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5146460 
Journal Article 
32P-Postlabeling Assay for Carcinogen-DNA Adducts: Nuclease P1-Mediated Enhancement of Its Sensitivity and Applications 
Reddy, MV; Randerath, K 
1987 
Yes 
Environmental Health Perspectives
ISSN: 0091-6765
EISSN: 1552-9924 
76 
A phosphorus-32 (P-32) postlabeling procedure was examined for its ability to detect and quantify DNA lesions produced by the anticancer drug mitomycin-C (50077) (MMC). Male and female Fischer-344-rats were treated with 9mg/kg MMC intraperitoneally, and various tissues were obtained after 24 hours and the DNA isolated. Following enzymatic digestion of DNA to 3'-phosphorylated normal and adducted mononucleotides (P-32 labeled at their 5'-hydroxyl groups), thin layer chromatography was used to resolve the labeled derivatives, which were then detected by autoradiography and quantitated by counting. The method using P-32 labeling proved to be very sensitive, especially when used in combination with nuclease-P1 enhancement due to resistance of aromatic and bulky adducts to dephosphorylation; this enabled detection and quantitation of such adducts at frequencies of 1 lesion per 10(10) DNA nucleotides. Nuclease-P1 dephosphorylated normal nucleotides but not most aromatic or bulky nonaromatic adducts, so the latter served as substrates for the kinase catalyzed labeling reaction. MMC showed ubiquitous binding to DNA in the various tissues, although differences in levels and adduct patterns were noted, with brain showing the lowest levels of adduction. Some sex differences were also seen. Nuclease-P1 enhancement of sensitivity was also found for adducts of benzo(a)pyrene (50328) from ICR-mice given 200 micromoles/kilogram (micromol/kg) orally, 4-aminobiphenyl (92671) from mice given 800micromol/kg, 7H-dibenzo(c,g)carbazole (194592) from BALB/c-mice given 80micromol/kg topically, and 7,12-dimethylbenz(a)anthracene (57976) from mice given 1.2 micromoles topically. The authors conclude that this method is useful for detecting DNA adducts in smokers, and it may be useful in measuring DNA adducts in experimental animals receiving single or mixed carcinogen exposures at low doses corresponding more closely to actual levels of exposure experienced by humans.