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Nuclease P1-mediated enhancement of sensitivity of 32P-Postlabeling test for structurally diverse DNA adducts
Department of Pharmacology, Baylor College of Medicine, Texas Medical Center Houston, TX 77030, USA
Exceedingly sensitive procedures are required to detect the presence of covalent DNA adducts in humans exposed to environmental genotoxicants because of low levels of such derivatives (1 adduct in 108-1010 DNA nucleotides). A 32P-postlabeling assay for detection and quantitation of DNA adducts with a sensitivity limit of 1 adduct in 107-108 nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3'-phosphorylated normal and adducted mononucleotides, which are quantitatively 32P-labeled at their 5'-hydroxyl groups by T4 polynudeotide kinase-catalyzed [32P]phosphate transfer from [
-32P]ATP. 32P-labeled derivatives are resolved by t.l.c, detected by autoradiography and quantitated by counting. We now report that a minor modification of this procedure, entailing the postincubation of DNA digests with Penicillium citrinum nuclease P1 before 32P-labeling, enhanced the technique's sensitivity to 1 adduct in 
1010 nucleotides for a 10-µg DNA sample. Nuclease PI cleaves deoxyribonucleoside 3'-monophosphates of normal nucleotides to deoxyribonudeo-sides which do not serve as substrates for polynudeotide kin-ase, while most adducted nucleotides were found to be totally or partially resistant to the 3'-dephosphorylating action of nuclease PI. The additional enzymatic step enabled specific labeling of adducts in up to 20 µg of DNA with excess carrier-free [-32P]ATP. The enzymatic digestion conditions were standardized to afford optimal adduct recovery. The new procedure was found to be simple, highly reproducible, and applicable to the detection and measurement of aromatic or bulky non-aromatic DNA adducts formed with such structurally diverse carcinogens as benzo[a]pyrene, 7, 12-dimethyl-benz[a]anthracene, dibenzo[c, g]carbazole, 4-aminobiphenyl, safrole and mitomycin C; most adducts were recovered quantitatively with a 500- to 1000-fold increase in 32P-count rates as compared with the standard procedure.
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J. I. Goodman, D. J. Brusick, W. M. Busey, S. M. Cohen, J. C. Lamb, and T. B. Starr Reevaluation of the Cancer Potency Factor of Toxaphene: Recommendations from a Peer Review Panel Toxicol. Sci., May 1, 2000; 55(1): 3 - 16. [Abstract] [Full Text] [PDF]
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H. Sato, H. Sone, M. Sagai, K. T. Suzuki, and Y. Aoki Increase in mutation frequency in lung of Big Blue(R) rat by exposure to diesel exhaust Carcinogenesis, April 1, 2000; 21(4): 653 - 661. [Abstract] [Full Text] [PDF]
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D. R. Lloyd and P. C. Hanawalt p53-dependent Global Genomic Repair of Benzo[a]pyrene-7,8-diol-9,10-epoxide Adducts in Human Cells Cancer Res., February 1, 2000; 60(3): 517 - 521. [Abstract] [Full Text]
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D. Renault, D. Brault, Y. Lossouarn, O. Perin-Roussel, D. Taras-Valero, F. Perin, and V. Thybaud Kinetics of DNA adduct formation and removal in mouse hepatocytes following in vivo exposure to 5,9-dimethyldibenzo[c,g]carbazole Carcinogenesis, February 1, 2000; 21(2): 289 - 294. [Abstract] [Full Text] [PDF]
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V. M. Lakshmi, F. F. Hsu, A. E. McGarry, B. B. Davis, and T. V. Zenser Hypochlorous Acid-Mediated Activation of N-acetylbenzidine to Form N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine Toxicol. Sci., February 1, 2000; 53(2): 202 - 212. [Abstract] [Full Text] [PDF]
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R. W.L. Godschalk, E. J.C. Moonen, P. A.E.L. Schilderman, W. M.R. Broekmans, J. C.S. Kleinjans, and F. J. Van Schooten Exposure-route-dependent DNA adduct formation by polycyclic aromatic hydrocarbons Carcinogenesis, January 1, 2000; 21(1): 87 - 92. [Abstract] [Full Text] [PDF]
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C.-L. Chen, C.-W. Chi, K.-W. Chang, and T.-Y. Liu Safrole-like DNA adducts in oral tissue from oral cancer patients with a betel quid chewing history Carcinogenesis, December 1, 1999; 20(12): 2331 - 2334. [Abstract] [Full Text] [PDF]
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D. H. Phillips, A. Hewer, M. N. Horton, K. J. Cole, P. L. Carmichael, W. Davis, and M. R. Osborne N-Demethylation accompanies {alpha}-hydroxylation in the metabolic activation of tamoxifen in rat liver cells Carcinogenesis, October 1, 1999; 20(10): 2003 - 2009. [Abstract] [Full Text] [PDF]
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A. Izzotti, A. Camoirano, C. Cartiglia, C. J. Grubbs, R. A. Lubet, G. J. Kelloff, and S. D. Flora Patterns of DNA Adduct Formation in Liver and Mammary Epithelial Cells of Rats Treated with 7,12-Dimethylbenz(a)anthracene, and Selective Effects of Chemopreventive Agents Cancer Res., September 1, 1999; 59(17): 4285 - 4290. [Abstract] [Full Text] [PDF]
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D.H. Phillips and M. Castegnaro Standardization and validation of DNA adduct postlabelling methods: report of interlaboratory trials and production of recommended protocols Mutagenesis, May 1, 1999; 14(3): 301 - 315. [Abstract] [Full Text] [PDF]
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H. A.J. Schut and E. G. Snyderwine DNA adducts of heterocyclic amine food mutagens: implications for mutagenesis and carcinogenesis Carcinogenesis, March 1, 1999; 20(3): 353 - 368. [Abstract] [Full Text] [PDF]
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F. A. Beland, L. P. McDaniel, and M. M. Marques Comparison of the DNA adducts formed by tamoxifen and 4-hydroxytamoxifen in vivo Carcinogenesis, March 1, 1999; 20(3): 471 - 477. [Abstract] [Full Text] [PDF]
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M. Ochiai, H. Nakagama, R. J. Turesky, T. Sugimura, and M. Nagao A new modification of the 32P-post-labeling method to recover IQ–DNA adducts as mononucleotides Mutagenesis, March 1, 1999; 14(2): 239 - 242. [Abstract] [Full Text] [PDF]
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M. R. Osborne, A. Hewer, W. Davis, A. J. Strain, A. Keogh, I. R. Hardcastle, and D. H. Phillips Idoxifene derivatives are less reactive to DNA than tamoxifen derivatives, both chemically and in human and rat liver cells Carcinogenesis, February 1, 1999; 20(2): 293 - 297. [Abstract] [Full Text] [PDF]
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F. Tombolan, D. Renault, D. Brault, M. Guffroy, O. Perin-Roussel, F. Perin, and V. Thybaud Kinetics of induction of DNA adducts, cell proliferation and gene mutations in the liver of Muta(TM)Mice treated with 5,9-dimethyldibenzo[c,g]carbazole Carcinogenesis, January 1, 1999; 20(1): 125 - 132. [Abstract] [Full Text] [PDF]
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F. J. Van schooten, A. Hirvonen, L. M. Maas, B. A. De mol, J. C. S. kleinjans, D. A. Bell, and J. D. Durrer Putative susceptibility markers of coronary artery disease: association between VDR genotype, smoking, and aromatic DNA adduct levels in human right atrial tissue FASEB J, October 1, 1998; 12(13): 1409 - 1417. [Abstract] [Full Text]
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G. Dirheimer Commentary on Ashby Human and Experimental Toxicology, April 1, 1997; 16(4): 188 - 188. [PDF]
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W. Feser, R. K. H Blode, and R. Reimann Formation of DNA-adducts by selected sex steroids in rat liver Human and Experimental Toxicology, July 1, 1996; 15(7): 556 - 562. [Abstract] [PDF]
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B. P. Dunn Detection of Aromatic DNA Adducts in Human Cells and Fish Liver International Journal of Toxicology, September 1, 1989; 8(5): 905 - 912. [Abstract] [PDF]
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K. Randerath, G.-D. Zhou, R. L. Somers, J. H. Robbins, and P. J. Brooks A 32P-Postlabeling Assay for the Oxidative DNA Lesion 8,5'-Cyclo-2'-deoxyadenosine in Mammalian Tissues. EVIDENCE THAT FOUR TYPE II I-COMPOUNDS ARE DINUCLEOTIDES CONTAINING THE LESION IN THE 3' NUCLEOTIDE J. Biol. Chem., September 14, 2001; 276(38): 36051 - 36057. [Abstract] [Full Text] [PDF]
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T. Takamura-Enya, M. Watanabe, Y. Totsuka, T. Kanazawa, Y. Matsushima-Hibiya, K. Koyama, T. Sugimura, and K. Wakabayashi Mono(ADP-ribosyl)ation of 2'-deoxyguanosine residue in DNA by an apoptosis-inducing protein, pierisin-1, from cabbage butterfly PNAS, October 23, 2001; 98(22): 12414 - 12419. [Abstract] [Full Text] [PDF]
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