The tenth anniversary of the Björn Ekwall memorial foundation.

The Björn Ekwall Memorial Foundation (BEMF) was initiated by the Scandinavian Society for Cell Toxicology in 2001, to honour the memory of Dr Björn Ekwall (1940-2000) and to establish a prize, the Björn Ekwall Memorial Award. The prize is awarded to scientists who have significantly contributed to the field of cell toxicology, and whose work is contributing toward the replacement of animal experiments by alternative toxicity tests. Over the past 10 years, the Björn Ekwall Memorial Award has been presented annually. Björn Ekwall, an outstanding Swedish cell toxicologist, was one of the pioneers in the development and application of alternative methods to animal tests in toxicology. All his scientific work was devoted to in vitro toxicology, and in particular, to the use of cultured human cells for the screening of toxic chemicals. In the middle of the 1980s, he initiated the international Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) project, to evaluate the usefulness of in vitro tests for the estimation of human acute systemic toxicity. To prove his "basal cytotoxicity concept", he established the MEMO database, in which data on the acutely toxic human blood concentrations of drugs and chemicals were collated from the literature and from clinical studies. He also initiated another project, Evaluation-Guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT). The ideas from the EDIT project, together with those from the MEIC project, became the basis for today's international EU projects, e.g. ACuteTox, Sens-it-iv and ReProTect. In this article, 10 years after the start of the BEMF, the scientific achievements of each of the award winners in the field of in vitro toxicology are presented, together with a brief synopsis of their careers.

Estimation of human blood LC50 values for use in modeling of in vitro-in vivo data of the ACuteTox project.

The main aim of the ACuteTox project, under EU 6th Framework programme, is to investigate whether animal toxicity tests for acute systemic toxicity could be replaced by a combination of alternative assays. Data for 97 reference chemicals was collected in the ACuteTox database (Acutoxbase), designed to handle invitro and invivo (human and animal) lodged data. The principal basis for demonstration of the applicability of invitro tests is the invitro-invivo modeling, by using statistical correlation between invitro IC50 molar values (the 50% inhibitory concentration for the endpoints measured) and human blood molar concentrations LC50 (50% lethal concentrations). The LC50 values were calculated from time-related sub-lethal and lethal blood concentrations determined from human acute poisoning cases. The 3T3 standard NRU assay (3T3 NRU) was chosen, among the various basal cytotoxicity assays, applied in the ACuteTox project, to demonstrate the applicability of the IC50/LC50 values for invitro-invivo modeling. Linear regression analysis between IC50 (x) and LC50 (y) gave an explained variance R2=0.56 for the 67 reference chemicals, for which both sets of data were available. The results demonstrated usefulness of human LC50 values for invitro-invivo evaluation of the predictability of basal cytotoxicity assays for human acute systemic toxicity. The R2 value of 0.56 shows, as in the MEIC study, that additional organ-specific and biokinetic tests are needed in order to improve the predictability.

The integrated acute systemic toxicity project (ACuteTox) for the optimisation and validation of alternative in vitro tests.

The ACuteTox project is designed to replace animal testing for acute systemic toxicity, as is widely used today for regulatory purposes, by using in vitro and in silico alternatives. In spite of the fact that earlier studies on acute systemic toxicity demonstrated a good correlation between in vitro basal cytotoxicity data (the 50% inhibitory concentration [IC50]) in human cell lines and rodent LD50 values, and an even better correlation between IC50 values and human lethal blood concentrations, very few non-animal tests have been accepted for general use. Therefore, the aim of the ACuteTox project is to adapt new testing strategies, for example, the implementation of new endpoints and new cell systems for toxicity screening, organ-specific models, metabolism-dependent toxicity, tissue absorption, distribution and excretion, and computer-based prediction models. A new database, AcuBase, containing descriptions and results of in vitro tests of the 97 reference chemicals, as well as the results of animal experimentation, and human acute toxicity data, will be generated within the framework of ACuteTox. Scientists from 13 European countries are working together and making efforts to find the most appropriate testing strategies for the prediction of human acute systemic toxicity, and also to select a robust in vitro test battery for cytotoxicity testing of chemicals.

Activity of epothilones.

Epothilones represent a novel class of anticancer drugs which inhibit the cell cycle and strongly influence cell division. The biological activity of epothilones is associated with their capacity to bind to the protein tubulin of microtubules and to disturb the dynamic equilibrium between microtubule assembling and disassembling. Consequently, in dividing cells, it leads to mitotic arrest and to apoptotic cell death. A number of epothilones demonstrate a potent inhibitory effect on cell proliferation in human tumor cell lines, as well as antitumor activity in experimental animals bearing human tumor xenografts. This review will focus on the recent preclinical studies of the in vitro and in vivo activities of four epothilones, EPO-906, BMS-247550, KOS-862 and BMS-310705. Several epothilones are undergoing clinical evaluation and are promising candidates for advanced cancer therapy.

BMS-310705 Bristol-Myers Squibb/GBF.

Bristol-Myers Squibb, in collaboration with the German Research Centre for Biotechnology, is developing BMS-310705, an epothilone analog, for the potential treatment of cancer. By April 2002, phase I trials of BMS-310705 were underway.

Epothilone D (Kosan/Roche).

Kosan and Roche are developing the microtubule inhibitor epothilone D (KOS-862 and NSC-703147), an analog of epothilone B for the potential treatment of cancer. Phase II trials in colorectal, metastatic breast and non-small-cell lung cancers were initiated in December 2003.

Aphidicolin induces 6-thioguanine resistant mutants in human diploid fibroblasts.

Cytotoxic and mutagenic effects of aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta, were studied in human diploid VH-10 fibroblasts. The cells were treated (2 or 4h) with APC at concentration ranges of 10-40 microM. The effect of APC on cell survival after 4 h treatment was significantly higher than after 2 h treatment. The mutagenicity of APC was investigated at the HPRT locus, and the frequency of HPRT mutants was estimated by selection in medium containing 6-thioguanine (6-TG). Treatment of fibroblast cells with 20 microM of APC for 2 or 4 h resulted approximately in 5 or 10 times increase of 6-TG resistant mutant frequencies, respectively, compared to untreated control cells. The cell cycle analyses performed during the expression time (9-12 days) have shown that after 2 and 4h treatment with APC the cells were blocked in G2 phase during the majority of the expression period, compared to control cells. Four days after the treatment, the amount of cells in G2 phase increased about two-fold (28.6-31.8% compared to 13.5% in the untreated cells). The mode of cell death during the expression time was via necrosis, rather than apoptosis, which was demonstrated by fluorescein-diacetate (FDA)-staining and terminal dUTP nick end labeling (TUNEL)-method.

Genotoxic effects of ethylene oxide, propylene oxide and epichlorohydrin in humans: update review (1990-2001).

Ethylene oxide (EtO), propylene oxide (PO) and epichlorohydrin (ECH) are important industrial chemicals widely used as intermediates for various synthetic products. EtO and PO are also environmental pollutants. In this review we summarize data published during the period 1990-2001 concerning both the genotoxic and carcinogenic effects of these epoxides in humans. The use of DNA and hemoglobin adducts as biomarkers of exposure and the role of polymorphism, as well as confounding factors, are discussed. We have also included recent in vitro data comprising genotoxic effects induced by EtO, PO and ECH in mammalian cells. The uncertainties regarding cancer risk estimation still persist, in spite of the large database collected.

Efaproxiral (Allos Therapeutics).

Allos Therapeutics Inc is developing the hemoglobin allosteric modifier efaproxiral as a potential radiosensitizer and chemotherapy enhancer in cancer therapy.

INVITOX 2004: 13th Workshop of the European Society of Toxicology In Vitro, organised in cooperation with the Scandinavian Society for Cell Toxicology.

INVITOX 2004, the 13th Workshop of the European Society of Toxicology In Vitro, was held on 8-11 September 2004, in Zegrze, Poland, in cooperation with the Scandinavian Society for Cell Toxicology. The workshop was attended by 112 participants from 19 countries. The programme included 11 main sessions and two round table discussions. The lectures of the invited speakers, together with short oral presentations and posters, covered the most important aspects of current research in the field of in vitro toxicology. ECVAM's strategies for the replacement of animal testing for toxic chemicals by alternative test systems were discussed.

New achievements in human cell toxicology: the 20th annual workshop on in vitro toxicology.

The 20th Annual Workshop on In Vitro Toxicology (Oxford, UK, September 22-24, 2002) was convened as part of a European meeting entitled Human Cell Culture 2002. The meeting was arranged by the Scandinavian Society for Cell Toxicology (SSCT), the European Tissue Culture Society and the British Prostate Group. Two sessions, which are summarised in this report, were devoted to in vitro toxicology: Human Cell Toxicology and The SSCT Free Paper Session. Outstanding experts in the field of toxicology outlined contemporary approaches in toxicity testing in their lectures. Short oral presentations demonstrated a variety of in vitro model systems and methodologies, which can be useful for investigating human toxicity, as well as for studies on mechanisms of toxicity.

Use of the PFGE assay for studies of DNA breakage induced by toxic chemicals.

The relevance of the pulsed field gel electrophoresis (PFGE) assay for the estimation of the DNA damaging effects of chemicals was studied. Four chemicals were randomly chosen from the list of 50 Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) reference chemicals with known human acute systemic toxicity: acetylsalicylic acid, paracetamol, ethylene glycol and sodium chloride. Human fibroblasts (VH-10) were used as a model system. For the estimation of cytotoxic effect, cell monolayers were treated with chemicals for 24 hours. Cloning efficiency (colony-forming ability) at different concentrations of the test chemicals was estimated, and the 50% inhibitory concentration (IC50) was determined. The IC50 values obtained demonstrated a correlation with human lethal blood concentrations. The induction of DNA double-strand breaks, measured by PFGE as the fraction of activity released, was detected after treatment with paracetamol. However, the other three chemicals tested mainly induced DNA degradation.

Development of an in vitro test battery for the estimation of acute human systemic toxicity: An outline of the EDIT project. Evaluation-guided Development of New In Vitro Test Batteries.

The aim of the Evaluation-guided Development of new In Vitro Test Batteries (EDIT) multicentre programme is to establish and validate in vitro tests relevant to toxicokinetics and for organ-specific toxicity, to be incorporated into optimal test batteries for the estimation of human acute systemic toxicity. The scientific basis of EDIT is the good prediction of human acute toxicity obtained with three human cell line tests (R(2) = 0.77), in the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. However, the results from the MEIC study indicated that at least two other types of in vitro test ought to be added to the existing test battery to improve the prediction of human acute systemic toxicity - to determine key kinetic events (such as biotransformation and passage through biological barriers), and to predict crucial organ-specific mechanisms not covered by the tests in the MEIC battery. The EDIT programme will be a case-by-case project, but the establishment and validation of new tests will be carried through by a common, step-wise procedure. The Scientific Committee of the EDIT programme defines the need for a specific set of toxicity or toxicokinetic data. Laboratories are then invited to perform the defined tests in order to provide the "missing" data for the EDIT reference chemicals. The results obtained will be evaluated against the MEMO (the MEIC Monograph programme) database, i.e. against human acute systemic lethal and toxicity data. The aim of the round-table discussions at the 19th Scandinavian Society for Cell Toxicology (SSCT) workshop, held in Ringsted, Denmark on 6-9 September 2001, was to identify which tests are the most important for inclusion in the MEIC battery, i.e. which types of tests the EDIT programme should focus on. It was proposed that it is important to include in vitro methods for various kinetic events, such as biotransformation, absorption in the gut, passage across the blood-brain barrier, distribution volumes, protein binding, and renal clearance/accumulation. Models for target organ toxicity were also discussed. Because several of the outlier chemicals (paracetamol, digoxin, malathion, nicotine, paraquat, atropine and potassium cyanide) in the MEIC in vivo-in vitro evaluation have a neurotoxic potential, it was proposed that the development within the EDIT target organ programme should initially be focused on the nervous system.

Björn Ekwall, an outstanding Swedish cell toxicologist.

Dr. Björn Ekwall (1940-2000) was an outstanding Swedish cell toxicologist who made pioneering contributions to the field of in vitro toxicology. In particular, he formulated the so called "basal cytotoxicity concept" (1983) which provided a conceptual basis for the estimation of acute systemic toxicity of chemicals in humans by the use of in vitro tests. Björn Ekwall formulated, initiated and, together with a group of dedicated Scandinavian toxicologists, guided the MEIC project (Multicentre Evaluation of In Vitro Cytotoxicity Programme, 1989-1999), in which 50 reference chemicals were voluntary tested in 100 laboratories worldwide by 61 different in vitro assays. This project was unique because human sub-lethal and lethal blood concentrations were used for a first time as a reference system for the evaluation of predictability of in vitro tests for human acute systemic toxicity. The results of MEIC project have shown good correlation between human LC50 values (50% lethal concentrations) and IC50 values (50% inhibitory concentrations from basal cytotoxicity tests), by the use a battery of three 24-h basal cytotoxicity tests (R²=0.77). The MEIC project paved the way to the present validation projects, under EU 6th Framework programme, such as ACuteTox, Sens-it-iv, and ReProTect.

The Escherichia coli RecA protein complements recombination defective phenotype of the Saccharomyces cerevisiae rad52 mutant cells.

The Saccharomyces cerevisiae rad52 mutants are sensitive to many DNA damaging agents, mainly to those that induce DNA double-strand breaks (DSBs). In the yeast, DSBs are repaired primarily by homologous recombination (HR). Since almost all HR events are significantly reduced in the rad52 mutant cells, the Rad52 protein is believed to be a key component of HR in S. cerevisiae. Similarly to the S. cerevisiae Rad52 protein, RecA is the main HR protein in Escherichia coli. To address the question of whether the E. coli RecA protein can rescue HR defective phenotype of the rad52 mutants of S. cerevisiae, the recA gene was introduced into the wild-type and rad52 mutant cells. Cell survival and DSBs induction and repair were studied in the RecA-expressing wild-type and rad52 mutant cells after exposure to ionizing radiation (IR) and methyl methanesulphonate (MMS). Here, we show that expression of the E. coli RecA protein partially complemented sensitivity and fully complemented DSB repair defect of the rad52 mutant cells after exposure to IR and MMS. We suggest that in the absence of Rad52, when all endogenous HR mechanisms are knocked out in S. cerevisiae, the heterologous E. coli RecA protein itself presumably takes over the broken DNA.