P-32 POSTLABELING OF BILE COMPONENTS - BULKY ADDUCT-LIKE BEHAVIOR IN POLYETHYLENEIMINE CELLULOSE THIN-LAYER CHROMATOGRAPHY

The P-32-postlabeling assay has been used widely in carcinogen-DNA add uct analysis because of its sensitivity and reproducibility. Cloned T4 polynucleotide kinase (PNK), routinely used in this assay, phosphoryl ates the 5'-OH groups of adducted nucleotides in the presence of [gamm a-P-32]ATP. However, as an exception to this property, PNK has been re ported to phosphorylate non-adducted carcinogen metabolites, such as t etrol derivatives of benzo[a]pyrene and chrysene. Also, PNK phosphoryl ates both 5'-OH and 3'-OH groups of safrole-adducted deoxydinucleoside monophosphates having an unmodified purine in the 3'-position. In the present study we show that T4 PNK catalyzed the transfer of [P-32]pho sphate from [gamma-P-32]ATP to rat bile components or purified bile ac ids (derivatives of 3 alpha-hydroxy-5 beta-cholanic acid) in the absen ce of nucleic acids or nucleases. However, labeling of the bile acids appeared over 100 000-fold less efficient than labeling of 2'-deoxyade nosine-5'-monophosphate. There was no reaction in the absence of bile components or PNK. Dehydrocholic acid, which lacks hydroxyl groups, wa s resistant to phosphorylation. On polyethyleneimine-cellulose TLC map s, P-32-labeled rat bile extract gave an array of non-polar radioactiv e spots which resembled carcinogen-DNA adducts, while P-32-labeled pur ified bile acids each gave a single spot. These P-32-labeled products liberated P-32(1) upon incubation with prostatic acid phosphatase. Two of the radioactive spots obtained from rat bile were identified as ph osphorylated taurocholic and taurodeoxycholic acids by co-chromatograp hy with P-32-labeled standards. These findings demonstrate for the fir st time that PNK is able to phosphorylate natural products other than nucleotides and further emphasize the need to rule out contamination w ith bile acids and possibly other bulky/hydrophobic alcohols when anal yzing DNA samples by P-32-postlabeling.