Llg. Mourato et al., P-32-postlabeling of N-(deoxyguanosin-8-yl)arylamine adducts: A comparative study of labeling efficiencies, CHEM RES T, 12(7), 1999, pp. 661-669
P-32-Postlabeling is an extremely powerful technique for the detection of D
NA adducts. Typically, the quantitation of DNA adducts by P-32-postlabeling
is achieved by relative adduct labeling, via comparison of the radioactivi
ty incorporated into the adducts to that associated with the normal nucleot
ides. This approach is based on a number of assumptions, the foremost being
that normal and adducted nucleotide 3'-phosphates are converted to 3',5'-b
isphosphates with similar efficiencies. To evaluate labeling efficiencies f
or specific DNA adducts, we conducted a comparative study of the kinetics o
f phosphorylation by T-4 polynucleotide kinase using 2'-deoxyguanosine 3'-p
hosphate (dG3'p) and a series of N-(deoxyguanosin-8-yl)arylamine 3'-phospha
te adduct standards (dCT3'p-C8-Ar, Ar being 4-aminobiphenyl, 3- and 4-methy
laniline, and 2,4- and 3,4-dimethylaniline). Phosphorylation of dG3'p and t
he dCT3'p-C8-Ar adducts followed Michaelis-Menten kinetics. The apparent tu
rnover numbers were 40-240-fold lower when labeling dG3'p-C8-Ar adducts com
pared to that when labeling dG3'p. The apparent specificity constant calcul
ated for dG3'p-C8-4-aminobiphenyl (1.4 mu M-1 min(-1)) was approximately 4-
fold lower than that (5.4 mu M-1 min(-1)) found for dG3'p. Apparent specifi
city constants for the monoarylamine adducts were even lower (0.043-0.23 mu
M-1 min(-1)) and decreased in the following order: 4-methylaniline > 3,4-d
imethylaniline > S-methylaniline > 2,4-dimethylaniline. Similar experiments
conducted with dG3'p-C8-Ar standards for 2-methylaniline and 2,3-dimethyla
niline produced very poor and irreproducible labeling. These results indica
te that P-32-postlabeling of dG3'p-C8-Ar adducts is less efficient than tha
t of dG3'p and suggest that normal nucleotides will be labeled preferential
ly to the arylamine adducts under kinetically controlled conditions. The da
ta also indicate a further decrease in labeling efficiency upon substitutio
n ortho to the amino group (e.g., 2,4-dilmethylaniline). In addition, the A
TP concentrations required for optimal labeling were found to be substantia
lly higher than those used in typical 32P-postlabeling assays. Since the hi
gh specific activity of carrier-free [gamma-P-32]- ATP precludes increasing
the ATP concentration to a significant extent, these data emphasize the ne
ed for using highly efficient adduct enrichment procedures when conducting
P-32- postlabeling analyses of DNA adducts.