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© 1986 Oxford University Press

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Nuclease P1-mediated enhancement of sensitivity of 32P-Postlabeling test for structurally diverse DNA adducts

M.Vijayaraj Reddy and Kurt Randerath

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 [{gamma}-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 [{gamma}-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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Toxicol SciHome page
N. R. Bordelon, K. C. Donnelly, L. C. King, D. C. Wolf, W. R. Reeves, and S. E. George
Bioavailability of the Genotoxic Components in Coal Tar Contaminated Soils in Fischer 344 Rats
Toxicol. Sci., July 1, 2000; 56(1): 37 - 48.
[Abstract] [Full Text] [PDF]


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CarcinogenesisHome page
S. J. Culp, A. R. Warbritton, B. A. Smith, E. E. Li, and F. A. Beland
DNA adduct measurements, cell proliferation and tumor mutation induction in relation to tumor formation in B6C3F1 mice fed coal tar or benzo[a]pyrene
Carcinogenesis, July 1, 2000; 21(7): 1433 - 1440.
[Abstract] [Full Text] [PDF]


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CarcinogenesisHome page
S. Nesnow, C. Davis, W. T. Padgett, L. Adams, M. Yacopucci, and L. C. King
8,9-Dihydroxy-8,9-dihydrodibenzo[a,l]pyrene is a potent morphological cell-transforming agent in C3H10T1/2Cl8 mouse embryo fibroblasts in the absence of detectable stable covalent DNA adducts
Carcinogenesis, June 1, 2000; 21(6): 1253 - 1257.
[Abstract] [Full Text] [PDF]


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Cancer Res.Home page
W. Davis, A. Hewer, K. M. Rajkowski, W. Meinl, H. Glatt, and D. H. Phillips
Sex Differences in the Activation of Tamoxifen to DNA Binding Species in Rat Liver in Vivo and in Rat Hepatocytes in Vitro: Role of Sulfotransferase Induction
Cancer Res., June 1, 2000; 60(11): 2887 - 2891.
[Abstract] [Full Text] [PDF]


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Toxicol SciHome page
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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CarcinogenesisHome page
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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CMAJHome page
C.-J. Liu, C.-L. Chen, K.-W. Chang, C.-H. Chu, and T.-Y. Liu
Safrole in betel quid may be a risk factor for hepatocellular carcinoma: case report
Can. Med. Assoc. J., February 1, 2000; 162(3): 359 - 360.
[Full Text] [PDF]


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Cancer Res.Home page
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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CarcinogenesisHome page
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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Toxicol SciHome page
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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CarcinogenesisHome page
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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CarcinogenesisHome page
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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CarcinogenesisHome page
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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Cancer Res.Home page
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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Hum Exp ToxicolHome page
J P Arnould, A Pfohl-Leszkowicz, V Bach, J P Libert, and J Belegaud
Biological monitoring exposure of workers from plant producing carbon electrodes: quantification of benzo[a]pyrene DNA adducts in leukocytes, by a 32P-postlabelling method and an immunoassay
Human and Experimental Toxicology, May 1, 1999; 18(5): 314 - 321.
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MutagenesisHome page
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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CarcinogenesisHome page
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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CarcinogenesisHome page
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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MutagenesisHome page
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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CarcinogenesisHome page
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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CarcinogenesisHome page
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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FASEB J.Home page
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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Hum Exp ToxicolHome page
G. Dirheimer
Commentary on Ashby
Human and Experimental Toxicology, April 1, 1997; 16(4): 188 - 188.
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J. Biol. Chem.Home page
C. Vaziri and D. V. Faller
A Benzo[a]pyrene-induced Cell Cycle Checkpoint Resulting in p53-independent G1 Arrest in 3T3 Fibroblasts
J. Biol. Chem., January 31, 1997; 272(5): 2762 - 2769.
[Abstract] [Full Text] [PDF]


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Hum Exp ToxicolHome page
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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International Journal of ToxicologyHome page
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.
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J. Biol. Chem.Home page
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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Proc. Natl. Acad. Sci. USAHome page
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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