Drug-induced liver injury is the major reason why studies investigating potential drug candidates become cancelled, and why already approved drugs are withdrawn from the market or modified. Liver toxicity is also the most common cause of acute liver failure and liver transplantation in industrialized countries. Although drugs have to be tested in numerous animal experiments, the risk of liver damage cannot be accurately predicted by animal models. Test systems based on human liver cells represent a promising alternative to animal experiments in toxicological risk assessment and management.
The predictive performance of the so far established test systems nevertheless remains limited. In addition, until now it has only been possible to measure the in-vitro-concentration of a substance that damages the cells. However, it is of interest to know the amount of drug a person can ingest without exhibiting toxic effects, or the dose at which there is an increased risk of developing adverse health effects. At IfADo, toxicologists have been investigating alternative methods to animal experiments for years. Together with an international research team, IfADo-scientist Wiebke Albrecht has now developed new methods that can be used to assess how well an in-vitro-system reflects the processes in the human body.
Performance metrics provide hints for better test systems
There is an urgent need to establish a level of quality control in the test systems. Many parameters need to be considered in the development of in-vitro-systems, for example whether important functions are impaired, such as the production of proteins. So far, it has not been possible to reliably assess which of the parameters are best suited to reflect the situation in the organism. To fill this gap, novel test performance metrics have been introduced in the now awarded study. With the help of mathematical methods, the researchers were able to calculate which of the numerous parameters enable the best distinction between liver-toxic and non-toxic substances. Since it is not only important whether a substance causes liver damage, but also the concentration at which liver toxic effects are likely, a second metric was established, which is used to assess how well the toxic blood concentration can be estimated in vitro.
The researchers started out by testing 28 training substances, mainly drugs, using an in-vitro-system based on human hepatocytes. Prior to testing, it was already known whether the substances caused liver toxicity in humans, and at which dose the risk of liver damage arose. „The measurements showed, for example, that an incubation period of 48 hours produced more reliable results than 24 hours or several days. In addition, we could demonstrate that the concentration at which ten percent of the cells die should be used as the lowest toxic in-vitro concentration. In past studies up to 50 percent were taken into account,“ explains Wiebke Albrecht.
The researchers used these references to optimize the test system. Importantly, all liver-toxic substances were subsequently correctly identified. For two non-toxic substances, the system caused a false alarm. „After this optimization phase, we tested eight more substances, all of which were correctly classified,“ says Albrecht. In order to find out how well the system works, the team is currently investigating further 200 substances.
Optimized test systems for safe daily intake
With the help of the new method, the researchers were also able to estimate an acceptable daily intake for a food additive, i.e. the amount of a substance that can be ingested on a daily basis without an appreciable health risk. Using the lowest toxic concentration in the cell culture, a computer program helped to calculate both the dose that will cause harm to humans when taken orally and the acceptable daily intake for humans. The program also took into account how the human organism processes a substance.
„Our calculated acceptable daily intake was similar to values previously established based on animal experiments,“ says Wiebke Albrecht. „However, we have to investigate many more substances in order to understand how accurate our method is.“ Despite the work to be done, it is always important to keep the major goal in mind: „With our established methods, we want to promote research into alternative methods to reduce and replace animal experiments in toxicological risk assessment and management. It is really great that Wiebke Albrecht receives this prize. She had some brilliant ideas and worked very hard to generate all the data”, says Prof. Dr. Jan Hengstler, head of IfADo’s toxicology research department and Albrecht’s PhD advisor.
About Wiebke Albrecht:
Wiebke Albrecht performed her undergraduate and postgraduate research in the Molecular Medicine department at the Georg-August-University of Göttingen. In September 2016, she joined the Leibniz Institute for Work Research at the TU Dortmund (IfADo) as PhD student. Wiebke Albrecht is part of IfADo’s research department „Toxicology“, which is headed by Prof. Dr. Jan Hengstler.
About the prize:
The Federal Ministry of Food and Agriculture (Germany) annually awards the “Animal Welfare Research Prize” for outstanding research works that aims to reduce or replace animal experiments in the long term. Researchers can apply with papers already accepted for publication, or with scientific papers or publications that were published no more than two years ago. The recipient of the animal welfare research prize is awarded 25,000 euros.
48 international researchers and representatives of federal authorities and companies under the auspices of the Technical University of Dortmund and the Leibniz Research Centre for Working Environment and Human Factors (IfADo) were involved in the publication. Wiebke Albrecht, Tim Brecklinghaus and Franziska Kappenberg (TU Dortmund) shared first authorship. The study was published in the journal „Archives of Toxicology“. This work received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 681002 (EU-ToxRisk), from TransQST (no. 116030), LivSysTransfer (BMBF, 031L0119) with contributions from StemCellNet (BMBF, 01EK1604A), LiSyM (BMBF, 031Loo45031L0045) and InnoSysTox (BMBF/EU, 031L0021A) and the European Regional Development Fund (CP16/00097).
contact for scientific information:
M. Sc. Wiebke Albrecht
PhD student, research department “Toxicology“
Phone: +49 231 1084-348/-349
Prof. Dr. Jan G. Hengstler
Head of research department “Toxicology”
Phone: +49 231 1084-348/-349
Albrecht, W., Kappenberg, F., Brecklinghaus, T. et al.: Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentration. Arch. Toxicol. 93: 1609-1637 (2019). doi: 10.1007/s00204-019-02492-9