effects of chemicals is important for the protection of exposed humans. Since sufficient information in humans is often lacking, experiments on laboratory animals must be performed. The LD₅₀ test, which requires large numbers of animals, has become the preferred procedure. It is now widely criticized on scientific and ethical grounds. This paper reviews the possibilities of using fewer animals to obtain relevant information on the acute hazards of chemical substances, but it also identifies the reasons why the traditional testing approaches cannot be changed immediately. An important problem is the practice of basing legal decisions on classification of chemicals in official lists of hazardous substances and for labeling purposes on LD₅₀ values. Proposals are presented on how pain and suffering of the animals included in acute toxicity tests can be reduced. The use of in vitro systems for the evaluation of the hazardous properties of chemicals is discussed.Abbreviations LD₅₀ lethal dose 50%, median lethal dose     

New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota

The development and validation of a new three-stage culture system with immobilized fecal microbiota to simulate infant colonic ecosystem is described. Two continuous cultures with different fecal inocula were used to assess the validity and stability of the intestinal model. The total anaerobe populations measured in beads and effluent fermentations reached high concentrations similar to infant feces. Fluorescence in situ hybridization analyses and denaturing gradient gel electrophoresis profiles of effluent samples from the three reactors revealed complex patterns similar to that observed in the inoculum, indicating that fecal bacterial diversity was well-preserved and that dominant bacterial populations showed good stability among reactors. For both experiments, the bacterial populations and fermentation product concentrations were in the range of published data for infant feces. These results demonstrate that this new three-stage continuous culture with immobilized cells provides a useful tool for studying the infant colon ecosystem.