DNA as an in vitro trapping agent for detection of bulky genotoxic metabolites.

The instability of electrophilic reactive metabolites in in vitro metabolism studies makes their accurate analysis challenging. To stabilise the reactive compounds prior to their analysis, different trapping agents, such as thiols, amines and cob(I)alamin, have earlier been tested depending on the metabolites to be analysed and the type of study. In the present work, DNA is introduced as a trapping agent for measuring the formation of bulky electrophilic metabolites. Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), was used as a model compound in a rat liver S9 metabolic system. Under physiological incubation conditions, B[a]P metabolises to diol epoxide (BPDE) metabolites which were trapped by DNA resulting in the formation of covalently bound DNA adducts. The methodology for analysis of these adducts included extraction of the DNA from the metabolic system, digestion of the DNA to yield nucleosides and analysis of the BPDE-adduct to deoxyguanosine (BPDE-dG) by liquid chromatography coupled to high resolution mass spectrometry (HRMS). The chromatographic conditions in combination with the high mass accuracy data (±3 ppm) was useful in resolving BPDE-dG in its protonated form from the complex set of ions present in the metabolic matrix. The method was validated in terms of sensitivity, specificity, accuracy, precision and recovery, and applied to provide a preliminary estimate of BPDE-dG levels from the metabolism of B[a]P in rat S9. The use of DNA as a trapping agent for in vitro metabolites has a potential to aid in cancer risk assessment procedure of PAHs, for instance, in inter-species comparison of metabolism to reactive metabolites and can be adapted for screening of genotoxic metabolites, e.g., from emerging environmental contaminants.