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Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling
Published on: March 20, 2018
DNA adduct formation by the anticancer drug ellipticine in rats determined by 32P postlabeling
Marie Stiborová1, Andrea Breuer, Dagmar Aimová
1Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030. 40 Prague 2, Czech Republic. stiborov@natur.cuni.cz
Abstract:
Ellipticine is a potent antineoplastic agent whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms covalent DNA adducts in vitro and that the formation of the major adduct is dependent on the activation of ellipticine by cytochrome P450 (CYP). Here, we investigated the capacity of ellipticine to form DNA adducts in vivo. Male Wistar rats were treated with ellipticine, and DNA from various organs was analyzed by (32)P postlabeling. Ellipticine-specific DNA adduct patterns, similar to those found in vitro, were detected in most test organs. Only DNA of testes was free of the ellipticine-DNA adducts. The highest level of DNA adducts was found in liver (19.7 adducts per 10(7) nucleotides), followed by spleen, lung, kidney, heart and brain. One major and one minor ellipticine-DNA adducts were found in DNA of all these organs of rats exposed to ellipticine. Besides these, 2 or 3 additional adducts were detected in DNA of liver, kidney, lung and heart. The predominant adduct formed in rat tissues in vivo was identical to the deoxyguanosine adduct generated in DNA by ellipticine in vitro as shown by cochromatography in 2 independent systems. Correlation studies showed that the formation of this major DNA adduct in vivo is mediated by CYP3A1- and CYP1A-dependent reactions. The results presented here are the first report showing the formation of CYP-mediated covalent DNA adducts by ellipticine in vivo and confirm the formation of covalent DNA adducts as a new mode of ellipticine action.
Insights
Ellipticine forms covalent DNA adducts in vivo, primarily in the liver, mediated by cytochrome P450 enzymes. This confirms a new mechanism of action for this antineoplastic agent.
Area of Science:
- Pharmacology
- Toxicology
- Molecular Biology
Background:
- Ellipticine is an antineoplastic agent primarily acting via DNA intercalation and topoisomerase II inhibition.
- Recent in vitro studies revealed ellipticine forms covalent DNA adducts following cytochrome P450 (CYP) activation.
Purpose of the Study:
- To investigate the in vivo capacity of ellipticine to form DNA adducts in various rat organs.
- To identify the specific CYP enzymes involved in ellipticine-DNA adduct formation in vivo.
Main Methods:
- Male Wistar rats were administered ellipticine.
- DNA from multiple organs was extracted and analyzed using (32)P postlabeling.
- Adducts were characterized by cochromatography and correlation studies with CYP activity.
Main Results:
- Ellipticine-DNA adducts were detected in most organs, with the highest levels in the liver.
- Testes DNA remained free of ellipticine adducts.
- The predominant adduct in vivo matched the in vitro deoxyguanosine adduct, mediated by CYP3A1 and CYP1A enzymes.
Conclusions:
- This study provides the first evidence of CYP-mediated covalent DNA adduct formation by ellipticine in vivo.
- Covalent DNA adduct formation represents a novel mode of action for ellipticine.
