A total of 1 500 000 SEK was awarded in Grants from the Swedish Fund for Research without Animal Experiments for 2007-2008. It was divided between 14 projekts. The recipients shortly describe their projects below.
Christel Bergström, Computational prediction of drug transport mechanisms. BMC, Uppsala University
In the cell membranes there are proteins that can protect the body from unknown compounds. These efflux proteins can also bind drugs and transport them out of the cell. If this happens in the intestine only a small fraction of the given drug will reach the systemic circulation, resulting in no or minor therapeutic effect.
Moreover, the presence of efflux proteins can give rise to side effects due to the variation in the amount of protein that is expressed between people. Furthermore, different drugs can compete for the binding to the protein. These mechanisms result in an unpredictable oral absorption of such drugs and a less understood distribution process within the body. We will set up experimental methods for screening of drug interactions with efflux proteins using cell models. The experimentally generated values will thereafter be used for development of computational theoretical models of the drug-protein interaction. If we are successful in the development of these models the use of animals in the drug discovery and development process will decrease markedly, since compounds with a bad absorption- and distribution profile are identified in the computer before they are even synthesised. Hence, such troublesome compounds will not reach the animal testing.
Anna Forsby, Neurotoxic mechanisms for acute, systemic toxicity studied in vitro
Department of neurochemistry and neurotoxicology, Stockholm university, firstname.lastname@example.org
All chemicals have to be studied in an array of tests in order to assess their risk for humans or environment. According to present rules, most tests shall be performed on animals (mostly rats, mice and rabbits) but there are strong forces working for their replacement with safe alternatives, especially because the REACH proposal will result that the 30000 chemicals to be tested require approximately 10 million animals. The objectives of the project are to develop and evaluate methods for acute, systemic toxicity, i.e. measuring how much an animal or human can be exposed to a chemical at one time before it dies. Since many chemicals are toxic because they affect the nervous system in some way, our theory is that these effects can be monitored in cells originating from nervous tissue. Effects of narcotic and pharmaceutical drugs, pesticides and industrial chemicals will be studied on nerve stimulation, signal transmission and energy supply in our cell model. It is our hope that the methods are valid and predictable so they can be integrated in a large set of different tests that can estimate a whole organism’s response on chemical exposure.
Anna Forsby, Neuronal cell model for identification of mildly eye-irritating compounds
Department of neurochemistry and neurotoxicology, Stockholm university, email@example.com
The Draize’s eye irritation test has been used during decades for identification of irritating and tissue damaging chemicals. The test, which is performed on rabbits, is quite painful and the scientific value of the results from the test has been questioned. Therefore, extensive efforts to find alternative methods have resulted in several validation studies. These studies have generated alternatives to the Draize’s test for chemicals, which induce severe eye tissue damage (corrosion), but there is still no method that is able to identify chemicals, which only generate mild eye-irritation, e.g. smarting pain. Mild irritation occurs when specific pain-mediating receptors (nociceptors) are activated. We will therefore in this project develop neuronal cell clones, which express specific nociceptors, in order to investigate the possibility to use them for the identification of mild eye-irritating chemicals. If the neuronal clones fulfil the criteria, there is a good chance that the alternative test batteries will be significantly improved. Today, when the EU chemical test strategy proposes an extensive eye irritation screening programme by using the Draize’s test, an optimised alternative test battery can result in that thousands of rabbits will be saved from painful experiments.
Anna Fyrberg, Use of RNA interference to study mechanisms of nucleoside analogue resistance. Linköping University, firstname.lastname@example.org
Nucleoside analogues (NAs) are cytotoxic drugs mainly used to treat different types of leukemias as well as some solid tumors. These dugs require intracellular activation by different enzymes to become cytotoxic. The two most important activating enzymes are deoxycytidine kinase (dCK) and deoxyguansoine kinase (dGK). Decreased activity of these enzymes is one mechanism of resistance to NAs. It is of great importance to learn more about these enzymes and the mechanisms behind resistance development in order to develop new, more effective drugs.
Studies on resistance have been performed in animal models where tumor cells have been inserted into mice with subsequent treatment with NAs and development of resistance. The mice have later been sacrificed and tumor cells taken for analysis. Another possibility to develop NA resistant cells is to treat cultured cell lines with NAs for several months up to a year. However, these processes are time-consuming and several compensatory mechanisms develop in the tumor cells, which make the data difficult to interpret.
We developed a method to use small interfering (si) RNA to decrease the activities of dCK and dGK within 24 hours in different cell lines. SiRNA are small molecules binding to a unique mRNA sequence in the cells and degrades it, leading to less protein produced from that specific mRNA sequence. It is then possible to perform toxic studies on the cells and to study the impact of the decreased activities of these enzymes. This will make it possible to test existing drugs and also new promising substances on cells already made resistant with this technique. In many cases this technique can replace the use of knock-out animals, were different genes have been deleted, at least in early drug development.
Roland Grafström, Culture of oral keratinocyte stem cells: gene expression and sensitivity to programmed cell death
Institute of Environmental Medicine, Karolinska Institute, Stockholm. email@example.com
The overall aim is study the growth of the stem cells underlying epithelial regeneration in oral mucosa. Culture methods will be established, including variants without serum, for assessment of gene expression. Profiling of transcript and proteins will be made to determine expression differences between stem cells and epithelial cells of higher maturity. The utilization of stem cells in biomedical research are likely to provide new alternatives to work with laboratory animals, including for cancer research.
Jan Grawé, The micronucleus test: from in vivo to in vitro. Towards an in vitro model for the study of
chromosome damage in human bone marrow cells.
Rudbeck laboratory, Uppsala University firstname.lastname@example.org
The numbers of laboratory animals used for risk estimation of chemicals has successively decreased. However, regarding their potential to induce damage on chromosomes, the in vitro test alternatives are not wholly statisfactory. This can lead to that a relatively large use of animals for this purpose in the extensive EU programme for risk assessment of chemicals (REACH).
A much used test for chromosome damage is the bone marrow micronucleus test. In the present project we aim to develop an in vitro test for the same purpose. The test will be based on human cells which are cultured in vitro, but but through special culture conditions are made to develop and react to chemicals in a similar manner as if they would be in the body.
Per Hultman, Reagents without animals: In Vitro test for prediction of allergenic hypersensitivity reactions.
Linköping University, email@example.com
Allergy is a common disease worldwide and is increasing in prevalence. There is no simple explanation to why the disease arises. The symptoms of an allergic reaction can vary from mild inconveniences to life threatening conditions. In order to increase the safety of commercial products and thereby to minimize the risk of allergic sensitization, more extensive demands coupled to higher requirements on the testing of products have been reinforced by authorities. The current method for identification of potentially allergenic characteristics of a substance involves animal testing (in vivo tests), and for many hypersensitivity reactions this method has a limited ability to predict allergic reactions in human. The aim of the present project is to develop an in vitro method (i.e. in test tubes) based on human cell cultures and modern gene expression technologies. The purpose is to completely replace today's allergy-related animal tests and to create a test system specific for human.
In order to create an in vitro test for routine use the test needs to be scientifically solid and should have undergone an accurate validation in collaboration with ECVAM (European Centre for the Validation of Alternative Methods). The effect by different allergens on the immune cells must be determined on a molecular level. The Institute of Molecular and Immunological Pathology at Linköping University will, in collaboration with Biovator AB, Stockholm, develop a so called "in vitro assay" for the predictive identification of potentially allergenic substances found in e.g. pharmaceuticals, chemicals, cosmetics or food additives.
The present project aims at providing more insight into the underlying mechanisms of the immunobiological reactions found in allergy and to increase the knowledge of the prerequisites for the development of an in vitro method. We have identified human cell lines that in pilot experiments, using known allergenic substances, have shown promising results for the predictive identification of allergens. The cell culture conditions should be further optimized, which also includes cell cultivation without the use of fetal calf serum (FCS). The continued optimization work also includes investigations of gene activation and cytokine production upon stimulation with known allergenic substances and control substances (known to be non-allergenic).
The assay should be accessible for the users as a validated test kit, containing all components and all information that is needed for the performance. The future users of the assay are laboratories performing immunotoxicological analyses and "screening" of new substances for the predictive identification of potential allergens.
Ada Kolman, Use of human toxicity data for the evaluation of in vitro tests, Expertrådet AB, Sollentuna, 140.000 SEK
Human toxicity data on different chemicals in our environment (drugs, industrial chemicals, herbicides etc) will be collected and analyzed with the purpose to evaluate in vitro data. Human data on acute toxicity and animal data (LD50 values) will be used as an important reference system, which may show that a combination of in vitro tests is enough successful to predict human toxicity. If the in vitro tests are as good as or better than the animal tests on predicting human toxicity of chemicals, the animal tests used today for acute toxicity testing could be replaced by in vitro tests.
Malin Lindstedt, Studies of the effect of allergens on dendritic cells - basis for an in vitro test of the potential of chemicals and proteins to cause sensitization
Lund University, firstname.lastname@example.org
The objective of the project is to identify the mechanisms involved when allergens are recognized by the central regulators of the immune response; the dendritic cells (DC). Understanding the interactions between epithelial cells (EC), dendritic cells and T cells during allergic response, and identification of the molecules involved, are important for the development of an in vitro test which can predict the allergenicity of new chemicals and proteins produced by the chemical, cosmetic and pharmaceutical industries. Allergy is a large social and economic problem today and up to 40 % of children in some industrialized countries are affected. Hundreds of chemicals and proteins cause contact hypersensitivity, rhinitis and asthma and there is thus a great need for identification and documentation of which agents lead to sensibilisation.
Before chemicals reach product level, they have to be tested for toxicity and this is currently performed in animal models. For some factors, e.g. skin irritability, there are validated alternative methods available, but no test is available for sensitization. Our group is part of a 6th framework EU program with the title Novel testing strategies for in vitro assessment of allergens - Sens-it-iv (www.sens-it-iv.eu ). The program consists of 28 European partners in institutions, organizations, academia and industry which all focus on the development of novel test systems. The test should be based on human EC- and DC-like cell lines, cultured together to mimic the environment in skin or lung, and should replace the use of animals for evaluating the ability of new agents to cause sensitization. The obvious advantageous of the in vitro test is reduced animal experimentation, but also increased relevance for man as human cell lines are used and that these in many instanced can be grown without fetal calf serum.
In order to select the optimal DC candidate for the in vitro assay, mimicking in vivo DCs the most, we are currently characterizing the available in vivo DC populations and in vitro models. In total, we have collected 17 different DC populations and their gene expression and cell surface markers are now being analysed and compared. The transcriptional analysis demonstrate that the different primary DCs express population-specific profiles, which means that the populations isolated from skin, tonsils or blood are unique and have specialized functions in the immune response.
Even if the in vitro models of DCs also express population-specific markers, they resemble their counterparts in vivo. The most "DC-like" in vitro models will now be stimulated with various allergens in order to determine if they respond in a "DC-like" fashion to sensitizers. Allergens such as DNCB, CIN, Derp1 and Phlp5 will be tested initially. To be able to identify the DC markers involved, we will use CD arrays (Medsaic Pty Ltd, Australia), by which you can measure the expression levels of 140 CD antigens simultaneously. When we have characterized the DC response to allergens, we will culture the cells with an EC line (Calu-3 or A549) in collaboration with Novozymes A/S, Denmark, to develop an assay which is based on the crosstalk between DC and EC. Novozymes have developed a method to culture ECs on membranes which resemble the environment in lung and skin, with air and culture medium on each side of the membrane.
We now want to bring our models together to determine the effect of allergens on migration, phenotype and production of soluble molecules. We have already started to culture these cells in serum-free medium. In conclusion, this project will give information about which in vitro cells to use for the test system. We also aim to determine the molecular markers involved when DC respond to allergens and do the first attempts to bring ECs and DCs together in a test system to determine the propensity of chemicals and proteins to cause allergy.
Tommy Linné, To develop methods for substitution of animal testing and straintyping of TSE agens (prions) with an alternative animal free cell-culture based system. SLU, BMC, Uppsala, email@example.com
Prion diseases or TSE (transmissible spongiform encephalopathies) can affect humans (CJD, vCJD), cattle (BSE), cervids (CWD) and sheep (Scrapie). Both Scrapie and BSE have been diagnosed in Sweden and recently 13 cases of an atypical Scrapie named Nor98 have been demonstrated.
The definition of prion diseases as transmissible clearly differ prion diseases from other disorders due to protein aggregation as for ex Alzheimers- and Parkinson disease. In prion diseases as Scrapie in sheep and Chronic Wasting Disease (CWD) of cervids a horizontal transmission is evident with a direct spread in the population of concern, resembling a classic infectious disease.
The infectious prion, PrPSc, can be transmitted to an individual from a diseased source or can be generated spontaneously in the nervous system. In both cases PrPSc will directly affect the normal cellular prion protein and convert it into the diseased prion conformation. The newly formed PrPSc will by unknown mechanisms be spread to neighboring cells.
In vitro system based on cell cultures present many obvious advantages including a possibility to analyze the biological properties of PrPC and PrPSc both on a molecular level and on a cellular level. In addition, the determination of the infectious principle, identifying factors important for it's propagating and the mechanism of shedding and transmission between cells can be elucidated. The most used cell culture is the mouse neuroblastoma cell line; N2a and studies in this and other cell-lines have outlined various chapters of prion biology. However, common to all these cell lines are that they support propagation of selected mouse adapted TSE strains and not propagating natural TSE cases as sheep Scrapie strains or BSE. Multiple distinct natural strains of Scrapie in sheep have been isolated by propagation in inbred mice and been distinguished by incubation periods and specific neuropathology. In Sweden both classical Scrapie and atypical Scrapie Nor98 have been demonstrated. We have earlier differentiated these two natural strains by Western immunoblot electrophoretic profiling from naturally diseased sheep victims.
Our aim is to find cell culture systems instead of animal interventions, which will faithfully propagate prion strains and preserve their associated biochemical characteristics and biological properties on a cellular level permitting straintyping without animal involvements.
Christina Rudén, An improved model for increasing the efficiency of toxicity testing in chemicals control
KTH Filosofi, Stockholmcr@infra.kth.se
The purpose of this project is to develop an improved model for tiered toxicity testing by combining methods from toxicology, decision theory, and ethics. The efficiency of a toxicity test, or combination of tests, is determined by the costs associated with performing the test and the amount of decision relevant information that it will generate. In the improved model we will supplement previously proposed models so that not only the monetary costs of performing a test is taken into account, but also the social cost of using experimental animals.
We will first review the existing literature to identify a useful and relevant way of quantifying animal suffering. If no such model is available, we will develop one. The model will provide the relative degree of animal suffering in different standardized toxicity tests as well as the number of animals used in each test.
This information will be combined with information about the usefulness of different tests for risk management, in particular for hazard identification, within the legal system for classification and warning labeling of industrial chemicals. The combination of these test parameters (their monetary cost, their social cost associated with animal use, and their usefulness for risk management) will generate knowledge that have the potential to lead to a re-evaluation of the efficiency of standardized tests. This could contribute to the increased regulatory acceptance of methods that refine, reduce, or even replace animal experiments without compromising the need for generation of knowledge about chemical risks towards the aim to protect human health and the environment.
Johan Rönnelid, Immune complex-mediated regulation of cytokine production in rheumatic diseases and cancer
Uppsala University, Uppsala, firstname.lastname@example.org
Antibodies produced by the immune system can aggregate into immune complexes. Such immune complexes composed of antibodies and substances from the body are essential in the pathogenesis of rheumatic disorders and may have importance in the regulation of the immune defence against tumours. Today basic research in both these areas utilizes substantial numbers of laboratory animals.
Our aim is to understand how immune complexes influence the production of cytokines, signal substances of the immune system. The work is entirely based on in vitro research on human tissues and blood. For these purposes we develop new sensitive laboratory techniques to detect immune complexes and their immunological effects on human cells in vitro. Important parts are e.g. to establish totally human cell culture conditions without addition of animal sera, and to develop sensitive techniques for the detection of cytokine production in vitro.
Therese Söderdahl, Human stamcellsmodell för utvärdering av läkemedels-inducerad toxicitet: Karaktärisering av biotransformationsenzymer i hepatocyt-lika celler från humana embryonala stamceller. Karolinska Institute, Stockholm, email@example.com
Foreign compounds, such as environmental agents and drugs, may damage cells and tissues via metabolic transformation, so called biotransformation, to reactive intermediates. Liver injury is a common side effect of e.g. drugs, since the liver is the main organ that comes into contact with the foreign compounds we are exposed to. The liver may also leak reactive compounds into the circulation which may lead to damage of other tissues. Toxicologists strive to identify compounds that are metabolized in the liver and assess the risk they may involve. For this predictive model systems are needed, and today animals or cells are used which cannot correctly predict the responses of the liver. Human embryonic stem cells (hESC) that may develop into liver cells is a potential new model system for studies of toxicology and liver injury, which may better reflect the metabolism in man and lead to a significant reduction of the use if experimental animals. However, for these cells to be used, we need to evaluate if the enzymes needed for metabolism into reactive intermediates are present in these cells. The aim of this project is to study a selection of these enzymes in liver-like cells derived from hESC. We have studied enzymes from the first two phases of the biotransformation in the liver. The results so far show that the liver-like cells have an expression pattern that closely resembles that of human liver cells; however more work is needed for the cells to be used as a model system. It is important to include cells from several donors since the expression pattern of the enzymes under study vary greatly between different individuals, and this may lead to different reactions to substances, including drugs. It is also of great interest to study the capability of these cells to respond to and repair damage of DNA.
Erik Ullerås, Studies of endocrine disruptors in the human adrenocortical cell line (H295R) SLU, Uppsala, firstname.lastname@example.org
Disruption of adrenal steroidogenesis has been demonstrated after treatment with a number of environmental pollutants and medical drugs. Adverse effects on adrenal function have several consequences, for example dimished capacity to cope with physical and psychological stress. The human adrenocortical carcinoma cell line H295R produces the three steroids, aldosterone, glucocorticoids and androgenic steroids, in a similar manner as in the normal human adrenal. The aim of the project is to characterize the gene regulation of the steroid synthesis in the H295R cell line. Furthermore, molecular mechanisms for some adrenotoxicants will be investigated. The H295R cell line has a potential to be developed into an in vitro test model for endocrine disruptors acting on adrenal steroidogenesis.