Professor Jan-Åke Gustafsson, cascade coordinator
Providing scientific facts to protect consumers In our global, increasingly complex world interaction and cooperation are more needed than ever before. Old rigid structures have to give way to new thinking, new coalitions, new attitudes. Our future health problems, many of them caused by environmental pollution, can only be dealt with by many brains working together towards the same goal. CASCADE Network of Excellence is a good example of the paradigmatic shift in European research that we are now experiencing. I am convinced that this new way of international scientific collaboration has come to stay and that it is the first step towards a big change in all major seats of learning in Europe.
100 000 chemicals in our environment 4 What is a hormone? 5 Some common – and harmful chemicals 6 Can phyto-estrogens protect against cancer? 8 Big CASCADE project to find out if bread is harmful
The brain seems to be well protected
Declining male fertility
Can red wine increase stress?
Karo Bio – industrial partner in CASCADE
School project found chemical pollutants in seal oil
Early estrogen exposure can affect brain functions
Fireflies are saving testanimals
CASCADE test methods already in industrial use
Risk assessment – a difficult task
Pesticides causes hormonal imbalance
Dioxin is affecting female reproduction
Computers instead of animals
Zebra fish – excellent tools for research
Are the Networks of Excellence at risk?
CASCADE and REACH
More than 50 junior scientists in CASCADE
Breast milk and baby-food – are they safe?
Long exposure to pesticides increases cancer risk
All CASCADE partners
Recycled food boxes are more harmful
Borders have to be crossed, doors have to be opened to let in a new culture in the scientific world. A culture with more clever collaboration and less prestigious competition. To establish changes like this, to brake new ground, takes a long time and the process is not always easy. But after almost four years with CASCADE I am convinced that we are on the right track and that our joint efforts eventually will make a difference. One of our foremost responsibilities as researchers in this field is to protect the consumers by providing scientific facts that serve as a foundation for new legislation. Facts that we achieve through highly qualified research using our own new test models combined with advanced risk assessment. The next step in our work for consumer safety will be to use those methods to search for chemical residues in bread and baby-food – food-stuffs that are impor tant to almost all of us. We strongly believe that closely connected networks of scientists like CASCADE is a force to be reckoned with in the development of our common future. This magazine, a cascade also in a literal sense with its many faces of junior and senior, female and male researchers from all over Europe, is an attempt to show the European public who we are, how we work and why we do it.
200 CASCADE scientists against 100 000 chemicals
How dangerous is our environment? The CASCADE Network of Excellence, fun ded via the European Commission, Sixth Framework Programme, Thematic Area 5: Food Quality and Safety, was formed in 2004. The acronym stands for “Chemicals as con taminants in the food chain, a network for research, education and risk assessment”. CASCADE consists of 24 research groups from nine European countries and is coordi nated by Professor Jan-Åke Gustafsson, of Karolinska Institutet, Sweden. The overall aim of the network is to bring together scientific experts from different dis ciplines in a joint action to identify and study the effects of food-borne environmental pol lutants. World production of chemicals has increa sed from 1 million tons in 1930 to more than 400 million tons today. With this fast acce leration it is extremely difficult to obtain suf ficient knowledge to carry out a risk assess
ment of the effects of the substances on health and the environment. But what we do know is that a vast number of chemical pollutants are bound to end up in food, accumulating along the food chain and eventually reaching the human population. The harmful effects of chemical contami
”The overall aim of the network is to bring together scientific experts from different disciplines in a joint action to identify and study the effects of food-borne environmental pollutants.” nants in food are a major health concern in Europe today. The research within CASCADE focuses on human health, in particular hormone disrup ting effects (so called endocrine disruption), of chemical residues in food and drinking water.
Important ethical principles
These residues can interfere, even at low le vels, with the function of hormone systems in the body. The chemicals imitate human hor mones by interacting with cellular structures called nuclear receptors. This family of recep tors includes receptors for hormones like estro gen, testosterone and thyroid hormone.
The letter R is fundamental to CASCADE’s ethical principles. “R for Refinement, R for Reduction and R for Replacement”, explains Professor Olle Söder, chairman of the Ethical Review Board (ERB), the ethical watchdog keeping track of all animal experimentation within the network.
Replacement – referring to the requirement that animal experiments must be replaced by other test models when ever possible.
CASCADE agrees with the general aim of EU to reduce the number of animal experiments. “We hope to be something of a role model in this, but unfortunately it’s not possible to do research without some level of animal experiments. All other test methods form an excellent complement, but cannot give answers to the same questions as animal tests”, says Professor Söder. The project subscribes to the
“We follow these rules rigidly in CASCADE, but there are also national laws that we cannot override. This said, all experiments carried out in the project must pass the scrutiny of the ERB and obtain our approval.” Professor Söder recommends everyone wishing to know more about ethics and animal experimentation to visit www.codex.vr.se (site also available in English).
following three international ethical principles: Refinement – referring to the requirement that animal experiments must be carried out as ethically correct as possible, avoiding all unnecessary suffering and with the provision of the best conceivable care. Reduction – referring to the requirement that scientists must always endeavour to use as few animals as possible.
A disrupted nuclear receptor function may be linked to increased risk of widespread con ditions, like cardiovascular disease, obesity, diabetes, reduced fertility, breast cancer, pro state cancer, colon cancer and neurodegene rative disease. Since its start in 2004, the network also has developed several standardised tests and models for endocrine disruption, not the least virtual screening methods which not only can reduce the costs of toxicological testing but also minimize animal experimentations. Further on, CASCADE has built up a new infrastructure of collaboration between Eu ropean laboratories, leading to broader and faster analyzing of test samples. CASCADE has also a strong focus on education and has estab lished a training programme at the European level in the field of endocrine disruption, food safety and health risk assessment. The multidisciplinary, cross-border team work within CASCADE is rather new in Eu ropean research. The intention is to keep close contact with consumer’s organisations and provide scientific advice and recommen dations to support regional, national and European decision-makers and authoritieswith the overall aim to protect the health and wellbeing of European citizens.
Vinclozolin is a fungicide used on fruits, vegetables, and ornamentals to control various blights and rots (caused by Botrytis, Monilinia and Sclerotinia). As an active ingredient in plant protection products vinclozolin is deliberately released into the environment through application to crops and ornamental plants. It is not persistent in the field and is readily degraded to several metabolites. Occupational exposure to vinclozolin may occur during production, the application process, or when crops and plants are harvested or handled. Consumer exposure via inhalation or skin contact could similarly occur via contact with turf sod or private use in home gardens. However, the main cause for concern regarding consumers is oral exposure from food products containing residues of vinclozolin or its metabolites. Vinclozolin is not acutely toxic. The most sensitive effects of chronic vinclozolin exposure are anti-androgenic effects characterized by non-neoplastic changes in liver, adrenals and genital organs in males. Chronic exposure to vinclozolin also result in increased incidence of tumours, primarily in the adrenals, liver and genital organs of male and female rats, as a result of continuous hormonal stimulation. The most dramatic anti-androgenic effects of vinclozolin exposure are signs of feminization and infertility observed in male rat and mice offspring after prenatal exposure of the foetus. These effects range from weight changes to complete absence (atrophy) of genital organs, such as testes and prostate. Thus, exposure to the unborn foetus occurs, may cause permanent adverse effects in off-spring. Vinclozolin was prohibited in Europe in 2006, after pressure from the Swedish Chemicals Inspectorate in collaboration with CASCADE among others.
Some common c Today, the world market includes more than 100 000 different chemicals. Some of these chemicals are pesticides used in the agriculture sector. DDT, chlordecone and vinclozolin are examples of pesticides that have been or are being used as pesticides. Other chemicals come from industrial production, such as bisphenol A, used in plastics, PCB, previously used in many industrial products and brominated flame retardants, used to prevent electronic devices from catching fire. A third group of chemicals are spread in our environment as by-products of manufacturing or combustion processes. Dioxin is one such example. CASCADE researchers have analysed if these types of chemicals are endocrine disruptors and could be linked to increased risk of diseases such as cancer. Read more about their results in the following pages.
Foto: Anna Kern/Etsa
QB rominated Flame Retardants are leaking into our food
Brominated Flame Retardants (BFRs) are agents that are added to different materials, such as electric wires, building material, electronic devices and textiles, to help prevent them catch fire. There are many chemical substances that can be used for this purpose, including chlorine and bromine compounds, which often make effec足 tive flame suppressants. About 25 per cent of the flame retardants manufactured every year around the world (over half a million tonnes) are BFRs.However, owing to leaching and diffuse leaking from goods and waste, BFRs are being absorbed into the environment and, subsequently, into the food we eat. Much of this could probably be prevented by incinera足足ting the products under controlled conditions.
Tests show that long-term exposure in animals affects the liver, the thyroid gland and the reproductive system, disrupts foetal development and causes allergic reactions. Much remains unknown about the health consequences of BFRs, especially in terms of their neurotoxic and hormonal effects. According to experts, there is reason to suspect that the substances can cause serious problems, not least considering their extensive proliferation and persistence in the environment, and the fact that the substances have structural similarities with more well-known chemical pollu足 tants, such as PCBs. This, and the fact that they accumulate in living tissue and become concentrated in the food chain, may one day lead to very serious problems.
BPA is not acute toxic. However, after chronic administration, effects on the development of reproductive organs in both sexes and reduced body weight gain are observed in rats. In the EU risk assessment the conclusions drawn regarding health effects is that more information is needed to establish the risk to developmental effects. BPA is structurally different from naturally occurring estrogens but can still mimic the actions of estrogens in the body. BPA binds to both the nuclear estrogen receptors, resulting in alteration in gene transcription. There are also indications that BPA can disturb other hormone systems, such as the thyroid hormone system and plasma levels of insulin.
QP CBs in food can cause cancer and behavioural disorders PCBs (polychlorinated biphenyls) have been used in condensers and transformers, hydraulic oils, paints, adhesives, etc. since the 1930s. Although the use of PCBs in manufacture has now been banned in many countries, they continue to leak into the environment from landfills, old buildings and elsewhere. The main human source of PCB exposure is fatty animal food-stuffs, such as fish, milk and meat, as well as breast milk. PCB levels in fish and breast milk have fallen since the 1970s, but the downward trend has started to level off in recent years. The average daily intake of PCBs is estimated to be 10 ng/kg body weight, but is much
higher for breast-fed babies. Different types of animal and human studies show that PCB exposure can cause cancer, impaired immune defence and behavioural disorders (hyperactivity and learning difficulties), and that the early stage of development (i.e. gestation) is the most sensitive period. The health risk assessment of PCBs is made complicated by the fact that the biological effects of different PCB congenes differ in both strength and quality. Certain PCB congenes operate through the same mechanisms as dioxins (dioxin-like PCBs) and are risk-assessed alongside them.
Foto: Nicho Södling/Johnér
Foto : N
Bisphenol-A (BPA) is one of the highest-volume chemicals produced worldwide. BPA is used as a monomer in the manufacture of polycarbonates and epoxy resins, as an antioxidant in PVC plastics and as an inhibitor of end polymerisation in PVC. BPA is produced in great amount, and sources of exposure are both from the production, processing and use of the substance, resulting mainly in occupational exposure, but exposure to consumers can also occur via environment through contaminated air from the industries. However, the major sources of consumer exposure are when BPA containing materials are in contact with food-stuffs, since BPA can migrate from the material into the foods and beverages. This migration increases when the material is exposed to high or low pH and at high temperatures, and consumption of contaminated foods and beverages will then result in ingestion of the substance. Exposure through contaminated foods and beverages are for example plastic baby bottles, food and beverage cans and wine vats.
Q Bisphenol-A (BPA)
Q Dioxin Dioxin is the general term used to denote PCDDs (polychlorinated dibenzo-p-dioxins) and PCDFs (polychlorinated dibenzofurans). The chemical and toxic properties of these two groups of compounds are similar. The dioxin that is most highly toxic and that has been most extensively studied is known as TCDD. Trace amounts of dioxins are formed during the manufacture of compounds containing chlorine and during combustion processes such as the manufacture of iron and steel. Vehicle exhaust fumes and chlorine bleaching of paper were previously significant sources of dioxins. Humans are exposed to dioxins through fatty animal foodstuffs such as fish, milk and meat, and through breast milk. The levels of dioxins in fish and in breast milk have fallen since the 1970s, but the rate of this fall has decreased in recent years. Cancer, damage to the immune system, and reproductive and developmental problems arise in several species when exposed to low doses of TCCD for long periods. Animal studies have shown that TCDD is a very effective tumour promoter, and the International Agency for Research on Cancer (IARC) classified TCDD as a human carcinogen in 1997. It negatively affects the fertility of both males and females and causes developmental disturbances in both foetuses and the young. Various types of study have shown that foetuses are particularly vulnerable, and that effects from exposure in the uterus can become apparent when the animal becomes an adult. Despite a high exposure to dioxin-like substances from breast feeding, the current opinion is that the advantages of breast feeding outweigh the risks associated with dioxins in breast milk. It is believed that TCDD (and other dioxin-like substances) are toxic through their effects on the basic regulation systems for cell growth, development and function. Examples of regulation systems that are affected are sex hormones, growth factors, thyroid hormones and retinoids (vitamin A).
cascade partner: sari mäkelä
The human hormonal system is affected not only by foreign man-made chemicals or pollutants, but also by substances such as the phyto-estrogens that occur naturally in the food we eat. Professor Sari Mäkelä’s research group in Finland has shown that lignan phyto-estrogens, which are phenolic compounds found for instance in cereals, linseed and certain berries, seem to protect against breast cancer.
Can phyto-estrogens in food
prevent cancer? products of this lignan metabolism. “In a project, which we’re conducting with Cascade partners Günther Gauglitz in Ger many and Ingemar Pongratz in Sweden, we’ve discovered that enterolactone activates the estrogen receptor alpha,” she explains. “In terestingly, if rats with mammary cancer are fed with enterolactone, or its plant precur sors, the growth of the tumour is inhibited. This suggests that enterolactone protects against breast cancer.”
Sari Mäkelä and her group work at Functional Foods Forum and Turku Center for Disease Modeling at the University of Turku. Like many other researchers, Sari Mäkelä, MD, PhD, Professor of Food Development, is passionate about her work. She developed a fascination for hormone research early in her career: “Research is like planting a garden. Some things take a long time to grow, others appear quickly, some never thrive, and there are al ways surprises; whatever, the garden’s never really finished as it’s always developing. That’s what it’s like in cascaDE, where I really do
feel that we’re doing something important for the tax-payers’ money. We’re conducting re search which, given that it’s focused on the major health issues, is valuable for all Euro pean citizens,” she says. Professor Mäkelä has devoted many years of her research to phytestrogens and how they affect human health. Lignans are one type of phytestrogens, and they occur for example in cereals, linseed and in the skins of grapes and other berries. After ingestion, lignans are metabolized by the intestinal microflora. Enterolactone is one of the important end
Pauliina Damdimopoulou, a PhD student in the research group of Sari Mäkelä, focuses her research on the effects of food on the estrogen signalling. Although estrogens, in proper amounts and timing, are crucial for health, a high lifetime exposure to estrogens is also associated with increasing a woman’s risk for breast cancer, she says. Their studies on mice that have a reporter gene for detection of estrogenic acitivity have shown that diet can attenuate estrogenic sig nals. If the mice are fed with fiber rich food before being treated with estrogen, the repor ter gene is not as active as in the mice given a nutritionally equivalent diet devoid of plant fibre. This could mean that the food we eat can protect us from the negative effects of a too high level of estrogen in the body, caused for example by exposure to chemical substances. “These studies suggest that eating food containing plenty of fibre could reduce estro gen signaling, which in turn, could reduce the risk of breast cancer or other diseases that are related to too high exposure to the female hor mone”, says Sari Mäkelä, adding that further studies are needed to demonstrate the effect in humans.
Research Site The University of Turku is one of the largest univer sities in Finland. The Functional Foods Forum (http://fff.utu.fi/) is an independent special unit, founded by the Faculties of Medicine and Natural Sciences. FFF is a research and development centre providing research expertise for the development of existing innovations in the func tional food area. It brings together the multi disciplinary expertise for the research and development of high-quality safe foods to promote human health and well-being. Turku Center for Disease Modeling (http:// tcdm.utu.fi/), a new research infrastructure in the Faculty of Medicine, provides state-of-art research facilities and expertise in experimental studies in vivo. TCDM is specialized in experimental animal models for studies on the pathogenesis, mechanisms of diseases, and for testing novel therapeutic and preventive interventions.
What’s in our bread? From chemicals and plastics to bread and baby food. Does the bread we eat and the food we give to our babies have any hormone-like activity? That will be the next challenge for the CASCADE Network of Excellence.
Phyto-estrogens are naturally occurring substances in such foods as soya beans, wheat, rye, linseeds and sesame seeds. There are two main groups of phyto-estrogens: isoflavones (e.g. genistein, daidzein and glycetein) and lignans (e.g. lariciresinol and matairesinol). Enterolactone is a lignan metabolite. Many phyto-estrogens have weak estrogenic or anti-estrogenic effects, which has led to the supposition that they might give a certain degree of protection from breast and prostate cancer. The fatality rates of certain cancers, prostate for one, are higher in Western societies than in Japan, where people eat more phyto-estrogen-rich food. Despite this, the cellular changes that constitute the first stages of developed prostate cancer are just as common, which suggests that diet can have a stronger protective effect on the development of cancer than on the initiation of the disease.
The network has, up until now, focused on deve loping test methods to detect hormone-like activity of chemicals interacting with so called nuclear receptors – methods that now will be used to find out whether whole food items, such as bread and baby food, can exert any nuclear receptor-modulatory activity when eaten as such. Professor Sari Mäkelä and her PhD student Pauliina Damdimopoulou, at the University of Turku, Finland, are the coordinators of the CASCADE Bread project, which together with the baby food project will be the “Proof of Principle” for the network. All partners will participate with their different skills and methods in this huge project. Professor Mäkelä hopes to receive answers to many of her questions once the bread samples have been tested. “Bread is of immense interest to everyone in Europe. We all want to know what bread contains and how it affects our health. Today, we know that grains and seeds commonly used in bread, such as wheat and linseed contain cadmium, which can have a hormone-disrupting effect, but we’ve also observed that the lignans that occur in both wheat and linseed seem to give a certain amount of protection against breast cancer.” Bread is thus an intriguing mix of beneficial and potentially harmful components, and only by testing the whole food item, not the indivi-
dual components, it is possible to get an idea what the “net effect” is. “We’re going to use all the advanced research tools and test models that we have in CASCADE to try to find the answers‚ says Professor Mäkelä, who is also responsible for making sure that the two bread samples, equivalent to some 200 kg of bread, are prepared by the appointed Finnish bakery and distributed to all the partners. “It’s important to point out that we conduct our research without any kind of preconceived notions,” she continues. “This means that we have no suspicions whatsoever that bread would be harmful to eat. But since we’re hormone researchers, we have a theory that there MIGHT be hormone-disrupting substances contaminating the bread. We have no idea of the results we’ll obtain. Maybe we’ll find out that bread is as healthy as anything, which, of course, would be good news.” The final goal of the bread and baby food projects is to provide the European Union with basic information about the impact of commercial type of bread and infant food on nuclear receptor based modulation. Also, the generation of this information on the baby food aims at protecting non-breastfed children’s health during early development. Read more about the baby-food-project at page 41.
cascade partner: jan-åke gustafsson
The idea that everything is governed by the brain seems also to apply to hormone research. Estrogen has proved more important than previously thought, and this includes brain development. “Being exposed to early estrogen influence, in the womb for instance, can have considerable consequences for different cerebral functions, such as reading skills, memory, worry and anxiety”, says Professor Jan-Åke Gustafsson at Karolinska Institutet in Stockholm, CASCADE coordinator and one of the world’s pre-eminent hormone scientists.
Estrogen important for
cascade partner: adriana maggi
Endocrine disrupters perturb physiological mechanisms by altering the activity of our hormones. Thus, to fully understand how these agents interfere with our normal functions it is important to study what happens in the ENTIRE organism when it is exposed to chemicals. “It’s not enough just to look at what happens in isolated cells if we’re to understand how the body’s affected by exposure to foreign substances,” says Professor Adriana Maggi in Milan.
Firefly gene shows estrogen activity Professor Maggi, who is both a molecular bio logist and pharmacologist, directs the Centre of Excellence on Neurodegenerative Diseases at the University of Milan. “It’s one of the very first Centres of Excel lence founded by the Italian Ministry of Re search, with an unusually large number of phar macologists, biologists and medical doctors all working together towards common research goals. This blend of competences is really need ed in the research on hormonal receptors, which are so hard to study. The multidiscipli nary approach is also one of the main advan tages of the CASCADE network – that it al lows us to combine our most varied know ledge and test models.” “It’s important that computer models and cell studies are combined with animal expe riments to generate a complete and predic tive picture on the toxicity of chemical com pounds when extrapolated to the human si tuation, she stresses. “If we’re to judge whether some substance or other is dangerous, we must consider the whole animal, and we must see what happens at low doses and over a long period of time. After all, that’s how we humans are exposed to dangerous substances in food and the en vironment,” she explains. 12
There are hormone receptors everywhere in the body. Estrogen, for example, is invol ved in a vast number of physiological proces ses carried out by numerous organs. “We have produced a special strain of mou se containing a light gene from the firefly to detect how chemicals affect the animal, says Adriana Maggi, “This gene, which is activated by estrogenic compounds, can be activated in all cells of the mouse. When this happens, we can see exactly where it lights up and there fore discover which its target tissues are. This technique means that we don’t have to sacrifice or cause distress to the animals, just photograph them. At the moment, we’re exami ning how our reporter mouse reacts to the sub stances bisphenol A and soy (which contains the phyto-estrogen genistein), which occurs in our food and environment.” She believes that CASCADE is a good ex ample of the impact that science can have on the society. “We’re civil servants, and it’s our duty to use our skills and knowledge in the service of society for dealing with the major health threats of the future. In CASCADE, we pro duce the new tools needed to protect our child ren’s health as well as using the ones that al ready exist,” says Adriana Maggi in closing.
Research Site The Centre of Excellence on Neurodegenerative Diseases (CEND) is housed by the University of Milan in Italy, and is directed by Adriana Maggi. Its devotion to the application of novel multidisciplinary technologies to science has made the CEND a member of several European Programs (EMIL, DiMI) committed to the development of innovative research in the field of modern imaging, and it is now placing its expertise at CASCADE’s disposal.
Q Estrogen and breastcancer
Gian-Paolo Rando and Balai Ramachandran are members of Professor Adriana Maggi´s research group.
Estrogen is essential for the normal growth and development of the breast and reproductive organs. It is important for childbearing and regulation of the menstrual cycles. It also helps maintain healthy bones and the heart. However, lifetime exposure to estrogen is also associated with increasing a woman’s risk for breast cancer. The incidence of breast cancer in the Western world has risen steadily since the 1930’s. In the 1980’s, the numbers of menopausal hormone therapy prescriptions increased dramatically, and menopausal hormone therapy expanded from estrogen to estrogen plus progestin. In July 2002, the Women’s Health Initiative (WHI) estrogen-plus-progestin trial, a randomized, double-blinded, placebo-controlled clinical trial of estrogen-plus-progestin use among postmenopausal women for primary prevention of chronic disease was stopped early because risks of cancer exceeded benefits of estrogen treatment. After the WHI findings, menopausal hormone therapy use has declined substantially. Together with a more widespread mammography screening, the breast cancer incidence has started to decline in recent years.
cascade partner: barbara demeneix
“We’re scientists AND citizens – discovering how we’re affected by all the chemicals inside and around us is in all our interests”, says neurobiologist and endocrinologist Professor Barbara Demeneix. “The creative research collaboration in CASCADE has allowed us to waste no time putting out results into practice. One of the things we’ve developed is an instant test for detecting hormone-disrupting chemicals for example in our drinking water.”
cascade test methods used by industries Working in her lab in central Paris, Professor Demeneix has produced tadpoles that illumi nate when coming into contact with chemicals that disturb the production of thyroid hor mone. “The metamorphosis of a tadpole into a frog is totally dependent on thyroid hormone. With out it, the process cannot start, and the tad pole remains a tadpole. This is what makes them such useful models for studying thyroid
hormone disrupting chemicals. Despite the immense difference between humans and frogs, many of the decisive molecular steps in our foetal development are the same as those in the tadpole as it develops into a frog,” ex plains Professor Demeneix. While the new EU REACH legislation de mands the testing of chemicals, there is also a desire to reduce the number of tests on mam mals (e.g. mice and rats). The tadpoles deve loped by Barbara Demeneix’s CASCADE re search group are deemed to lie on the bor derline between in vivo and in vitro research, and can therefore be a more ethical way of conducting toxicological tests.
“We cooperate rather than compete here, and this lifts scientific quality.”
The company Watchfrog is based on the tadpole and frog models developed by Barbara Demeneix and her research group. www.watchfrog.fr
“Obviously we must reduce and refine ani mal testing, even replacing animal tests wher ever possible, but we shouldn’t forget either that today, many chemicals are actually already being tested on unwitting humans. In a totally uncontrolled way”, she adds dryly. “Our tests on larvae are a way of finding out what effect at least a fraction of the thousands of substan ces to which we are exposed daily have on all living organisms, including people.
“We have succeeded in showing that ap parently innocuous substances can act on hormone signalling in vivo. When our small tadpoles, which have all the vital organs of a mature animal, are exposed to certain chemi cals – such as Bisphenol A – their endocrino logical systems are modified, including thy roid hormone signalling”, she continues. The OECD already has tadpole-based test models, but they take a long time, around three weeks, to analyse. “With our model we can measure how che micals affect a tadpole’s thyroid hormone levels in just 72 hours, owing to the way that they quickly signal with a green fluorescence if they are swimming in ‘contaminated’ wa ter”, she says, adding that they have discove red that the carcinogenic herbicide aceto chlor also increases thyroid hormone signal ling. “This herbicide was introduced in 1994 in the USA to protect corn crops. Given that aceto chlor persists in water and exists in the na
“ So much
to learn from senior scientists ”
tural environment, it’s a potential chemical pollutant. Acetochlor can also pose a danger to farmers and their families. Children of far mers in the USA have been found to have acetochlor in their urine”, she continues. Last year, Professor Barbara Demeneix started up Watchfrog, a company that today has contracts with several major European industries seeking to test their products using the tadpole models developed by her group. “It wouldn’t have been possible to opti mise our test system and achieve such quick results without the deep and broad collabo rations made available by CASCADE.” — We cooperate rather than compete here, and this lifts scientific quality. It’s thanks to these idea exchanges that we’ve been able to waste no time applying our scientific results”, stresses Professor Demeneix, who is also the coordinator of the EU´s CRESCENDO pro ject, focusing on the function of hormone receptors in development and aging.
Research Site Professor Barbara Demeneix heads the UMR 5166 “Evolution of Endocrines Regulations”, a CNRS/MNHN unit placed within the Museum of Natural History, which is to be found in the exotic Jardin des Plantes park in Paris. The Muséum is a leading research institute in France with expertise in many areas of natural science ranging from palaeontology, systematics, chemistry of natural substances and physiology. The CASCADE team headed by Barbara Demeneix is interested in the molecular basis of thyroid hormone action.
– I have become passionate about environmen tal issues since I started to work with CASCADE. It is very rewarding for a PhD student like me to participate in a network like this. There is so much to learn from the senior scientists and it has been very exciting to get to know so many researchers from different countries. This would never have been possible for me without CASCADE, says French PhD student Dr JeanBaptiste Fini, 29, who has worked in the lab of Barbara Demeneix in Paris for almost 3 years, two of them financed by CASCADE.
cascade partner: patrick balaguer
Tadpoles, zebrafish, mice. In the scientific struggle for a healthier environment, a certain amount of animal experimentation is still necessary. However, CASCADE partner Dr Patrick Balaguer in Montpellier uses human cells in his research on endocrine disruption.
Banana pesticide a hormonal balance His latest discovery is that exposure to the biocide Kepone/Chlordecone disrupts the estrogen balance, increasing the risk of di sease. Dr Balaguer, who works at INSERM, a French public institution dedicated to human health, has developed human reporter cells for endo crine disruption. These cells can be used for screening large amounts of chemicals in an animal-free procedure, but they can also be injected into mice, to generate reporter ani mals to identify the target organ for a speci fic chemical. “We have produced human breast cancer cell lines, which we use in mice to trace chemi cally induced hormonal changes”, explains biologist Patrick Balaguer. “Thanks to the Dr
The bioluminescent light gene in the mouse shows what is happening when itis exposed to foreign substances, explains Dr Arnaud Pillon who works with Patrick Balaguer and is engaged in CASCADE research.
special bioluminescent light gene that we use in our mice, and using advanced camera tech niques, we can see exactly what happens when they are exposed to foreign substances.” The primary object of his research is antiestrogens, substances that can prevent breast cancer. There are two estrogen receptors in the body: ER beta, which is thought to give beneficial effects, and ER alpha, which in some instances acts as a repressor of these effects. Imbalance between these two recep tors is believed to cause a variety of diseases, including breast cancer. “From our cell models for disrupted hor mone signalling, we’ve now found that Ke pone/Chlordecone, a pesticide used on bana na plantations, stimulates the alpha receptor and inhibits the beta receptor. This means that the positive effects of estrogen are coun teracted by Kepone/Chlordecone and the negative effects boosted”, says Patrick Bala guer, adding that high concentrations of the substance have been found in plantation workers. Patrick Balaguer is conducting his estrogen research in tandem with a number of CASCA DE partners, including Nicolás Oléa in Spain, Lars-Arne Haldosen and Olle Söder in Swe den. To date, he has co-published four scien tific articles with three of CASCADE’s research groups. “We’ve also started a joint project on me tabolism with Jean-Pierre Cravedi in Tou louse thanks to the CASCADE project, which has really opened the way for new networks and research projects”, says Patrick Balaguer,
Kepone, also known as chlordecone, is a carcinogenic insecticide, was used between 1966 and 1975 in the USA for ant and roach baits. It produced a nationwide pollution controversy due to improper handling and dumping of the substance into the James River. Its use was banned in 1975. Chemically, Kepone is a chlorinated polycyclic ketone insecticide and fungicide with the chemical formula C10H2Cl10O. The dry powder is readily absorbed through the skin and respiratory tract. Some unprotected production workers exposed to Kepone powder suffered tremors, jerky eye movements, memory loss, headaches, slurred speech, unsteadiness, lack of coordination, lost of weight, rash, enlarged liver, decreased libido, sterility, chest pain, anthralgia, and the increased risk of developing cancer. Kepone persists in the environment, with a half-life of about 30 years. In France, Kepone was still used in large quantities in the French isles la Guadeloupe and Marti nique until 1993 for the treatment of banana plantations. Due to this massive use and persistence of the compound, it is still present in the environment (water, ground) and in humans (blood, adipose tissue).
who is also working together with a large local cancer hospital and different sectors of French industry. “CASCADE can actually help the industry by testing more substances, and then informing about which are dangerous and which are less harmful. It is possible to test more substances than we do today. And I’m still optimistic, even though there are 100,000 chemicals in our environment to be tested.”
Research Site Created in 1964, INSERM is a public institution with a scientific and technical vocation operating under the dual auspices of the Ministry of Health and the Ministry of Research. INSERM is the only French public research body entirely dedicated to human health. Its researchers are committed to studying all diseases, common or rare, through its research within the fields of biology, medicine and public health.
cascade partner: emilio benfenati
– Something evolving. That is really the basic meaning of research. And for CASCADE this is really true. The target is not simply to reach predefined research results but to check and verify the targets of the research and to feed the process with our experiences, says Professor Emilio Benfenati in Milan, who has developed new testmodels using computers instead of animals.
Chemistry in computers Professor Benfenati is Head of the Labora tory of Environmental Chemistry and Toxi cology at Mario Negri Institute for Pharma cological Research in Milan, Italy. He is a chemist with a curiosity for discovering so mething new behind every observation. – I chose chemistry because it is a good com bination of theory and reality for me. You can translate reality into theoretical aspects which can then be assessed and studied. His curiosity has led him into the field of environmental pollutants and health effects. And the chemistry has now moved into com puters, which is in line with one important mission in the CASCADE project – to mini mize animal testing. – We are exploring chemicals that could be present in food and which may be players in the endocrine systems in human. For instance
we are searching for compounds in the food and then try to understand how they interact with the cells in the body. And all this is done in computer models, says Emilio Benfenati. Emilio Benfenati is active in several inter national collaborations. He was, for instance, coordinator of the EU-project RAINBOW, where several other CASCADE partners also participated. — The possibility to identify good interac tions and new collaborations is a key feature of CASCADE. It is not purely about the scien tific goals, but as much about opportunities, links, connections and sharing methods on how to reach the goals. And since the partners come from a multitude of fields, we have com plementary skills and experience allowing us to approach the research in a new way.
Research Site Mario Negri Institute for Pharmacological Research is a non-profit biomedical research organization, established in 1961 in Milan. Research is mainly concentrated on the fight against cancer, nervous system and mental illnesses, cardiovascular and kidney diseases, rare diseases, and the presence and potential toxic effects of environmental contaminants.
QV irtual screening reduces animal testing CASCADE is dedicated to contribute to the efforts of decreasing the number of laboratory animals used for testing of chemicals. This is tackled by developing a system for virtual experiments in computers, called in silico screening. Hereby, CASCADE researchers make use of known nuclear receptor structures in order to find chemicals which could possibly interact with them. By making computer models
for endocrine disruption, the enormous number of compounds within our environment can be screened for binding to the relatively large number of hormone receptors in a fast, cheap and ethically sound way. During the first project year, CASCADE researchers Konrad Koehler at Karo Bio and Emilio Benfenati ran a list of about 400 chemicals through this in silico test system, and 12 of them
came up as hits. This means they are identified by the system as potential endocrine disrupters, which could be tested further in cell studies and animal studies according to existing legislation and rules for risk assessment. Without this in silico screening all of the 400 chemicals would have had to be tested in cells and animal studies.
cascade partner: vincent laudet
Zebrafish might be small but they contain everything a molecular biologist needs. Professor Vincent Laudet at the Ecole Normale Supérieure in Lyon thinks that these fish fry are the best scientific tool for understanding how organisms work and react to chemicals.
Zebra fish – small but very useful for science They are more relevant for human health than fruit flies, easier to use than mice, cheap to buy; and they also have an impressive rate of reproduction. Furthermore, their trans parency makes all changes directly visible. “Indeed, you can dissect them with the na ked eye.” Professor Laudet, head of the research team “Molecular Zoology” at the Ecole Nor male Supérieure in Lyon has only praise for the thousands of zebrafish he has studied over the past decade.
“What drives me is to understand how things actually work, and that’s why we try to do research with an open mind. Other wise you’ll just find what you’re looking for. A result from studying effects objectively is that we’ve suddenly discovered that the in ner ear of the zebrafish can be damaged by Bisphenol A, a component of several plastics. This finding could mean that exposure to certain substances in the environment could cause impaired hearing in humans”, says Vin cent Laudet, who is mainly devoting his CAS CADE work to endocrine disrupters that bind to nuclear receptors. “Roughly twenty per cent of today’s medi cines are based on nuclear receptors, which places our research very close to people and their health. What we’re doing, in other words, is highly result-orientated research.” Vincent Laudet works closelywith CAS CADE partners Barbara Demeneix in Paris and Jean-Pierre Cravedi in Toulouse. Like Barbara Demeneix and her fluorescent tad poles, Vincent Laudet’s laboratory uses fish with fluorescent protein to reveal endocrine disruptors and understand their mechanisms of action. “We work together with cross-species com parison. This means that we compare our fin dings on tadpoles, mice and zebrafish, to gene rate results that can be extrapolated for human health.” In addition, the group of Laudet has built a public database containing bioinformatics and comparative genomics with a particular emphasis on nuclear receptors (NuReBase www.ens-lyon.fr/LBMC/laudet/nurebase/
nurebase.html). The database now also con tains information on hormone disrupting compounds that often are food contami nants. A new and expanded version of the database, called NurXbase, will be online early 2008. “Food is important to people, especially for us French, and everyone wants to know what their food contains. We scientists have a hea vy responsibility in this respect as we’re the ones with the knowledge. CASCADE is a very good forum for making our voices heard in the public debate.”
Research Site The research group is located at Ecole Normale Supérieure de Lyon (ENSL), a high-rank institution in France, within the Laboratoire de Biologie Moléculaire de la Cellule. More than 100 researchers focus their work mainly on basic biological questions, on a variety of model systems and have access to several technical facilities. Nuclear receptors are studied by several groups.
â€œThe harmful effects of chemical contaminants in food are a major health concern in Europe today.â€?
Professor Nicolás Oléa, from Granada, Spain and post doc nutrionist Krista Power from Toronto, Canada, working with Professor Sari Mäkelä in Finland, is one of the ten mentor/mentee pairs in the CASCADE mentor programme.
Investment in junio
– high on the Cascade a
PhD students Nguyen Ba Tiep from Vietnam and Panida Loutchanwoot from Thailand work with Professor Wolfgang Wuttke in Göttingen, Germany.
PhD student Sana Sassimessai from Tunisia has worked with CASCADE partner Professor Jean-Pierre Cravedi in Toulouse and is currently at Professor Vincent Laudet´s lab in Lyon, France.
How do you become a successful scientist? How can you reconcile your work and private life? And how do you find a mentor? These are among the questions that the CASCADE junior scientist programme brings up. “We in CASCADE decided early on to invest in Europe’s future scientists, not least by fi nancing 20 postdocs and PhD students,” says Teaching Manager Dr Johanna Zilliacus at Karolinska Institutet, who is responsible for coordinating all training activites within CASCADE. Today, about 50 junior scientists are invol ved in CASCADE, which has offered them a
wide range of activities over the years since its start. “We’ve had many junior scientist sessions, including workshops on presentation skills and ethics in research, and group discussions on everything from career development to life in general”, says Johanna Zilliacus, endo crinology researcher and director of postgra duate training at the Department of Bioscien
t nior scientists
n the de agenda
Marchela Pandelova, post doc from Bulgaria is working with the Baby-foodproject at the lab of Professor Karl-Werner Schramm in Neuherberg, Germany.
ces and Nutrition at Karolinska Institutet in Stockholm. CASCADE has been able to provide a twoyear salary for 20 PhD students and postdocs, who work on joint research projects supervi sed by two senior scientists from different CASCADE partners – all within a specially established and highly appreciated exchange programme. “It’s fantastic to watch these young people develop into skilled scientists at an interna tional level. All the contacts they make nur ture future integration and research collabo ration in Europe”, says Johanna Zilliacus. Since April this year, around a dozen PhD
Open training courses are important for CASCADE. So far 12 courses have been orga nized on important health subjects. This photo is from the course ”Health Risk Assessment – Focus on Cancer, Developmental Neurotoxicity and Endocrine Disruption as Critical Effects” – that took place in Stockholm in May 2007 and attracted more than 58 applicants from all over the world, among which 30 were accepted.
– I am happy to get the opportunity to meet colleagues from other EU countries and discuss ideas and possible collaborations, says Cigdem Akdemir from Ankara Provincial Control Laboratory in Turkey who so far has attended two CASCADE courses.
students and postdocs have also each been appointed a senior researcher as a mentor. “We were pleased to see that many of our senior researchers signed up as willing men tors. They’ll serve as independent soundingboards whose job it will be to support their mentees’ personal and professional develop ment”, says Johanna Zilliacus. The personal mentoring is the last phase of a multi-step mentor programme that has been developed within CASCADE. CASCADE has also developed a course pro gramme and organised 12 courses on subjects that are important for European researchers working on health risks associated with food
contaminants. The courses are open for all researchers and have attracted a large number of applications. “We are very pleased that the courses we have organized are appreciated and it is obvious that there is a very large need for this kind of training”, says Johanna Zillia cus. “We have also recently received further support for the training programme in health risk assessment from EU-funded Marie Curie Actions in a project called RA-COURSES”, adding that this gives possibility to continue the effort with the ultimate aim to provide train ing in this important field on a long term basis. Fore more information about CASCADE courses see: www.cascadenet.org and choose Education.
cascade partner: jean-pierre cravedi
The fact that a substance is classed as non-toxic in certain tests does not necessarily mean that it is harmless to human life. During the complex process of degradation in nature, food and, finally, in the body it can transform and become toxic. Dr Jean-Pierre Cravedi, whose work straddles both biology and chemistry, has discovered a correlation between exposure to pesticides and certain cancers.
Exposure to pesticides can cause cancer Doctor Cravedi is leading a research group of some 40 scientists at the Laboratory of Xeno biotics at the INRA (the French National Insti tute for Research in Agronomy) in Toulouse. Xenobiotics are chemicals not naturally found in a biological system, in this case the human body. “The science of poison, if you like”, ex plains Jean-Pierre Cravedi, adding that one of his first findings in xenobiotics showed that most of the petroleum hydrocarbons re leased into the aquatic environment after an oil spill accumulated dramatically in fish. “We were able to show that certain substan ces were accumulating and being stored in the liver, fat and several tissues. This means that the fish were to a large extent unable to break down and eliminate all these foreign chemicals. We also found that this elimination occurred
to a lesser extent in fish living in cold waters as compared to their warm water cousins.” For many years, Jean-Pierre Cravedi’s team of scientists, which has the use of one of Euro pe’s most advanced laboratories for investiga ting the fate of xenobiotics, has been concentra ting its efforts on how food contaminants, in cluding agricultural pesticides, affect human health. What Doctor Cravedi and his colleagues have discovered is that xenobiotic substances like plasticizers, brominated flame retardants or pesticides, can affect the body at a later stage. “We’re translating results from animals to human studies on the substances that accu mulate throughout life. During metabolism, the body ‘restructures’ the chemicals, and they can become toxic. What we’re doing is trying to find out exactly how this transformation ta
es place and what are the harmful compounds k produced within the body, as we would then be able to do something about the problem”, explains Jean-Pierre Cravedi, who has recent ly completed a study showing that long-term exposure to pesticides may play a role for dif ferent kinds of cancer. “Pesticide exposure may have both acute and chronic effects on health. Our study shows that there’s an increased risk for non-Hodgkin lymphoma in people who have worked with pes ticides in farming, agriculture or the chemi cals industry. A long period of exposure (more than 10 years) gave an increase in the risk of all hematopoietic cancers. The study has been pub lished recently in the scientific journal Cancer Causes Control.” Jean-Pierre Cravedi’s research group has also been working alongside CASCADE part ner Patrick Balaguer in Montpellier on the effects of Vinclozolin, a fungicide commonly used in vineyards. Vinclozolin is one of the che micals being studied under the CASCADE pro ject, and the results suggest that the substan ce primarily affects the male hormone balan ce, and that exposure can lead to infertility. “We have a long way to go before we know all the answers. There are more than 100 000 chemicals in the environment, the effects of which are still a complete mystery to us. Nor do we know very much about what happens when different substances are combined. The challenge is to find the tools to answer these questions, and it is here that CASCADE has helped us become more efficient, as all the different test models found in the network have given us a picture of the whole process. Research is like piecing together a puzzle, and if we can collaborate both vertically and ho rizontally, we’ll hopefully be able to com plete the picture more quickly.”
cascade partner: nicolás oléa
We have long assumed that recycled paper products are more environmental friendly than new paper. New findings from the research group of professor Nicolás Oléa in Granada, now suggests that recycled containers for fatty take away food, like pizza boxes, could be harmful to our health because recycled cardboard contains more hormone disrupting chemicals than new cardboard.
Recycled pizza boxes
oily food make fat-soluble chemicals travel more easily into the food. “This research may in the future orientate consumers to old ways of preparing food and the modern style of packaging might go down. Traditionally we have used ceramics, glass and metal, not so much plastic and cardboard. This could feed into the debate on ‘slow food’ for example”, says Nicolás Oléa. But it is not self-evident that the alterna tive is automatically better. “Different kinds of packaging that comes into contact with food needs to be tested be
”This is just a pilot study, but it does show that the issue is more complex than just that recycled equals good.” fore we know that. This is just a pilot study, but it does show that the issue is more com plex than just that recycled equals good” closes Nicolás Oléa. The findings have been published in the scientific journal Food Packaging.
French fries, hamburgers, pizza and other popular fatty take away food are often pack ed in boxes made of recycled cardboard. Warm fat increases the release of endocrine disrupting chemicals from take away food containers made of recycled cardboard. CASCADE partner Professor Nicolás Oléa from University of Granada, Spain, has now shown that recycled cardboard contains bisphenol A and phtalates to a higher extent than new cardboard. “We looked at pizza boxes and wrappings for French fries and hamburgers and found chemicals in the cardboard that are not pre sent in new paper. Recycled paper is seen as friendly for environment, so it must be good, right?” “The impression we have is that it is not so good since it has not been tested properly for foreign chemicals. We have found a new ap proach to look at these chemicals”, says Nico lás Oléa. But he emphasises that it is too early to discourage people from using the recycled food containers. And it is the role of the na tional food administrations, not of the re searchers, to give recommendations to the consumers. “We indicate that there is a need for explo ring new ways of involuntary exposure to chemicals and these issues of food safety need to be considered by the authorities.” It has previously been shown that hot and
contain harmful chemicals
Q Thyroid hormone needed for normal development Thyroid hormone (TH) is secreted from the thyroid gland and controls a number of functions: body temperature, body growth, small intestine function and normal embryonic development. It acts directly on gene expression by
binding to receptors present in the nucleus of most cell types. Congenital hypothyroidism, usually linked to iodine deficiency, can lead to dwarfism, deafness and severe mental retardation. Thus, if chemicals present in food
are able to interfere with TH signalling, they might be dangerous to development during the foetal period and in childhood. Present data are not sufficient to rule out or to confirm such a possibility. It is of the utmost impor
tance to evaluate the possibility. Thus, it is of the utmost importance to eva luate the possibility that chemicals can irreversibly damage the developing brain in different cell and animal models.
The brain more protected than expected As a scientist, one must always be prepared for the unexpected. When the Lyon-based scientist Dr Frédéric Flamant in his animal tests discovered that some of the chemicals studied in CASCADE, including bisphenol A, do not seem to affect the development of the mouse brain, he was surprised. “We know, you see, that these chemicals can disrupt thyroid hormone production, which is critical to foetal development. So it was sur prising to discover that they don’t seem to cause any brain damage.” “Thyroid hormone is necessary for the proper development of the foetus, and the brain is a particularly important target for it. The brain seems to be more protected than we had believed”, says Dr Flamant of Lyon’s Eco le Normale Supérieure, whose CASCADE partners include Vincent Laudet of the same institute, Barbara Demeneix in Paris and Ma ria Bondesson of Karolinska Institutet in Stockholm. Frédéric Flamant holds a PhD in molecu lar and cellular biology, and later specialized in virology. His specialist field is develop ment biology. “One of the objectives of our research is to find the molecules that are harmful to the deve lopment of the mouse brain. Since the nerve cells are dependent on thyroid hormone, we are studying the effects of chemical pollu tants so that we can identify target genes for this hormone in the brain and nerves. “We’re attempting to establish the com plete list of thyroid hormone target genes in immature neurons to later gain a more precise picture of chemical exposure consequences. We’re also using advanced transgenic techno
logy to identify the indirect impact on mouse brain development of the metabolic disorders which are induced by impaired thyroid hor mone signalling in other organs”, he conti nues, adding that the work he has done under
“We scientists can no longer work alone. International researcher networks are vital these days, but we mustn’t forget that the main role of the scientist is to think rather than just produce lots of data.” CASCADE has, in a way, changed his ap proach to research. “We scientists can no longer work alone. Vast international researcher networks are vital these days, but we mustn’t forget that the main role of the scientist is to think rather than just produce lots of data. This can ea sily rob you of your creativity”, says Frédéric Flamant, who believes that CASCADE is a very good platform for starting new collabo rative projects. “The engagement in young scientists, with the summer schools and possibilities for young researcher to work in several labs around Europe, is an excellent initiative.” 29
cascade partner: olle söder
Male hormones in focus Active paediatrician, researcher, supervisor, unit manager and head of department. Professor Olle Söder at the Department of Paediatric Endocrinology at Karolinska Institutet in Stockholm enjoys the mix of patient-contact and research. His day-to-day work involves everything from growth problems, puberty disorders and thyroid disorders to patients of indeterminable sex. Professor Söder’s research is centred on the pathogenesis of hormone diseases, especi ally on the different cell types that produce male and female sex hormones, how they are regulated and how they are affected by ex ternal factors like hormone-disrupting che micals in food. Hormone-producing cells are found in, for example, the testicles, the adre nal glands and the ovaries. “We can see an increase in what’s known as ‘testicular dysgenesis syndrom’ in boys and young men”, he says. “The syndrome comprises a number of symptoms/diseases such as testicle cancer, low sperm counts, and abnormal testicle development. On ac count of this, fertility in young men has de clined over the past 50 years. “One theory of why we’re seeing this trend is our exposure to hormone-disrupting sub stances that interfere with the normal hor mone signals for the male sex hormones or androgens”, he explains. Phthalates are one such androgen-disrup ting substance. These esters are used as plas ticisers, but as they do not bond directly with the plastics they soften, then can leak out into food, for instance, from the packaging. Current human biomonitoring data prove that the tolerable intake of children is ex 30
Professor Söder receives patients at his clinic every Monday. The string of beads, which is actually an instrument for measuring testicular volume called an orchidometer, is what he uses to explain the situation to children and their parents.
ceeded to a considerable degree, in some in stances up to 20-fold. Olle Söder has much experience of inter national research collaborations. His current group brings scientists from Denmark, Greece, Argentina, Finland, Thailand, China, Cambodia, Russia, Iran, Pakistan and Swe den. He considers his CASCADE activities an important and personally developing fea ture of his international work. “CASCADE is a network that really does work”, says Olle Söder, who is engaged in se veral joint projects with CASCADE partner Professor Wolfgang Wuttke in Göttingen. “It’s given rise to collaborations and new pro jects with scientists I’ve never been in con tact with before.” Söder’s and Wuttke’s research groups have together found that foetal and young mice are very much more sensitive to phthalates than adult mice. This means that male foe tuses or premature and newborn baby boys who are exposed to phthaltes might develop irreversible testicle damage, which might eventually lead to testicular dysgenesis syn
drome and reduced fertility. Another example of international research collaboration is the EU’s PIONEER project, of which he is the vice coordinator and Sari Mäkelä, she too of CASCADE, the coordina tor. The focus of this project is premature puberty. “The collaborative set-up and contact net work of CASCADE was a basis of the PIO NEER project.” International collaboration is also mani fest in the day-to-day activities, in that seve ral scientists in Olle Söder’s group have been visiting CASCADE’s labs in Turku and Göt tingen to exchange experiences and know ledge with their colleagues, who, in turn, have been coming to the lab in Stockholm. “Before CASCADE, I had no contact with Wolfgang Wuttke and his team in Göttingen, and the partnership we have now established has proved very successful. Since we have different kinds of expertise, we complement each other well, which is something we both benefit from scientifically.”
Q Androgens Androgens is the collective term for the male sex hormones. Androgens stimulate and control for example the development and function of the male sex organs.The most well-known androgen is testo sterone. Androgens are produced in the testicles and the adrenal glands and can be converted into estrogens through the agency of the enzyme aromatase. Testosterone and dihydrotestosterone bind to the androgen receptor; this receptor is what is known as a ‘transcription factor’, which regulates androgen target gene expression. Disrupted androgen signalling can impair embryonic and pubertal development, and can cause reduced fertility in the adult male.
Research Site The Department of Clinical and Experimental Endocrinology belongs to the Faculty of Medicine of the University of Goettingen. The clinical obligations of the department comprise determination and interpretation of hormonal constellations of patients. The 3 medically trained staff members see outdoor patients primarily with endocrine problems related to dysregulation of the hypothalamo-pituitary-gonadal axis.
cascade partner: wolfgang wuttke
Can red wine make us more stressed? CASCADE scientists from Sweden and Germany have discovered that resveratrol, a natural remedy found in grape skins, seems to reduce the body’s ability to tackle stress, thereby increasing the risk of burnout. Resveratrol is marketed in the USA and else where as a preventative agent against cancer and heart disease. “This is a surprising result that suggests that resveratrol can reduce stress tolerance to infection etc., which might eventually affect the risk of burnout”, says Professor Olle Söder. Olle Söder and Professor Wolfgang Wuttke have published their unexpected findings in the scientific journal Hormone Research. Resveratrol is a phyto-estrogen naturally occurring in blue grapes and red wine. The CASCADE scientists’ research can help to balance our understanding of resveratrol and, in the long run, to improve usage recom
mendations. The effects were observed in an animal study where rats were treated with resveratrol and then their hormone levels were measured. Then the researchers exa mined different hormone-producing organs from the rats, studying their function at an organ and cellular level. It was in these studies that the researchers discovered the unexpected effect: resveratrol inhibits cortisol production in the adrenal glands. The adrenal glands secrete hormones and are located just above the kidneys on either side of the spinal column. Cortisol is an endogenous steroid hormone, the anti-inflam matory properties of which are important in
the regulation of the body’s stress response. However, the mechanism behind this effect is still a mystery, and is the next challenge fa cing Söder and Wuttke. “CASCADE has allowed us to establish a working test model for studying what causes this effect”, says Olle Söder. “The body’s hor mone regulation system is complex and oc curs at different levels. We’re now carrying out tests to determine where in the chain resveratrol is active.”
cascade industrial partner: karo bio, stockholm
“The CASCADE network is of great value” Access to an academic network of highly skilled and experienced scientists; constructive discussions and new partnerships that stimulate new ideas for early discovery projects that could lead to the development of new substances. Dr Stefan Rehnmark, principal scientist at Karo Bio, sees many advantages in being an industrial partner in CASCADE. The Swedish drug discovery and develop ment company Karo Bio has been involved in the CASCADE project from the start. “Gaining a contact network containing so many competent academic groups in Europe is of great value for Karo Bio. Thanks to the regular CASCADE meetings, which give eve ryone the time and opportunity to socialise, we’ve got to know the scientists so well that we now can call anyone in the network to discuss research.” The focus of Karo Bio is on nuclear recep tors as drug targets for the development of new drugs for metabolic diseases, a research field that parallels the activities of the CAS CADE network. In collaboration with Emilio Benfenati in Milan, Karo Bio has developed in silico (vir tual) test models, that hopefully will minimize the use of animals when testing substances, may they be pollutants or potential drugs. Dr Rehnmark also mentions the work be ing done in Paris with Barbara Demeneix de veloping novel chemical screening models in the tadpole. “Due to the CASCADE postdoc financing system, we could recruit Andrew Tindall to further develop the tadpole test models at a small company called Watchfrog. Today, the 32
tadpoles are used to screen for hormone-dis rupting substances, such as chemicals that af fect the thyroid and estrogen hormone system, but one day we may even be able to use the same model to screen for effects on the heart.”
Research Site Karo Bio is a drug discovery and development company specializing in nuclear receptors for the development of novel pharmaceuticals with focus on metabolic diseases. The company has expanded from being a drug discovery company by adding in-house preclinical and clinical development resources and competence. The company has a strong project portfolio with inno vative molecules that primarily targets diseases such as diabetes, atherosclerosis and dyslipidemia. In all of these areas there are significant market opportunities and a growing need for new pharmaceuticals with new mechanisms of action. Karo Bio has three clinical and four preclinical projects and in addition to the proprie tary projects Karo Bio has two strategic collaborations with international pharmaceutical companies and one biotech collaboration for the treatment of common diseases. www.karobio.se
cascade partner: helen håkansson
Predicting the risks of chemicals on human health is absolutely necessary for the control of and legislation on chemical pollution. And it is also very difficult since it is not possible to do experiments on humans. – But the enhanced knowledge of mechanistic behaviour of chemicals, generated from improved basic research during the last decade, can serve as a new basis for risk assessment, says Helen Håkansson, Professor of toxicology at Karolinska Institutet.
Q Health risk assessment Risk assessment is the process of quantifying the likelihood of, for example, chemical exposure having a harmful effect on individuals or populations. In this case, a risk assessor would weigh the toxicity of a substance against how much people are exposed to it, and produce a ceiling value for exposure to this particular substance. Risk assessment is a crucial part of risk management that ultimately feeds into legislation and official recommendations on the protection of human health. Health risk assessments of chemicals are based largely on data from toxicological studies on animals that are evaluated and extrapolated to represent the human situation. New molecular biology methods, such as the ones developed within CASCADE, can play an important part in risk assessment by clarifying the mechanisms of toxicity for a chemical and the relevance for human health.
How is risk estimated? Professor Håkansson has many years’ expe rience of health risk assessment at an inter national level. One of her appointments is on the WHO Expert Panel for the health risk assessment of biocides. In CASCADE, Helen Håkansson is leading the work on improving and developing new methods for assessing the health risks of che micals. “For instance, we develop existing mathe matical modelling methods for toxicological and exposure data to ensure that research results are put to optimal use and give a risk assessment that is as reliable as possible. We’ve also started a European network in CASCADE called MSTnet (Mathematical and Statistical Tools net), which, in being open for non-CASCADE researchers, will hope fully initiate further cooperation in this field”, she says. 34
One of the objectives of CASCADE is to integrate the experimental research being conducted within the network with the risk assessment activities. One such example is that the whole CAS CADE consortia are involved in the produc tion of risk assessment documentation on CASCADE’s model substances, namely bis phenol A, vinclozolin, dioxin and genestein. “By engaging all CASCADE researchers in this, we make sure that the very latest results on a compound are described in the docu ment and can be evaluated for risk assess ment. Another gain is that basic researches, who have no previous experience of risk as sessment, become involved in and gain a bet ter understanding of what risk assessment entails. This will facilitate that experiments will be designed and performed in such a way that the results can be used for predicting
risks”, says Professor Håkansson and conti nues “The risk assessment documents are pub licly available on the CASCADE website”. Helen Håkansson has also been involved in the education of a new generation of sci entists, who will have a background both in molecular biology or chemistry and risk as sessment. “We have recently launched a unique course programme in health risk assessment that we’ve developed in CASCADE”, she explains. The programme is called the Advanced Inter national Training Courses in Health Risk Assessment and has attracted a great deal of interest. For more information on the risk assessment of model chemicals see www.cascadenet.org
cascade partner: ingemar pongratz
A very vulnerable and sensitive time in human development is in the womb and during the first five years of infancy. Unfortunately, it is during this time that we take in between 70 and 80 per cent of all the toxicants accumulated by the body during our lifetimes. In his research on dioxin, Dr Ingemar Pongratz in Stockholm, CASCADE’s vice coordinator, is studying how this chemical can cause different developmental problems such as female infertility.
Just how the body is affected by environmen tal pollutants like dioxin is one of the ques tions that the scientists of the CASCADE network are attempting to find answers to. Dioxin is ubiquitous in our environment and in food like fish, meat, grain and vegetables. All of us carry in our bodies traces of this toxic substance, which can disrupt hormonal signal ling and promote cancer. “Dioxin has an exceptionally long half-life, which basically means that the body can never rid itself of the dioxin it has absorbed. The substance can leave the body through the breast milk, but then unfortunately it passes on to the baby”, says Ingemar Pongratz, and explains that dioxin is stored in the fat tissue. Dioxin was discovered to disrupt the hor monal balance twenty years ago. “Yes, we know that the substance is clo
Research Site Karolinska Institutet (KI), founded in 1810, is Sweden’s only university to focus exclusively on the field of medicine. Situated in Stockholm, it is Sweden’s largest medical research centre, and accounts for roughly 45 % of the country’s universitybased medical research. It is also home to the country’s largest medical school. 2500 postgraduate students and 2800 research staff work in over 600 research units of KI. KI is responsible for awarding the annual Nobel Prize in Medicine or Physiology. Research at KI has a strong European dimension, with around 400 contracts within EU Framework Programmes. Of these contracts, about 100 are, or have been coordinated from KI.
sely linked to a variety of reproduc tive problems in women, such as endometriosis, a disease affecting the uteral lining that can prevent women from becoming pregnant. There is also a possible connection between dioxin and obesity, since high-dose exposure is thought to interfere with the metabolism.” Scientists have a relatively good understanding of how humans and animals react to high doses of di oxin (i.e. the actual condition of intoxication). For example, studies have been conducted of Vietnam veterans who were exposed to large amounts of dioxin during the war and who now display a higher inci dence rate for certain cancers than the average population. “However, what happens on pro longed low-dose exposure, as we get during our lifetimes, is still unknown. In CASCADE we are studying this with the help of novel molecular biology techniques”, explains Ingemar Pongratz, whose CASCADE part ners include Jean-Pierre Cravedi in Toulou se and Sari Mäkelä in Turku. “Thanks to the CASCADE network, we have access to advanced technologies and methods that we hardly knew existed in Europe. Cross-border collaboration fueled by Networks of Excellence produces better stu dies, more comprehensive publications – bet ter research, quite simply. And hopefully, it will also help improve the health of future generations. That’s our main objective in CASCADE – to make sure that our collective
Dioxin disturbs hormonal balance
knowledge about the risks to which we’re ex posed gives rise to new legislation that can bet ter protect human life in the years to come.”
H cascade collaborations CASCADE and WWF collaborate on chemical pollutants in our food A study conducted by the WWF has shown that common dairy products, meat and fish sold in seven different EU countries contain biocides, softening agents and Brominated Flame Retardants (BFRs). The highest concentration of the banned chemical PCB in Europe was found in Baltic herring. CASCADE and WWF are concerned by effects of chemical pollution and work together to spread information on the risks. The WWF’s food study was ba sed on samples from 27 different producers from seven EU count ries, purchased in high street supermarkets. The samples were analysed at Holland’s TNO labo ratory for chemical pollutants. Dairy products, meat, fish, bread, honey and olive oil were tested for eight groups of chemi cals: chlororganic pesticides, PCB, BFRs, perfluorinated che micals (e.g. PFOS), phthalates, tin organic compounds, alkyl phenols, and synthetic musk chemicals. Toxic residues were found in all tested food-stuffs. Olive oil,
cheese and meat contained phtha lates. Fish and reindeer meat contained banned chlororganic pesticides, BFRs were found in meat and cheese, and synthetic fragrances and tin organic com pounds were found in fish. Swedish minced meat had the highest concentrations of BFR and Swedish herring the highest concentrations of PCB and per fluorinated chemicals (e.g. PFOS). “All the chemicals found in the food have also been found in human blood and in the natural environment”, says Secretary General of WWF Sweden Lars
Kristoferson. “This shows that what we eat influences how chemicals get into our blood stream. We’re surrounded by a toxic cocktail, which has worry ing long-term implications.” The WWF’s report, entitled Chain of Contamination: The Food Link, shows that food is an important link in the chain, from manufacture to how the chemicals end up in our bodies and environments. This is par ticularly true of persistent and bioaccumulative substances such as DDT, PDC and BFRs. Chemicals spread to our environ ment through industrial waste, transport and waste storage, and leak from computers, TV sets and hygiene products. “The fact that herring has remarkably high concentrations shows that the Baltic Sea is still extremely contaminated”, says Mr Kristoferson. “Even though
levels pose no immediate threat to our health, we’re concerned about the long-term effects. More and more animal research is demonstrating that even low doses can be carcinogenic and can disrupt reproduction and arrest foetal and child develop ment.” “Since we’re at the top of the food chain, we’re particularly vulnerable to chemicals in food”, says KI’s Ingemar Pongratz, vice coordinator of the EU’s CASCADE network. “Some of them can affect certain hormo ne systems and increase the risk of obesity, diabetes, cancers, infertility and other common diseases.” (This article is from the WWFwebsite.) For more information, see http://www.panda.org/ about_wwf/what_we_do/poli cy/toxics/index.cfm.
OECD and the US Environmental Protection Agency (U.S. EPA) has recently launched world wide programmes to develop systems to determine toxicity of chemicals. The aim is to set up a global col laborative effort to use state-ofthe-art high-throughput screen ing, toxicogenomics, and com putational chemistry tools to screen a very large number of chemicals for toxic properties, such as endocrine disruption. U.S. EPA estimates that more than 10 000 environmental che 36
micals need prioritizing for further testing. For example, antimicrobials, inert pesticide ingredients, high-productionvolume chemicals, drinking wa ter contaminant candidates, and endocrine disruptors, general ly have limited toxicological da ta available for hazard and risk assessments.
The U.S. EPA is very interes ted in engaging other organiza tions in collaborative research arrangements in which expe riences, efforts, and best practi ces can be shared. CASCADE has generated several different screening methods for endocrine disrup ting chemicals and on the 1st of October 2007, researchers from CASCADE and U.S.EPA met in Brussels to discuss future colla borations.
Under the umbrella of a joint workshop, representatives from the U.S. EPA and CASCADE researchers opened the doors for a so far unique worldwide chemical safety programme. The large amount of produced data will be made available through deposition into a publi cly available database, called Toxicology Reference Database (ToxRefDB). A preliminary sche me for the database is available at www.epa.gov/ncct/toxcast.
CASCADE test methods in world wide chemical safety programmes
“Since we’re at the top of the food chain, we’re particularly vulnerable to chemicals in food.”
Ingemar Pongratz, vice coordinator of the EU’s CASCADE network.
Collaborations before CASCADE
CommNet CommNet is a Network for Communication consisting of 21 EU-projects and net works that do research on food quality and safety. About 3 000 scientists are fully or partly engaged in the consortia forming the CommNet, which is chaired by CASCADE. CommNet members are responsible for managing communication of EU food and health research with non-governmental organizations, authorities, and other policy-makers, as well as journalists and the public community. To contact CommNet go to: www.fp6commnet.eu.
CASCADE Governing Council agrees on continued collaboration A collaboration agreement is being signed between the universities and research insti tutes in CASCADE Network of Excellence. This was the result of the first Governing Council meeting when presidents and directors of CASCADE partner organiszations met to discuss future collaborations. The collaboration agreement will facilitate a durable collaboration on research and education at the institutional level. CASCADE is one of very few EU-projects that has been able to bring together the highest level of university management to discuss durability issues. CASCADE coordinator Jan-Åke Gustafsson was very pleased with the outcome of the meeting: – We consider this as a breakthrough and an important step towards integrated European research and training collaborations. A durable integration is the overall aim of the network and the collaboration agreement shows that CASCADE takes this assignment seriously, says Jan-Åke Gustafsson.
Current European research strategy may put the future of Networks of Excellence at risk
Established collaborations within CASCADE
Science and technology are absolutely necessary for find ing solutions to the major problems facing the world today such as climate change, pollution, the cleaning-up and replacement of fossil fuels, globalization, threats to human health, migration and lack of social cohesion. They are also required to se cure Europe’s leading position in economic and technologi cal fields. However, as science has reached a very advanced level, it has become clear that it is increasingly difficult for a single research organization to master all the methods re quired in its field of research, or have the capacity to pro duce breakthrough results to their full potential for the benefit of society. The European Commission realized this during the de sign of FP6 (frame work pro gramme 6 on research fun ding) when it created a new instrument, the Network of Excellence. The main objectives of the Network of Excellence were to strengthen Europe’s scien
tific impact in a particular research area, tackle the fragmentation of European re search and spread excellence within and beyond the confi nes of the Network. The first Networks of Ex cellence were formed in 2004, and now 170 Networks have been created. All of them have been selected on a highly competitive basis and provide state of the art research based on close collaborations of leading European research groups (including those from industry), the interdiscipli nary training of a new genera tion of scientists and the spreading of research results to the European public. Thus, Europe has taken the initiative and created for the first time an efficient, inter disciplinary infrastructure to lead research in key areas and to solve current and future problems. However, many scientists are currently feeling concer ned about the long-term future of Networks of Excellence and for good reasons. In the new research programme from the European Commission,
the number of Networks of Excellence has been substan tially cut back in the first calls, with only 17 Networks being funded, as compared to 101 in previous calls. In addi tion, no support for existing Networks of Excellence has been announced. This reduction in the oppor tunities for funding will jeo pardize the future of the Euro pean research infrastructure. Realizing this, a group of 55 leading Network of Excellen ce representing more than 13 000 researchers through out Europe have now written an opinion paper describing why the new strategy change is mistaken and recommen ding practical interventions to avert future scientific and technical losses that Europe can ill-afford. Since nothing has been an nounced in terms of support to existing networks, we fear a huge waste of investment, says Ingemar Pongratz, vice coordinator of CASCADE and one of the initiative takers of the opinion paper. The Lisbon European Council held in March 2000, set the goal for the Union to become the most competitive and dynamic knowledgebased economy in the world by 2010. One of the key elements for this development will be sci entific and technological re search, and will require a con tinued support for Networks of Excellence. For more information www.supportresearchnoe.eu.
REACH protects our environment The new EU legislation REACH from June 2007 gives the industry the main responsability for the testing of 30 000 chemicals. The EU has produced new community-wide legislation on chemicals in order to come to grips with the problem of chemicals in our environment. The objective of the new law, which is termed REACH – Registration, Evaluation, Authorization of Chemicals – is to enforce the registration, evaluation and authorisa tion of industrial chemicals and to restrict the spread of toxins. The industry will have the main responsibility for the test ing of estimated 30 000 chemicals before the year 2016. REACH supersedes around forty dif ferent national laws, making it one of the most important measures to remove harmful chemicals from the environ ment throughout Europe. The legisla tion was passed by the EU parliament in December 2006 and entered into force on June 1 2007 when ECHA, the European Chemicals Agency, opened a new office in Helsinki, Finland. ECHA will run a central database and act as the central point in the REACH system.
CASCADE scientists published this article in Financial Times shortly before the legislation was passed by the EU Parliament in December 2006.
World production of chemicals has increased from one million tons in 1930 to more than 400 million today.
cAScADE this is
Jan-Åke Gustafsson, Karolinska Institutet Helen Håkansson, Karolinska Institutet Olle Söder, Karolinska Institutet Stefan Rehnmark, Karo Bio AB I ta ly
Adriana Maggi, Università degli Studi di Milano Emilio Benfenati, � Istituto di Ricerche Farmacologiche “Mario Negri” Catherine Leclercq, Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione Alberto Mantovani, Istituto Superiore di Sanità Germany
Christian Behl, Johannes Gutenberg Universität Mainz
Wolfgang Wuttke, Bereich Humanmedizin GeorgAugust Universität Göttingen Karl-Werner Schramm, GSF Forschungszentrum für Umwelt und Gesundheit GMBH Martin Göttlicher, GSF Forschungszentrum für Umwelt und Gesundheit GMBH Günter Gauglitz, Eberhard Karls-Universität Tübingen
Jean-Pierre Cravedi, Institut National de la Recherche Agronomique Patrick Prunet, Institut National de la Recherche Agronomique Patrick Balaguer, Institut National de la Santé et de la Recherche Médicale
The Netherl ands
Wout Slob, Rijksinstituut vor Volksgezondheid en Milieu Fr ance
Vincent Laudet, École �������������� Normale Supérieure de Lyon Frederic Flamant, École Nor- male Supérieure de Lyon Barbara Demeneix, Centre ���������������������� National de la Recherche Scientifique Hector Escriva, Centre National de la Recherche Scientifique
S pa i n
Nicolás Oléa, Universidad de Granada Sari Mäkelä, University of Turku Olli Jänne,�������������������� Biomedicum Helsinki Hungary
Janos Garai, University of Pecs S l o va k R e p u b l i c
Julius Brtko, Slovak Academy of Sciences
All CASCADE partners from nine European countries will use their different skills and methods to find out if there are any hormone disturbing substances in our bread. This project is the Proof of Principle for the network.