PhD conferral Bernardo Espadinha de Brito Palma

10/16/2017

13.45h

Aula, Vrije Universiteit Amsterdam

Development of human Cytochrome P450 competent genotoxicity tester bacterial systems for high throughput screening

Bernardo Espadinha de Brito Palma

Prof. N.P.E. Vermeulen and Prof. J. Rueff, copromotor Dr M. Kranendonk

Amsterdam Institute for Molecules, Medicines and Systems

Sciences

PhD conferral

Development of human Cytochrome P450 competent genotoxicity tester bacterial systems for high throughput screening

Humans are continuously exposed to a variety of xenobiotics, such as, pharmaceuticals, cosmetics, dietary, occupational or environmental chemicals. Some of these have been classified as carcinogens. Most genotoxic carcinogens are chemically inert, but upon metabolism or biotransformation they can form chemically reactive metabolites (CRMs). CRMs may subsequently covalently bind to DNA (genotoxic), RNA and proteins. A diversity of metabolic pathways has been recognized in the bioactivation of carcinogens, with a prominent role of cytochrome P450 enzymes (CYPs).

During his PhD research, de Brito Palma designed and engineered the novel SOS-CYPtest system. This system uses several E. coli and S. typhimuriumtester strains, made competent in human CYP1A2 metabolism and appropriate for in vitro HTS genotoxicity testing of chemicals. For proof of concept, human CYP1A2 was chosen as model biotransfomation enzyme. Although applied for a restricted number of compounds, the newly developed SOS-CYP test system seems to have the appropriate characteristics for the detection of DNA damaging CRMs in a HTS manner. HTS characteristics include a short assay time (max 120-180 min), the need of only small amounts of test compound (low micrograms), the adaptability to a microplate reader format and real-time measurement.

Genetic variants
During his PhD, de Brito Palma also investigated the role of genetic variants of human CYPs in their biotransformation capacities, to characterize their activity towards a diverse group of substrates and the role of the accessory redox partner cytochrome b5 in their activity. Due to the huge amount of data produced, and to scrutinize all results appropriately, multivariate analysis methods were applied and demonstrated to be a powerful and very useful tool regarding data analysis. It was concluded that 5 of the 8 different CYP1A2 variants analyzed, namely T83M, G299S, I386F, C406Y and R456H, were of relevance in terms of altered metabolism. Cytochrome b5 seems to compensate for structural deviations of the CYP1A2 variants, most likely through allosteric effects. 

Alltogether, application of the novel SOS-CYPtest system to study genotoxicity will help improving the selection of safer drug candidates, but also the identification of safer pollutants and/or hazardous environmental and occupational agents.

PhD_Brito_Palma