Co-culture blood-brain barrier models and their use for pharmatoxicological screening.

The availability of an in vitro blood-brain barrier model would represent a powerful alternative to experimental animals in pharmacological and toxicological research. This overview collects the various current approaches to build an in vitro model of the blood-brain barrier for these purposes. Purified bovine, porcine and human brain microcapillary endothelial cells as well as several immortalized cell lines have been used to model the blood-brain barrier in vitro, partly in co-culture with astrocytes of various species, or various cell lines such as C6 glioma or N2a neuroblastoma cells. The collected data indicate that functional parameters often can be induced by soluble and membrane-bound factors in such cell systems. Relevant barrier-specific parameters are reviewed: electrical resistance, and structure and function of the multidrug resistance P-glycoprotein and the y-glutamyl transpeptidase. Both P-glycoprotein and gamma-glutamyl transpeptidase have great influence on the pharmacodynamics, toxicology and metabolic capacity of the blood-brain barrier (drug efflux, oxidative damage, detoxification of endotoxins, etc.). Several available in vitro models appear to be suited for pharmacotoxicological screening, if the functional parameters gamma-glutamyl transpeptidase, P-glycoprotein as well as transendothelial resistance are monitored.

Glutamine synthetase activity as a marker of toxicity in cultures of embryonic chick brain and retina cells.

With the goal of developing a fast and sensitive primary cell culture assay for the determination of neurotoxic potential of compounds, the effect of various toxins on the morphology, cell number (estimated as total cell protein), and glutamine synthetase activity of chick embryonic neural cells has been tested. Isolated retina or brain cells, grown as reaggregates in suspension cultures or as monolayers in 24-well plates, were treated with the substances from day 2 to day 6 after the start of culture. Concentrations causing 50% reduction in protein content in brain cell monolayers were as follows: MeHgCl (0.8 mum), CdCl(2) (1 mum), 3-acetyl pyridine (0.1 mm), penicillin (above 0.1 mm), diazepam (0.25 mm), acrylamide (0.3 mm), 2,4,5-T (0.8 mm), lindane (1 mm). In general, retina cells were more sensitive than brain cells. The reaggregate cultures were less sensitive to 1-methyl-4-phenylpyridinium ion (MPP(+)) and cadmium than monolayer cultures, which may be attributable to their metabolic stability or to diffusional limitations. Glutamine synthetase (GS) activity, measured as glutamate production from glutamine, was a more sensitive indicator of toxicity than total protein. Retinal cells grown as reaggregates or monolayer cultures, produced two to four times more glutamate than brain cells grown in a similar fashion. This indicates that retinal glial cell (Müller cell) differentiation proceeds in vitro faster than brain astrocyte differentiation, which is consistent with the in vivo developmental pattern. In some cases (methylmercury, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPP(+), 3-acetyl pyridine, and lindane) a significant increase (as much as 30% of the basal level) was seen in the GS units/mug cell protein at concentrations of toxins below that reducing total cell protein. Thus, generation of neurotoxic glutamate might play a role in the cell destruction caused by the chemicals. Other substances (e.g. diazepam and cadmium) decreased the GS activity considerably, relative to decreases in total protein. This suggests that these xenobiotics act in a more general fashion to reduce metabolic activity and viability.

The ECITTS integrated toxicity testing scheme: The application of in vitro test systems to the hazard assessment of chemicals.

A multicentre project has been designed, with the purpose of developing the concept of integrated in vitro testing. The aim is to use non-animal methods to assess the toxicological properties of chemicals, and to improve this assessment through the use of knowledge of mechanisms of toxic action. A number of tests or test batteries were selected within eight areas: basal cytotoxicity, irritancy, developmental toxicology, hepatotoxicity, nephrotoxicity, immunotoxicity, neurotoxicity and biokinetics. For each area, a number of calibration chemicals were selected. In an initial pilot study, the neurotoxic properties of five chemicals will be studied in combination with a biokinetic analysis, in which blood and brain concentrations will be predicted from biokinetic modelling.

Neurodevelopmental toxicity in vitro: primary cell culture models for screening and risk assessment.

Robust models for the evaluation of developmental toxicity are briefly reviewed with emphasis on embryonic brain and retina cells in vitro. Organ slice and aggregate cultures under constant gyratory movement as well as high cell density monolayer ("micromass") cultures are considered as robust models. An in vitro model using high cell density monolayer and re-aggregated cells isolated from embryonic chick brain (ED 6) is presented. Cell development and differentiation of the astrocytes and nerve cells are monitored by marker proteins and cytotoxicity was quantified by neutral red uptake and protein content. Four human teratogens, six possible human teratogens and six unlikely human teratogens were tested in brain and retina cells for their cytotoxic and morphologic effect. All 16 substances were classified correctly except the neurotoxicants MPTP and MPP+, both of which are strong dopaminergic toxicants in vitro as well as in humans and are therefore proposed to be classified as human neuroteratogens. Preliminary data on the lowest effect levels of four potential neurotoxicants (cadmium chloride, Ara-C, Phenytoin, MPTP) in chick brain aggregate cultures correlate surprisingly well with known toxic human plasma levels. Further validation has to be undertaken to confirm these promising results. A battery of such robust in vitro models is proposed that could cover neurodevelopmental toxicity of drugs and chemicals for screening and risk assessment purposes.

Differentiation of embryonic chick brain cells in monolayer and reaggregate cultures: A potential model in vitro for neurotoxicity.

In order to develop a model for potential neurotoxicity and teratogenicity, chick brain cells (embryonic day 7) were mechanically dissociated and cultured for up to several months. Differentiation of nerve and glial cells (monolayers in petri dishes and reaggregates in suspension cultures) were monitored with monoclonal antibodies against 68 kDa neurofilament protein (anti-NF) and glial fibrillary acidic protein (anti-GFAP). Anti-NF stains neurons in vitro as they differentiate morphologically; at day 1 many nerve processes already are feebly stained. In monolayer cultures extensive neural networks develop at day 6, which are strongly stained by anti-NF. At day 8 staining fades, days before deterioration becomes visible. In reaggregates a stable differentiation of nerve cells could be observed by intensive anti-NF staining for as long as 3 wk in culture; 5-wk-old cultures still showed substantial staining. The differentiation of GFAP-positive cells takes 3 days in vitro in monolayers as well as in reaggregate cultures. Small groups of marked cells continually increase in number; after 2 wk in culture they are present throughout the reaggregates. The differentiation of astrocytes as measured by anti-GFAP immunostaining is considerably faster in vitro than in vivo. It is suggested that the expression of NF in nerve cells could be used as a sensitive cytotoxicity endpoint, whereas GFAP expression could serve as an endpoint for monitoring differentiation of neural tissue.

In vitro predictive tests for eye irritants.

The developments and attitudes towards in vitro testing since the first major workshop on irritation testing five years ago (Reinhardt et al., 1985) are summarized. Many test systems have been described and an increasing number of compounds tested. However, the in vivo data basis used for comparison is still heterogeneous and a proper analysis of most in vivo/in vitro correlations is difficult. Some progress has been achieved with various controlled validation programmes on a national and international level, all of which have not been completed. In order to improve this slow progress the following measures are proposed: (1) expectations for a uniform test need to be discouraged when heterogeneous chemical actions may occur, and rigid test guidelines have to be replaced by flexible ones (differentiated approach); (2) many practical small steps need to be taken rather than one theoretical large step, that is by accepting simple in vitro tests for restricted groups of chemicals such as severe irritants (adaptive approach); and (3) the threshold for acceptance of in vitro tests needs to be lowered by regulatory bodies and routine laboratories (optimistic approach).

Do we find relevant parameters for in vitro cytotoxicity testing?

The current strategy for the design of cytotoxicity tests is briefly reviewed in light of goals that need to be reached, and in the capacity of in vitro tests to fulfill the high public expectations for alternative methods to animal testing. Various cytotoxicity tests and parameters used for the assessment of topical toxicity and of neurotoxicity are chosen as examples and their relevance is discussed. Past experience with in vitro and short-term tests for mutagenicity shows not to look for one single supertest. A proposition for reasonable safety testing implies that a combined approach must be developed that integrates the results from a battery of cell tests and from structure-activity analyses as well as from kinetic and metabolic studies. The relevance of such an integrated approach must be aimed directly at the organisms that may be exposed.

Comparison of in vitro cell toxicity with in vivo eye irritation.

The effects of 26 different cosmetic ingredients (e.g., permanent wave and hair dye compounds, emulsifiers, resins, and detergents such as quats) were assessed by four end points indicative for qualitatively and quantitatively different cytotoxicity: (1) neutral red uptake reduction after 24 h of treatment (NR-90 and NR-50); (2) cell detachment from culture dish after 4 h of treatment (CD-25); (3) growth inhibition after 48 h of treatment (GI-50); and (4) membrane permeability measured by fluorescent dye retention (fluorescence shift FS-25) and dye exclusion (viability ratio VR-25). The cytotoxicity potentials of the test agents were ranked for each in vitro test and compared with the in vivo eye irritation in guinea pigs (Draize test) after application of 5 or 2.5% (w/v) solutions of the same test batches. Strong irritants could be easily detected by most of the in vitro tests, but the neutral red uptake assay (especially NR-50) was the only one that was able to distinguish the minimally irritating test agents from strong irritants as well as from nonirritants. (I) All three extremely irritating quaternary ammonia compounds were identified as the strongest cytotoxic agents. (II) Nine out of 12 minimally irritating substances (mainly emulsifiers and resins) were ranked in the intermediate group. (III) Eight out of 11 non-or practically nonirritating chemicals (mainly permanent wave compounds) showed cytotoxic effects at very high concentrations only. The distinction of these three groups was better by means of NR-50 than by NR-90 data. At least two of the other cell tests (CD-25, GI-50, FS-25, and VR-25) had to be considered to allow an adequate interpretation of in vitro cytotoxicity.

Fast and sensitive screening method for detection of trichothecenes in maize by using protein synthesis inhibition in cultured fibroblasts.

A fast and sensitive bioassay with hamster (BHK-21 C13) fibroblasts for the detection of toxic trichothecenes in maize is described. Cells are exposed to pure toxins or crude maize extracts for 30 min. The mixture is then incubated with [1-14C]-leucine for an additional 60 min and the radioactivity incorporated into the protein of the washed cells is determined. The sensitivity of the assay was in the range 1-10 ng/mL (or 50 ppb in maize) for T-2, HT-2, and diacetoxyscirpenol. At least 1000-fold higher concentrations of non-trichothecene mycotoxins and plant toxins were necessary to cause an inhibition of protein synthesis in the cells. Of 24 maize samples tested, 14 gave a positive response in this assay and the presence of trichothecenes could be confirmed chemically in 11 samples. Therefore, the described bioassay is proposed as a useful screening method for cytotoxic trichothecenes in maize.

A rapid cell membrane permeability test using fluorescent dyes and flow cytometry.

A reliable and rapid test to detect cytotoxic chemicals which affect cell membranes is described. Fluorescein diacetate freely penetrates intact cells where it is hydrolyzed to its fluorochrome, fluorescein, which is retained in the cell due to its polarity. On the other hand, ethidium bromide is known to be excluded from the intact cell, staining only nucleic acids of membrane-damaged cells. The combination of both fluorochromes results in counter-staining: intact cells fluoresce green (cytoplasm) and membrane-damaged cells fluoresce red (nucleus and RNA). Rat thymocytes freshly isolated without enzyme treatment were incubated simultaneously with test substance and dye solution fluorescein diacetate and ethidium bromide. A two-parameter analysis was performed on a flow cytometer with an on-line computer. Concentration-dependent effects of various detergents and solvents were quantified by measuring the amount of dye retention, i.e., the decrease or increase in fluorescein--fluorescence (peak shift), and the decrease in dye exclusion (increase in ethidium bromide-staining) relative to the untreated control. The assay can be used for rapid monitoring of chemical insults to cell membranes which precede the decrease of the viability measured by pure dye exclusion techniques.

Collagen synthesis in growing human skin fibroblasts.

Collagen and noncollagen protein synthesis in cultured human skin fibroblasts was studied in relation to different growth phases. In order to quantify collagen synthesis, we determined the release of incorporated radioactivity using purified bacterial collagenase. Collagen as well as noncollagen protein synthesis markedly decreased during fibroblast growth. On the other hand, we found a 3-fold increase in relative collagen synthesis (i.e. collagen synthesis compared to total protein synthesis) comparing cells in the log growth phase with cells in the stationary growth phase.

Acute irritation tests in risk assessment.

The two most important elements in assessing the risk of topical injury from a chemical are its biological properties, in the context of skin and mucous membrane damage, and the likelihood and likely nature of topical contact with the chemical. Appropriate biological tests in model systems should be based on the probable circumstances of exposure. Topical contact takes place under two distinct sets of circumstances--intentional and accidental. Chemicals that are intended to come into contact with skin and mucous membranes include cosmetics and dermatological preparations. For such compounds the frequency and extent of skin contact is predictable and any irritant effects are unacceptable. The absence of irritant effects is established by testing in human volunteers or experimental animals. Since animal skin and mucous membranes are more susceptible to irritants than those of man, the amounts or concentrations tested need not be greater than those intended for human use. It is hoped that validated alternatives to animal models will soon be available. For household or industrial chemicals where skin and/or mucous-membrane contact occurs accidentally, topical contact should generally be avoided. In such cases the objective of irritancy testing should be to establish which compounds are particularly irritant and therefore need extra care in handling. We are convinced that this latter objective can be achieved by simpler and less cruel tests than the Draize eye-irritation test.

Interpretation of cell toxicity data for the estimation of potential irritation.

Three cytotoxicity assays were evaluated using 57 chemicals of various classes (inorganic and organic metal salts, solvents, detergents, reagents, drugs) which have widely different mechanisms of cytotoxicity. Baby hamster kidney fibroblasts (BHK-21/C13) and early (Keller) and late (MRC-5) passage human fibroblasts were used to measure cell detachment, cloning efficiency, and growth inhibition under subconfluent culture conditions. For the majority of chemicals, for which comparisons were made, the ranking order was roughly the same in all three tests and with all three cell types. However, for some chemicals specific growth effects could either be detected or excluded because the relationship between the data from the detachment assay and that from one of the growth assays was characteristically altered. The ranking order resulting from our in vitro data correlated better with threshold limit values for human workroom air (TLV/TWA) than with LD50 values (rat, oral). Correlations with data from Draize skin and eye irritation tests were not determined since the available in vivo values were derived using various different scoring systems. However, when our in vitro data were used to divide the chemicals into three crude classes, (i) non-irritant, (ii) mild to moderate irritant, or (iii) strong irritant or corrosive, and the results were compared with the known irritation potential for skin and mucous membranes derived from human exposure data, the in vitro data were more than 80% predictive of the in vivo classifications.

Cell detachment and growth of fibroblasts as parameters for cytotoxicity of inorganic metal salts in vitro.

Eight inorganic metal compounds (AlCl3, Al(OH)3 gel, Al(OH)3 salt, SnCl2, ZnSO4, K2Cr2O7, CdCl2, HgCl2) were tested for their cytotoxic effect on an established hamster fibroblast line (BHK-21/C13) in vitro using a cell detachment assay and two different growth assays, the cloning efficiency and the cell number after 2 days subconfluent culture as parameters. The test conditions for these assays were optimized, including incubation period, application of test substance, growth conditions and data analysis. Aluminum, zinc and tin compounds showed low cytotoxic effects when compared to potassium, cadmium and mercuric compounds. Potassium dichromate was highly toxic in both growth assays (0.0001-0.01 mM, with a clear dependency on the incubation time), whereas it proved to be only slightly toxic in the detachment assay (0.1-5 mM). Cadmium and mercuric chlorides were the most toxic compounds in the growth (0.00001-0.001 mM) and the cell detachment assays (0.01-0.1 mM). Variable incubation periods barely affected the cytotoxicity of mercuric chloride. Ranking of these cytotoxicity data was found to be identical to the ranking of LD50 values (oral, rat) as well as to the ranking according to threshold limit values for human workroom environment, and of human eye irritation data.

Actin III and myofilaments in different muscles of wildtype and the mutant raised of Drosophila melanogaster.

The mutation raised (rsd, 3-95.4) of Drosophila melanogaster causes flightlessness as a consequence of abnormalities in the fibrillar flight muscles (FFMs). In this muscle type actin III is neither synthesized nor accumulated while adult tubular muscles of rsd flies are indistinguishable from wildtype. This paper demonstrates ultrastructural defects in rds FFMs and extends the biochemical comparison of adult wildtype and rsd muscles to larval muscles and to embryo cells differentiating in culture. The FFMs of mature rsd flies contain thick filaments in irregular bundles, but no thin filaments. Normal Z-discs are virtually absent. Instead, a large number of Z-disc residues are present in stacks attached to short filaments on either side. In newly emerged rsd flies the disorganization is less pronounced. The adult tubular muscles and the supercontracting muscles of third-instar larvae of rsd can ultrastructurally not be distinguished from wildtype. The present biochemical results indicate that not only FFMs of mature and newly emerged adults are affected by the rsd genotype. Synthesis of actin III is not detectable in rsd FFMs which corresponds to the heavy structural defects. In addition to the lack of actin III synthesis in rsd FFMs, three unidentified proteins (52 kDa, 80 kDa, 90 kDa) which are specific for wildtype FFMs are also not synthesized in rsd flies. Among all other muscle types studied, all of which are morphologically unaffected, only adult tubular muscle of rsd genotype showed no biochemical effect. Larval supercontracting muscle as well as embryo cells differentiating in culture failed to synthesize actin III in the case of rsd cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations affecting the indirect flight muscles of Drosophila melanogaster.

The development of the indirect flight muscles of Drosophila melanogaster was studied by analysing mutations that cause flightlessness. Twenty-five mutations on the X-chromosome and two on the third chromosome were examined. The X-chromosomal mutations form ten complementation units. The ten loci were assigned preliminary map positions by meiotic recombination and deficiencies and duplications. The two autosomal mutations represent two genes. Gynandromorph analyses suggest that many of these mutations have their primary effect in the presumptive thoracic muscle region of the embryo. The mutations cause a variety of characteristic defects, such as absence of the bulk of the thoracic muscle mass, or absence of only one of the two fibrillar muscle groups. Electronmicroscopic studies of sixteen mutants representing all twelve loci reveal abnormal myofibrillar organization in some of these mutants, e.g. aberrant or missing Z-bands, or absence of the thin filaments. Mutant protein patterns, obtained by SDS-polyacrylamide gel electrophoresis, show the following differences from wild type: ten mutants are characterized by absence of reduction of the 54 K protein, and most mutants exhibit a reduction and modification of the 80 and 90 K proteins. The absence of reduction of the 54 K protein was strongly correlated with aberrant Z-bands.

Cell detachment and cloning efficiency as parameters for cytotoxicity.

Cell detachment and cloning efficiency of Baby Hamster Kidney cells (BHK-21 C13) were used as parameters to quantify cytotoxicity in vitro of 3 endogenous chemicals (glutathione, L-methionine, L-cysteine HCl) and 4 organotin compounds (tributyltinoxide, tributyltinchloride, tetrabutyltin, tetraphenyltin). IC50 values (inhibitory concentration at which the cloning efficiency was reduced to 50%) were estimated to be larger than 10(-3) M for all 3 endogenous substances, which served as a calibration of the cell culture system for non-toxic chemicals. For the 2 tributyltin salts the IC50 values were estimated to be near 10(-6) M and for the 2 tetraalkyltin compounds near 10(-5) M. The estimated CD50 values (concentration at which 50% of the cells detach) were at least twice as large as the corresponding IC50 values for all 7 chemicals tested. For the toxic tributyltin salts the cell detachment assay was 30-60 times less sensitive than the cloning efficiency assay. However, both assays rank all compounds tested in the same sequence of toxicity as that known from in vivo studies.