Evaluation of a proposed in vitro test strategy using neuronal and non-neuronal cell systems for detecting neurotoxicity.

The European Commission White Paper, "Strategy for a future chemicals policy" (EC, 2001) is estimated to require the testing of approximately 30,000 "existing" chemicals by 2012. Recommended in vitro tests require validation. As the White Paper (EC, 2001) requires neurotoxic data, this study evaluated an in vitro testing strategy for predicting in vivo neurotoxicity. The sensitivities of differentiated PC12 cells and primary cerebellum granule cells (CGC) were compared to undifferentiated PC12 cells which can indicate basal cytotoxicity. Cytotoxicants and neurotoxicants selected for testing covered a range of mechanisms and potencies. Neurotoxicants were not distinguished from cytotoxicants despite significantly different cell system responses using all endpoints; cell viability/activity, ATP depletion, MMP depolarisation, ROS production and cytoskeleton modifications. For all chemicals tested, neuronal-like cell systems were generally less sensitive than undifferentiated PC12 cells. Acute oral rodent LD(50) values correlated with cytotoxicity IC(50) values for the respective chemicals tested in each cell system. This study concluded that although simple non-specific assays are required to distinguish basal cytotoxicity from specific neurotoxicity by using different cell systems with different states of neuronal differentiation, further work is required to determine suitable combinations of cell systems and endpoints capable of distinguishing neurotoxicants from cytotoxicants.

Impaired T cell functions during amphibian metamorphosis: IL-2 receptor expression and endogenous ligand production.

T cell functions are impaired during defined developmental stages of amphibian metamorphosis (Marx et al., 1987). Here we show, using a fluorescent anti-human IL-2 receptor antibody and flow cytometry, that during these stages, the splenocytes of Xenopus laevis, the South African clawed toad, have a progressively diminished capacity to express IL-2 receptors (IL-2R), after in vitro lectin stimulation. Preincubation with human rIL-2 specifically blocks binding of the anti-IL-2R antibody. Separation of an endogenous ligand bound to the IL-2R leads to a substantial increase in available epitope recognized by the anti-IL-2R antibody when pre- and postmetamorphic splenocytes are employed, but not when splenocytes of the prometamorphic stages are treated similarly. Thus, the cells from the prometamorphic stages are not producing significant quantities of the ligand. Finally, we demonstrate that human rIL-2 is not by itself mitogenic in the toad, but it can act as a co-stimulator of antigen-induced mitogenesis. Thus, an absence of an endogenous ligand (autologous IL-2?), coupled with a reduced capacity to express IL-2 receptors may be responsible for impaired T cell clonal expansion in metamorphosing Xenopus. Inhibition of T cell functions during this period is vital, since adult cells forming within the larval body bear surface proteins not found on larval cells (Flajnik et al., 1986).

An improved MTT assay.

The MTT assay for cell viability and cell proliferation has been modified to improve its reproducibility and accuracy. The modified test is performed on MultiScreen filtration plates, which permits the removal of the culture medium prior to formazan solubilisation, without loss of cells or formazan crystals. A 1:1 mix of DMSO and ethanol is used as the solvent, since this has the same optical refraction index as the filters, making it possible to measure the optical densities directly on the MultiScreen plate. Data obtained in the assay method using the MultiScreen plate were compared with data from studies employing the normal flat-bottomed plate, by using cells which grow in suspension. Two cell lines were used in the study. CTLL-2, which are IL-2 dependent cells of murine T cell origin, and Jurkat E.6.1 which are IL-2 producing cells of human lymphoma origin. CTLL-2 cells and the modified MTT assay were also used for evaluating the effects of different IL-2 concentrations on cell proliferation.

The characterization of the toad splenocytes which bind mouse anti-human IL-2 receptor antibody.

We have utilized two depletion protocols to characterize the spleen cells of the South African clawed toad, which bind specifically mouse-anti-human IL-2 receptor (anti-Tac) antibody and recombinant DNA produced IL-2. When the selectively lymphotoxic reagent, N-CH3-N-nitrosourea (NMU), is injected into adult toads, it removes the thymic cortex and lymphocytes throughout the body required for helper and cytotoxic cell functions. We have also used a monoclonal mouse anti-Xenopus IgM antibody to deplete toad splenocyte populations of surface (s) Ig+ cells. Freshly biopsied and cultured spleen cells were compared with respect to their capacity to bind fluorescent (Fl*-) anti-Tac antibody and its ligand, rIL-2, after a depletion protocol. The results clearly show that a phytohemagglutinin (PHA)/IL-2 sensitive splenocyte population is removed by NMU injection. While many of the remaining NMU insensitive, previously cultured cells are Tac+, they fail to bind rIL-2. Freshly biopsied spleen cells with constitutive IL-2 receptors are found in both the sIg- (T cell enriched) and sIg+ (B cell enriched) populations following panning. Moreover, both populations are able to bind the ligand with equal efficiency. Thus, constitutive IL-2 receptor bearing cells are not restricted to either the T or B cell populations. The predominant PHA activatable, rIL-2 binding cell populations of the toad appear to be T cells which are involved with helper and cytotoxic functions.

Apoptosis and the cell cycle in Xenopus laevis: PHA and PMA exposure of splenocytes.

T cell receptor (TCR) ligation and protein kinase C (PKC) activation stimulate proliferation and modulate apoptosis in both mammalian and amphibian lymphocytes. The potential relationship between apoptosis and the cell cycle in mature Xenopus laevis splenic lymphocytes is addressed by monitoring apoptosis and DNA synthesis over time, using incorporation of propidium iodide (PI) and flow cytometry. Aliquots of the same populations of cells are followed after exposure in vitro to phytohemagglutinin (PHA) or phorbol 12-myristate 13-acetate (PMA). Significant increases in apoptosis preceed those in DNA synthesis by 12 to 16 h following exposure to both reagents. Since apoptosis preceeds DNA synthesis, these dying cells clearly do not need to enter the S phase of the cell cycle before becoming apoptotic, in contrast to mammalian T cells. Another striking difference is that the reagent with weaker mitogenic properties in this species, PHA, is significantly a more potent apoptogen, than the strong mitogen, PMA. The two phenomena then appear to be inversely related in Xenopus cells. Data on DNA synthesis suggest independence of the two phenomena, as DNA synthesis is stimulated in direct proportion to the strength of each reagent as a mitogen. Mature mammalian T-cells undergo apoptosis only when previously activated. The Xenopus lymphocytes examined were not deliberately activated by exposure to antigen or lectin. PMA, a cancer promoter in mammals, usually 'rescues' mammalian cells from apoptosis, but stimulates apoptotic increases in Xenopus cells. Thus, mature Xenopus lymphocytes may be more readily stimulated to die by cancer inducing agents than mammalian lymphocytes. This could make them less susceptible to transformation into immortalized cancer cells. This characteristic may considerably contribute to the observed resistance to spontaneous and chemically-induced neoplasia in wild type, non-isogeneic or non-inbred Xenopus.

A monoclonal mouse anti-human IL-2 receptor antibody (anti-Tac) will recognize molecules on the surface of Xenopus laevis immunocytes which specifically bind rIL-2 and are only slightly larger than the human Tac protein.

We have made visible the binding of a mouse monoclonal anti-human interleukin 2 (IL-2) receptor (anti-Tac) antibody on the surface of phytohemagglutinin (PHA)-stimulated Xenopus thymocytes using a colloidal gold-conjugated goat anti-mouse antibody and transmission electron microscopy. No binding was found when a different mouse monoclonal antibody (mAb) of the same isotype and subclass was tested, or when the anti-Tac antibody was omitted from the procedure. After metabolic radiolabeling of the IL-2 receptors with [35S]methionine using PHA-stimulated thymocytes of Xenopus laevis, the South African clawed toad, we show that a concentrated preparation of the mouse anti-human Tac antibody will immunoprecipitate a radiolabeled molecule just slightly larger than 55 kDa. Phorbol dibutyrate (PDB), an effective T cell mitogen, and cyclosporin A, an inhibitor of T cell mitogenesis in this species, are both capable of regulating the expression of this IL-2-binding molecule on Xenopus immunocytes. Here, we use the calcium ionophore A23187 to show that the relationship between IL-2 receptor expression and mitogenesis, which was previously established in X. laevis, is associated with a calcium ion flux. Flow cytometry is used for assaying alterations in epitope expression after binding the lectin-stimulated cells under test with a fluorescence (Fl*) conjugate of the anti-Tac antibody or a control mAb, which is either anti-DNP or anti-keyhole limpet hemocyanin (KLH) in specificity, but of the same mouse isotype and subclass as the anti-IL-2 receptor antibody.

The time course, localization and quantitation of T- and B-cell mitogen-driven apoptosis in vivo.

Apoptosis has very recently been visualized in situ in a mammalian thymus and spleen. We report here the first in situ visualization, localization and quantitation of the time course of mitogen-altered basal levels of apoptosis within the thymus and spleen of a vertebrate. Adult Xenopus leavis, South African clawed toads, were injected intraperitoneally with either the T-cell mitogen, Concanavalin (Con) A, or the B-cell mitogen, lipopolysaccharide (LPS). Controls, reflecting the basal level of apoptosis of both organs, were injected with isotonic phosphate-buffered saline for amphibians (APBS). ConA and LPS failed to enhance the nearly 2% apoptotic rate in the thymic cortex, which is made up largely of immature lymphocytes, but it did double the base level of 2% apoptosis in the mature lymphocytes of the medulla, particularly along the corticomedullary boundary. In the lymphoid splenic white pulp, the 2% basal level was exceeded slightly after ConA treatment, while the 6% basal lymphoid apoptotic rate in the red pulp was enhanced 7-fold in 12 h. LPS induced lymphocytic apoptosis in the partly erythropoietic red pulp of the spleen after 12 h but did not effect the white pulp. Extensive macrophage engulfment of apoptotic cells was apparent in both the thymus and the spleen.

Expression of a Fas-like proapoptotic molecule on the lymphocytes of Xenopus laevis.

Ligation of the externally expressed Fas (APO1/CD95) molecule will initiate programmed cell death (apoptosis), in many mammalian developing and adult cells. Fas-induced apoptosis has not been demonstrated with the cells of any non-mammalian vertebrate. We immunostained suspensions of splenocytes from adult Xenopus laevis, the South African clawed toad, with a polyclonal rabbit anti-human Fas antibody raised against the amino acid residues 321-335 of human Fas. The binding was specific, as it was dramatically reduced by preincubation of the antibody with the Fas peptide used to make it, but not with a Fas-ligand (FasL) peptide. The binding was enhanced after in vitro exposure of the splenocytes to phytahemagglutinin (PHA), a T cell mitogen and apoptogen in this species. Sections of developing Xenopus larval tissue were also immunostained with the polyclonal rabbit anti-human Fas antibody. Consistent binding of thymocytes and splenocytes was not observed until early metamorphosis in these immunological sites. A monoclonal mouse anti-human Fas antibody, previously used to stimulate apoptosis in mammalian cells, induced significant levels of apoptosis in adult Xenopus splenocytes and additionally, bound specifically to a splenocyte extract, as assayed by ELISA. Thus, a molecule on Xenopus splenocytes shares both structural and functional homologies with human Fas, indicating the evolutionary conservation within vertebrates of this means of initiating apoptosis.

Two cellular pathways regulate the response to TNP-Ficoll in Xenopus laevis.

The initiation of the anti-TNP response to TNP-Ficoll in the amphibian Xenopus laevis has been studied. Although the response to this antigen is thymus independent in mammals, it is thymus dependent for the first three days following immunization in Xenopus. This thymus regulation is not MHC restricted, since it can be substituted for by thymus xenografts, and by prior or co-injection of heterologous red blood cells or Concanavalin A. The pathway which is activated by the Con A to substitute for the thymus is NMU sensitive, unlike the thymic pathway. The peripheralised alternative pathway is activated by particulate but not soluble TNP-Ficoll. The thymus-dependent and alternative pathways are discussed in terms of their possible nature, regulation and evolutionary significance.

In situ lymphocyte apoptosis in larval Xenopus laevis, the South African clawed toad.

During Anuran metamorphosis larval structures regress, adult structures form anew and impaired T cell immune functions are noted, as are alterations in endogenous glucocorticoid titers. In situ histological data, after staining for DNA fragmentation, reveal patterns of lymphocyte suicide in the thymus and spleen of non-antigenically challenged, laboratory bred, developing larvae, that do not correlate with either impaired immune functions or plasma glucocorticoid titers. Apoptotic levels in the thymus are high in premetamorphic stages, low during prometamorphosis and high again, after metamorphic climax, reflecting a periodic removal of thymocytes. Lymphocytic apoptosis in the spleen is low during premetamorphosis, rises in prometamorphic stages, principally within the red pulp, reaching a peak at climax, before declining as metamorphosis is completed.

Differential cyclophosphamide sensitivity of suppressor function in Xenopus, the clawed toad.

Thymocyte and Splenocyte cultures from in vivo immunised Xenopus were assayed to test their suppressive capacity. Immunisation with TNP-Polyvinylpyrrolidone induced suppression. Suppression induced by the haptenated antigens, TNP-Red blood cells, TNP-Lipopolysaccharide, and TNP-Ficoll affected the anti-TNP antibody response of splenocytes from TNP-PVP immunised animals. Pretreatment with cyclophosphamide revealed both a sensitive and insensitive suppression capacity in Xenopus laevis.

The development of peripheral TNP-tolerance and suppressor function in Xenopus laevis, the South African clawed toad.

In adult Xenopus laevis, inducer- and effector-suppressor functions are located in the spleen. These peripheral suppressor functions must be established at this location near the end of metamorphosis, since both functions are in the thymus in premetamorphic and in developmentally-blocked metamorphosing larvae. This study examined whether TNP-conjugated self-antigens resulting from exposure to trinitrobenzene sulfonic acid (TNBS), will stimulate TNP-tolerance in premetamorphic, metamorphic, and in developmentally-blocked metamorphosing larvae. Premetamorphic and developmentally-blocked larvae produce little TNP-tolerance or peripheral suppressor function. However, when TNBS exposure includes the late stages of the metamorphic period, both TNP-tolerance and splenic anti-hapten suppressor function are demonstrable. Removal of suppressor function with cyclophosphamide prevents expression of tolerance, thus, they are functionally related. Suppressor function and tolerance both differentiate during the late metamorphic stages when new adult antigens are being expressed and incorporated into a library of self.

Apoptosis in the thymus of developing Xenopus laevis.

Metamorphosis in Xenopus laevis is a time when thyroxine and glucocorticoid levels rise, dramatic morphological and physiological changes take place, and tolerance is established to newly expressed adult antigens. In vitro exposure of thymocytes tested at different metamorphic stages, to the T-cell lectin, phytohemagglutinin (PHA), stimulates increased apoptosis, but incubation with the synthetic glucocorticoid, dexamethasone (DEX), fails in this regard. Altered-self antigenicity, following trinitrobenzene sulfonic acid (TNBS) treatment, increases apoptosis only in the late stages of metamorphosis. Developmentally blocked metamorphosing larvae demonstrate low thymic apoptotic rates that are also unaffected by in vitro exposure to DEX or by in vivo exposure to thyroxine, but are increased by PHA and in some individuals by TNBS. When released from blockade, their thymic apoptotic rates rise as progress through metamorphosis is renewed. Larval thymic apoptosis is glucocorticocoid- and thyroxine insensitive, but is lectin and altered-self antigen activated, particularly during postclimax stages.

Apoptosis in thymus of adult Xenopus laevis.

Thymocyte apoptosis in adult Xenopus laevis is demonstrated on agarose gels and is quantified by propidium iodide incorporation using flow cytometry. Basal apoptotic levels are increased after in vitro exposure to a glucocorticoid, dexamethasone (DEX), and to the lectin, phytohemagglutinin (PHA). To determine the role that newly introduced antigenic determinants may play in this regard, a repertoire of altered-self antigens was created by exposing thymuses in vitro to trinitrobenzene sulfonic acid (TNBS) thereby derivatizing self-cells and proteins via 2,4,6-trinitrophenyl-acetic acid conjugation. An increase in apoptosis in TNBS-treated thymuses is observed. Thus, the thymocytes of adult Xenopus laevis are susceptible to apoptosis when induced by a glucocorticoid, a lectin, and by altered self, antigen activation.

Phosphatidylserine expression on apoptotic lymphocytes of Xenopus laevis, the South African clawed toad, as a signal for macrophage recognition.

Inflammation is avoided in apoptosis by early removal of dying cells by macrophages (MOs). In mammalian cells, an early aspect of apoptosis is the translocation of phosphatidylserine (PS) from the inner leaflet of the cell membrane to the surface. PS recognition can serve as a signal for triggering removal of dying cells. PS expression on splenocytes and thymocytes of Xenopus laevis was quantified using FITC-Annexin and flow cytometry following exposure in vitro to several known apoptogens for this species. All apoptogens used induced PS expression. Dose dependency and the kinetics of PS expression following exposure to the calcium ionophore, A23187, were also examined. Peritoneal exudate cells (PEC's) were cultured with A23187-treated thymocytes to test MO capacity for recognition of PS. MO binding to apoptotic thymocytes was reduced following exposure of PEC's to a water soluble analogue of PS, phospho-L-serine. The presence of a phagocytic PS-dependent recognition system in amphibia is supportive of the evolutionary conservation of this function in mammals that is crucial in limiting inflammation induced by dying cells.

Stimulation of dichlorofluorescin oxidation by capsaicin and analogues in RAW 264 monocyte/macrophages: lack of involvement of the vanilloid receptor.

In studies into the oxidative burst in RAW 264 monocyte/macrophages, it was observed that capsaicin, a vanilloid receptor agonist, stimulated dichlorofluorescin (DCFH) oxidation in a concentration-dependent manner, which could be blocked by capsazepine, a vanilloid receptor antagonist. However, by use of a number of vanilloid agonists (including N-octyl-3-chloro-4-hydroxyphenylacetamide, 4m), we demonstrated that there was no relationship between vanilloid agonist potency and the capacity to stimulate DCFH oxidation. The oxidative burst stimulators Tween 20 and phorbol myristyl acetate (PMA) also stimulated reactive oxygen species generation, which again was inhibited by capsazepine. Use of the selective inhibitor diphenyliodonium iodide ruled out a role for plasma membrane NAD(P)H oxidase as the site of capsaicin- and 4m-stimulated DCFH oxidation. However, this DCFH oxidation was modulated by a number of inhibitors of mitochondrial respiration. Rotenone enhanced DCFH oxidation induced by capsaicin and 4m, whilst malonic acid and potassium cyanide inhibited this response. 2,4-Dinitrophenol, an inhibitor of oxidative phosphorylation, was without effect. The antioxidant trolox c inhibited DCFH oxidation stimulated by capsaicin, 4m, and PMA, whereas N-acetylcysteine, a precursor of glutathione, was without effect. Capsazepine inhibited DCFH oxidation in unstimulated cells and in cells treated with menadione, a redox-cycling quinone. Capsazepine was also a potent antioxidant when measured in a Fe3+ reduction assay. We concluded that DCFH oxidation stimulated by vanilloid analogues was not mediated via a vanilloid receptor, but rather by impairment of mitochondrial electron transport.

Amphibian organ culture in experimental toxicology: the effects of paracetamol and phenacetin on cultured tissues from urodele and anuran amphibians.

Organ cultures of various tissues from urodele amphibians deacetylate paracetamol to p-aminophenol, which polymerises to form a brown precipitate. Paracetamol addition results in a loss of glycogen and lactate dehydrogenase (LDH) from urodele liver cultures and an increase in glucose release, and in LDH loss from kidney cultures. Organ cultures from anuran amphibians are unable to metabolise paracetamol and are not affected by its presence in the culture medium. The addition of unpolymerised p-aminophenol resulted in a loss of LDH from urodele and anuran organ cultures, whilst the addition of polymerised p-aminophenol had no such effects. This suggests that the toxic effects which follow the addition of paracetamol to urodele organ cultures are caused by unpolymerised p-aminophenol, a known toxicant in mammals. Cultures from both urodele and anuran amphibians are able to deacetylate phenacetin to p-phenetidine, but p-phenetidine was found to be much less toxic to amphibian tissues than p-aminophenol, causing LDH loss from kidney cultures only at very high dose levels.

Assessment of primary eye and skin irritants by in vitro cytotoxicity and phototoxicity models: an in vitro approach of new arginine-based surfactant-induced irritation.

Extensive efforts have been made, recently, to find surfactants with lower irritation potential than those presently commercially available, for use in pharmaceutical and cosmetic preparations. Cytotoxic and phototoxic effects of a novel family of dicationic arginine-diglyceride surfactant compounds, 1,2-diacyl,3-O-(l-arginyl)-rac-glycerol with alkyl chain lengths in the range from 8 to 14 carbon atoms, were compared to three commercial surfactants. The end-points used to assess toxicity were the red blood cell lysis assay and uptake of the vital dye neutral red 24h after dosing (NRU), respectively. Two immortalized cell lines, murine fibroblast cell line, 3T3, and one human keratinocyte cell line, HaCaT, were used as in vitro models to predict the potential phototoxicity which could result in irritation, determined by resazurin reduction to resorufin and neutral red uptake (NRU). All tested surfactants had cytotoxicity effects as demonstrated by and decrease of NR uptake, which showed a clear concentration-response relationship. Concentrations resulting in 50% inhibition of NR uptake (IC(50)) range from 1 microM(-1) (hexadecyl trimethyl ammonium bromide) to 565 microM(-1) (12,12-l-arginine). Erythrocyte haemolysis also showed a clear concentration-response relationship, the 50% of haemolysis ranged from 37 microM(-1) (10,10-l-arginine) to 151 microM(-1) (sodium lauryl sulphate). Phototoxicity was performed with 12,12-l-acetyl-arginine, the most stable chemical structure. The validated 3T3 NRU photoxicity assay was used and revealed a phototoxic potential.

Comments on the scientific validation and regulatory acceptance of in vitro toxicity tests.

The 1980s saw widespread acceptance of the Three Rs (reduction, refinement, replacement) concept of alternatives, and a great deal of effort was put into the development of non-animal toxicity tests. Yet, although new methods are gaining acceptance as pre-screens and as adjunct methods, there is little sign that any of them will be accepted as genuine replacements for current animal test procedures. This is partly because, until recently, the question of scientific validation for relevance, reproducibility and transferability had not been properly addressed, and partly because, despite national and international laws that require that replacement alternatives be used wherever possible, little attention had been paid to promotion of their formal acceptance into regulatory toxicology. Reference is made to the recommendations of two workshops organized by members of ERGATT (European Research Group for Alternatives in Toxicity Testing) early in 1990-on validation (with the Johns Hopkins Center for Alternatives to Animal Testing) and on regulatory acceptance (with the support of the Commission of the European Communities)-which could provide a basis for meeting this challenge in the 1990s. It is argued, however, that primary responsibility for the effective and orderly development, validation, independent assessment and regulatory acceptance of non-animal toxicity tests should rest with in vitro toxicologists themselves.

Validation of alternative toxicity tests: Principles, practices and cases.

In vitro toxicity tests must be properly developed and scientifically validated for relevance and reliability before they are independently evaluated for possible inclusion in toxicity testing schemes and promoted for regulatory and legal acceptance. Some lessons learned in the administration of the FRAME Alternative Test Validation Scheme are reported, using as an example correlations between the results obtained in cell growth inhibition tests, both with each other and with rat oral and mouse intraperitoneal LD(50) values. Some recommendations are given for consideration for the design of future validation schemes.