EFFECTS OF THIOUREA AND AMMONIUM BICARBONATE ON THE FORMATION AND STABILITY OF BIFUNCTIONAL CISPLATIN-DNA ADDUCTS - CONSEQUENCES FOR THE ACCURATE QUANTIFICATION OF ADDUCTS IN (CELLULAR) DNA

Cisplatin reacts with DNA by forming mainly bifunctional adducts via r eactive monofunctional intermediates. When freshly platinated DNA was postincubated with thiourea (10 mM, at 23 or 37 degrees C) for periods of up to 24 h, followed by determination of mono- and diadducts, a ra pid initial decrease was seen in the fraction of diadducts, followed b y a much slower decrease. About 40% diadducts were found after 10-min postincubation at 23 degrees C, which dropped to some 14% after 24 h a t 37 degrees C; total platination was hardly affected. Postincubation of ''aged'' platinated DNA (no reactive monoadducts) only showed the s lower decrease. The rapid process is likely to represent monoadduct in activation, preventing the formation of diadducts, whereas the slow re action must, be interpreted as diadduct-to-monoadduct conversion. Simi lar reactions, but less efficient than with thiourea, occurred during dialysis against NH4HCO3 (0.1-1 M). Pt-containing (di)nucleotides in d igested DNA were hardly affected by thiourea. Rapid reduction of the m easured level of bifunctional adducts also occurred when cisplatin-tre ated Chinese hamster ovary cells were postincubated with thiourea, wit h concomitant increase in survival. It is concluded that quantificatio n of the real levels of mono- and diadducts in freshly platinated DNA requires a posttreatment with thiourea of 30-60 min at 37 degrees C.