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Putative identification of functional interactions between DNA intercalating agents and topoisomerase II using the
Ronald D Snyder1, Marc R Arnone
1DuPont Pharmaceuticals Company, Stine-Haskell Research Center, P.O. Box 50, H1/1710, Newark, DE 19714, USA. Ronald.snyder@spcorp.com
Abstract:
Clastogenicity is frequently observed following treatment of mammalian cells with new chemical entities. This clastogenicity, unless proven otherwise, is assumed to result from the imperfect repair of DNA lesions produced from covalent chemical/DNA interaction. However, clastogenicity can also arise via other mechanisms such as non-covalent chemical intercalation into DNA resulting in poisoning of cellular DNA topoisomerase II (topo II) and stabilization of DNA double strand breaks. We have recently reported modifications to the V79 in vitro micronucleus assay which allow an indirect evaluation of both the intercalative and topoisomerase-interactive activities of chemical agents. In the present studies we have used these modified assays to further assess the validity of this approach in an evaluation of a number of intercalating and non-intercalating polycyclic compounds. It is shown that intercalating agents may be catalytic topo II inhibitors (e.g. chloroquine (CHL), tacrine (TAC), 9-aminoacridine (9AA), ethidium bromide (EB)) or topo II poisons (e.g. proflavine (PROF), auramine O (AUR) and curcumin (CURC)). Still other intercalators are shown to lack detectable topo II-interactions, (e.g. imipramine (IMP), quinacrine (QUIN), 2-aminoanthracene (AA), iminostilbene (IMN) and promethazine (PHE)). It is concluded that (1) the clastogenicity of three agents, PROF (a typical DNA intercalating agent), and AUR and CURC (both structurally atypical intercalating agents, with unknown clastogenic mechanisms), may be due to topo II poisoning; (2) other intercalating agents may either act as catalytic topo II inhibitors or exhibit no functional topo II interaction; (3) The use of these cell-based approaches may provide a logical first step in determining if unexpected clastogenicity associated with test article exposure is due to a topo II interaction.
Insights
Clastogenicity from new chemicals may stem from DNA damage or topoisomerase II (topo II) poisoning. Modified assays can distinguish these mechanisms, aiding chemical safety assessments.
Area of Science:
- Toxicology
- Molecular Biology
- Genetics
Background:
- Chemicals can cause clastogenicity (DNA damage) through covalent interactions or by interfering with DNA repair enzymes.
- Topoisomerase II (topo II) is crucial for DNA replication and repair; its inhibition or poisoning can lead to DNA strand breaks and clastogenicity.
- Distinguishing between direct DNA damage and topo II interaction is vital for accurate chemical safety evaluations.
Purpose of the Study:
- To evaluate modified V79 in vitro micronucleus assays for distinguishing chemical mechanisms of clastogenicity.
- To assess the topo II interaction profiles of various intercalating and non-intercalating polycyclic compounds.
- To determine if clastogenicity arises from direct DNA damage, catalytic topo II inhibition, or topo II poisoning.
Main Methods:
- Utilized modified V79 in vitro micronucleus assays to indirectly assess intercalative and topoisomerase-interactive activities.
- Tested a range of polycyclic compounds, including known intercalators and non-intercalators.
- Categorized agents based on their effects on topo II activity (inhibitor, poison, or no interaction).
Main Results:
- Identified intercalating agents as catalytic topo II inhibitors (e.g., chloroquine), topo II poisons (e.g., proflavine), or lacking detectable topo II interaction (e.g., imipramine).
- Demonstrated that clastogenicity of proflavine, auramine O, and curcumin may be due to topo II poisoning.
- Showed that other intercalators function as catalytic topo II inhibitors or have no topo II interaction.
Conclusions:
- The modified micronucleus assay effectively differentiates mechanisms of chemical-induced clastogenicity.
- Clastogenicity can result from topo II poisoning, catalytic inhibition, or lack of topo II interaction, depending on the chemical agent.
- These cell-based assays provide a valuable initial step in identifying topo II-mediated clastogenicity for novel chemical entities.