Treatment of DNA with ammonium bicarbonate or thiourea can lead to underestimation of platinum-DNA monoadducts.
Thiourea and NH4HCO3 are widely used to block the conversion of Pt-DNA monoadducts to diadducts prior to the enzymatic digestion of DNA and subsequent analysis of the relative proportion of the different types of Pt-DNA adducts. Our data show that NH4HCO3 (100 mM, 18 h, 25 degrees C) is much less effective than thiourea (10 mM, 10 min, 25 degrees C) at blocking monoadducts, apparently because considerable monoadduct-to-diadduct conversion occurs during the incubation of platinated DNA with NH4HCO3. Under these incubation conditions, neither NH4HCO3 nor thiourea treatment causes significant diadduct-to-monoadduct conversion. At 25 degrees C, thiourea causes no significant removal of either ethylenediamine(en)- or diaminocyclohexane(dach)-Pt monoadducts. However, at 37 degrees C, both en-Pt and dach-Pt monoadducts are selectively removed. Pt-DNA diadducts are stable to 10 mM thiourea at either temperature. These data suggest that previous experiments using NH4HCO3-blocked DNA are likely to have underestimated Pt-DNA monoadducts and to have overestimated diadducts. As a consequence, such studies are likely to produce inaccurate estimates for the repair of individual adducts. The data also show that although thiourea treatment is suitable for blocking Pt-DNA monoadducts under the conditions generally used (10 mM, 10 min, 25 degrees C), it can selectively remove Pt-DNA monoadducts at higher temperatures.
Gibbons
G R
GR
Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill 27599.
Page
J D
JD
Chaney
S G
SG
D001639:Bicarbonates / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D002945:Cisplatin / Q000032:analysis* / Q000378:metabolism; D004247:DNA / Q000032:analysis* / Q000187:drug effects* / Q000378:metabolism; D018736:DNA Adducts; D013890:Thiourea / Q000494:pharmacology*
D001639:Bicarbonates; D018736:DNA Adducts; C057270:cisplatin-DNA adduct; C027043:ammonium bicarbonate; D004247:DNA; D013890:Thiourea; D002945:Cisplatin