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 reactive 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 rapid initial decrease was seen in the fraction of diadducts, followed by 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 at 37 degrees C; total platination was hardly affected. Postincubation of "aged" platinated DNA (no reactive monoadducts) only showed the slower decrease. The rapid process is likely to represent monoadduct inactivation, preventing the formation of diadducts, whereas the slow reaction must be interpreted as diadduct-to-monoadduct conversion. Similar reactions, but less efficient than with thiourea, occurred during dialysis against NH4HCO3 (0.1-1 M). Pt-containing (di)nucleotides in digested DNA were hardly affected by thiourea. Rapid reduction of the measured level of bifunctional adducts also occurred when cisplatin-treated Chinese hamster ovary cells were postincubated with thiourea, with concomitant increase in survival. It is concluded that quantification 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.