Pyrrolizidine alkaloids crosslink DNA with actin.

Pyrrolizidine alkaloids (PAs) are toxic constituents of hundreds of plant species, some of which people are exposed to in herbal products and traditional remedies. The bioactivity of PAs are related, at least in part, to their ability to form DNA-protein complexes (DPC). Previous studies from our laboratory indicated a possible role for actin in PA-induced DPCs. Nuclei prepared from Madin-Darby bovine kidney (MDBK) and human breast carcinoma (MCF-7) cells were treated with the pyrrolic PAs dehydrosenecionine (DHSN) and dehydromonocrotaline (DHMO). DPCs were purified and then analyzed by Western immunoblotting. Actin was found in DPCs induced by both DHSN and DHMO, but not in those from control nuclei. Actin was also present in DPCs induced by cisplatinum and mitomycin C, two bifunctional cross-linkers. In separate experiments, DHSN and DHMO were crosslinked to a mixture of HindIII digested lambda phage with varying amounts of glutathione (GSH), cysteine, or methionine to identify the stoichiometry of competition between DNA and alternate nucleophiles for crosslink formation with pyrroles. GSH and cysteine, but not methionine, competed with lambda phage for DNA crosslinking, indicating that reduced thiols may have a role in nucleophilic reactions with pyrroles in the cell. While actin involvement in cisplatinum-induced DPCs is documented, the discovery of actin crosslinking in PA or mitomycin C-treated cells or nuclei is, to our knowledge, novel. Pyrrole-induced DPC formation with actin, a protein with structural and/or regulatory importance proteins, may be a significant mechanism for PA toxicity and bioactivity. Copyright 1999 Academic Press.