Protection against carcinogenic substances has become an important and much discussed topic. People are exposed to drugs and chemicals in both their private and professional lives, which means that the carcinogenic potential of drugs and chemicals needs to be assessed and excluded from the very start of the development process. At present, this is done through time-consuming animal experiments. Dr. Kerstin Stemmer from the University of Konstanz has now found a way to identify the carcinogenic potential of substances in just a few days using state-of-the-art technology and new investigation methods. This is the first time that researchers have been able to reduce the time and the number of animals used in carcinogenesis studies.
Current legislation requires the carcinogenic potential of certain substances to be intensively investigated in long-term studies. Carcinogenesis studies involving animals, particularly rodents, are used to test the effect of high doses of a substance under investigation in order to assess the extent of tumour formation in certain body organs. The results of these investigations are then used to assess the substances' carcinogenic potential. However, the currently used method is very costly and can take up to two years. In addition, widespread age-related pathologies and rodent-specific carcinogenic effects can confound the outcome of these studies.
Dr. Kerstin Stemmer, a scientist at the University of Konstanz, has now developed a method that enables the identification of renal carcinogens in short-term in vivo studies. She has used in vivo assays in combination with sensitive microarray transcriptome analyses to elucidate the early processes associated with renal carcinogenesis induced by genotoxic and non-genotoxic substances. Stemmer's results contribute to faster and more reliable in vivo analyses as well as providing encouraging results that may lead to the reduction and perhaps even replacement of animal experiments with other methods.
Pre-stages of carcinogen-induced tumours are the basis of Stemmer's research
"The causes that lead to the development of renal cancer have not yet been identified in detail. However, certain factors are known to increase the risk of developing renal cancer," explains Dr. Stemmer referring to hereditary factors, adiposity, contact with heavy metals and solvents, as well as smoking. The biologist has tested two renal carcinogens: the genotoxic renal carcinogen aristolochic acid (AA) and the non-genotoxic mycotoxin ochratoxin A (OTA). Wild-type rats and TSC2-mutant Eker rats (mutation of the TSC2 tumour suppressor gene) were used as experimental animals. "Due to the mutation of TSC-2, the Eker rats are very sensitive to renal carcinogens and therefore develop larger numbers of tumours than normal in a relatively short time," said Dr. Stemmer.
Stemmer's investigations were based on the fact that carcinogenic substances lead to cellular alterations and are always associated with gene expression changes. "We therefore asked ourselves whether the initial gene expression changes are able to reliably predict the carcinogenic effect of a substance within a few days of exposure, without having to wait the considerable period of time it takes for solid tumours to develop."
RNA analysis in three steps
Three experimental steps were used to analyse the genetic signature of tumour pre-stages and their importance for tumour development: "In an initial short-term experiment, the two rat strains were gavaged the renal carcinogens AA and OTA," said Dr. Stemmer summarising the experiment. Renal histopathology and cell proliferation were evaluated using paraffin slices between one and fourteen days after treatment. In addition, Dr. Stemmer used highly sensitive microarray transcriptome analyses to simultaneously investigate more than 31,000 genes and derive gene expression profiles. "Gene expression profiles of different stages of preneoplastic lesions provide information about the transcriptional processes that occur at a specific point in time in a specific tissue. Alterations in the animals' renal transcriptomes showed that the renal carcinogens had already led to the activation of cancer-related signalling pathways as early as one day after administration of the carcinogens. "While aristolochoic acid led to nearly identical gene expression changes in both rat strains, ochratoxin A led to a much larger number of deregulated genes in Eker rats than in wild-type rats," said Dr. Stemmer who believes that these findings show that the "ochratoxin A signalling pathways, which are normally slowed down by the tumour suppressor gene TSC2, are actually enhanced". The researchers have thus been able to show that the carcinogenic substances tested actually led to gene expression changes within a very short time.
RNA isolation using transcription analyses
Subsequent experiments were carried out to find out whether the early carcinogen-induced gene expression changes were only temporary or whether the changes actually led to the development of solid tumours. The exposure of Eker rats to AA and OTA for between three and six months allowed the researchers to assess the number of preneoplastic lesions formed. Dr. Stemmer used transcription analyses to find out how these preneoplastic lesions, which occurred in higher numbers in carcinogen-treated animals than in control animals, differ from healthy tissue and what effects AA and OTA have on the formation of preneoplastic lesions. "Using ‘laser capture microdissection' we were able to identify the microscopically small lesions under the microscope, cut them out using a computer-controlled laser beam and catapult them into reaction vessels. We succeeded in isolating RNA from these tiny tissue samples and this RNA was then used for microarray transcriptome analyses," said the biologist explaining the procedure. The researchers were able to verify individual signalling pathways using immunohistochemical staining. Traditional long-term studies are carried out under standardised conditions using rodents. However, these studies do not take into account the effect of risk factors such as adiposity and being overweight, conditions which are known to promote the development of tumours in humans. In a third step, Dr. Stemmer studied the effect of adiposity and high-calorie food on the development of early stages of renal carcinoma. The researchers were mainly interested in finding out whether high body adiposity per se or a high-calorie diet was the cause of increased renal cancer risk. "In collaboration with Prof. Daniel Dietrich's team at the University of Konstanz and Prof. Matthias Tschöp's and Prof. Paul Pfluger's groups at the Metabolic Diseases Institute at the University of Cincinnati, we were able to collect very promising data. Our results were awarded the Journal of Clinical Investigation poster prize at a recent scientific meeting," said the scientist.
Early processes provide information about mechanisms of action
All in all, Dr. Stemmer's research has led to some surprising results. The investigations showed that early gene expression changes after short-term exposure to carcinogens were predictive for the incidence and number of preneoplastic lesions. In addition, gene expression profiling enables the differentiation between genotoxic and non-genotoxic substances. It is worth noting that the altered gene expression profiles were very similar to spontaneous and substance-induced lesions. "It appears that early tumour stages no longer respond to long-term exposure to toxins," Dr. Stemmer explains.
Another interesting discovery was the fact that although aristolochic acid (AA) and mycotoxin ochratoxin A (OTA) led to early preneoplastic lesions, the surrounding kidney tissue seems to adapt to the toxic substances during long-term exposure: "Hardly any effects could be observed in healthy kidney tissue after a six-month exposure to the toxic substances."
It is the early processes in particular that can provide information about the carcinogenic mechanisms of action of a certain substance. "We assume that the application of new and more sensitive detection technologies such as microarrays will be able to considerably reduce the duration of long-term cancerogenesis studies and the number of experimental animals used for this purpose," said Dr. Stemmer highlighting the significance of her investigations. In July 2010, Dr. Stemmer was awarded the Nycomed Junior Investigator Award in recognition of her achievements.
Validation of new test systems and improved cancer therapies resulting from the collaboration between science and industry
In April 2010, the University of Konstanz offered Kerstin Stemmer the opportunity to spend a two-year research period at the University of Cincinnati Metabolic Diseases Institute. In 2012, Dr. Stemmer will return to the University of Konstanz and set up her own junior research group.
Dr. Stemmer's research at the Metabolic Diseases Institute focuses on the effects of adiposity and diabetes on elevated cancer risk. "Using state-of-the-art imaging methods such as bioluminescence and CT scanning, we are currently comparing the growth of tumours in slim and overweight rats and mice," said Dr. Stemmer describing her current research. "We are extremely interested in clarifying whether the therapeutic outcome of currently used cancer drugs differs between adipose and normal-weight patients." In addition, Dr. Stemmer is working with Ethicon Endo-Surgery, the global leader in the development and marketing of medical products for bariatic surgery. The partners are focusing on clarifying whether surgical methods to reduce adiposity such as gastric banding, gastric bypass or sleeve gastrectomy have an effect on the remission of solid tumours and are able to improve current cancer therapies.
"Generally speaking, there is huge potential for close collaboration between universities and industry in issues involving protection against carcinogenic substances and cancer," said Dr. Stemmer going on to add "I did my doctoral thesis in collaboration with Bayer-Scherin AG's Department of Special Toxicology in Wuppertal". The scientist believes that she will be able to expand her research in collaboration with industry and that microarray-based short-term investigations will some time in the future be validated as test systems. In addition, the in-depth understanding of cancer development in adiopose and normal-weight people will help to improve cancer therapies.
Dr. Kerstin Stemmer studied biology at the University of Konstanz. After a six-month practical training period in the medical laboratory service department of the Ministry of Health and Social Affairs in Windhoek, Namibia, she did her degree thesis in the field of human and environmental toxicology. She then spent some time at the German Institute of Human Nutrition in Potsdam and at the Max Delbrück Centre for Molecular Medicine in Berlin before returning to the University of Konstanz to complete her PhD thesis on the "molecular characteristics of renal cancerogenesis" in 2008. Since 2008, she has been participating in training given by the German Society of Pharmacology and Toxicology to become an expert in toxicology. Dr. Stemmer is deputy head of the Human and Environmental Toxicology research group at the University of Konstanz and is currently working at the Metabolic Diseases Institute at the University of Cincinnati before returning to Konstanz in 2012. In July 2010, Dr. Stemmer was awarded the Nycomed Junior Investigator Award.
Dr. Kerstin Stemmer
University of Konstanz
Human- and Environmental Toxicology
Metabolic Diseases Institute, University of Cincinnati OH, U.S.A.