Mutagenesis, Vol. 14, No. 3, 301-315,
May 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Standardization and validation of DNA adduct postlabelling methods: report of interlaboratory trials and production of recommended protocols
Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey SM2 5NG, UK and 1 International Agency for Research on Cancer, 150 cours Albert-Thomas, 69372 Lyon Cedex 08, France
The aim of this project was to devise and test improved protocols of the 32P-postlabelling assay for the detection of carcinogen–DNA adducts. The intention was to reverse the drift of different investigators using increasingly divergent experimental conditions. This would lead to a more standardized assay that can be used in future applications by different investigators for the monitoring of human exposure to genotoxic agents, permitting more meaningful comparisons between different studies or between different participants in the same study. As part of this process, there was perceived to be a need for carcinogen-modified DNA standards of known levels of adducts for use as positive controls, as standards for normalization of results with unknown samples and to assist interlaboratory comparisons. The preparation of characterized DNA standards modified by benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), 4-aminobiphenyl (ABP), an aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine, and N-methyl-N-nitrosourea (MNU), a methylating agent yielding DNA containing O6-methylguanine, was carried out. A critical appraisal of all aspects of the 32P-postlabelling procedure and investigations to examine the influence of a number of key variations on the assay were conducted. There followed testing of a consensus protocol in a first interlaboratory trial involving 25 participants in Europe and the USA, conducted on the prepared synthetic DNA standards, the assessment of interlaboratory variability and the reasons for it. Revision of the protocols was followed by further testing in a second interlaboratory trial in which liver DNA from mice treated with BaP or ABP were assayed together with the synthetic DNA standards. Adduct levels were found to be significantly lower by 32P-postlabelling than by 3H incorporation. A recommended set of procedures has been developed for the detection and quantitation of DNA adducts formed by PAHs, aromatic amines and methylating agents. These trials have led to a much clearer idea as to what are the critical features and procedures of the 32P-postlabelling assay and there is a set of standard DNA samples for use in quality control and against which biological samples can be normalized. Use of these standards and procedures has reduced interlaboratory variability in quantitation of DNA adducts.
2Trial participants: H.Autrup4 and P.S.Nielsen4 (University of Aarhus, Denmark); R.A.Baan4 (TNO Nutrition and Food Research, Rijswijk, The Netherlands); A.Barbin (IARC, Lyon, France); B.Binkova (Institute of Experimental Medicine, Prague, Czech Republic); M.Castegnaro (IARC, Lyon, France); S.J.Culp (NCTR, Jefferson AR, USA); W.Feser Zugner and R.S.Kerdar (Schering AG, Berlin, Germany); R.C.Gupta and J.Arif (University of Kentucky, Lexington, KY); A.Hewer and D.H.Phillips (Institute of Cancer Research, Sutton, UK); F.F.Kadlubar (NCTR, Jefferson, AR); A.Pfohl-Leszkowicz (ENSA, Toulouse, France); J.Lewtas and D.Walsh (US EPA, Research Triangle Park, NC); J.Nair (DKFZ, Heidelberg, Germany); K.Peltonen and K.Savela (Institute of Occupational Health, Helsinki, Finland); M.Peluso (Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy); F.PeĀrin and O.PeĀrin (Institut Curie, Orsay, France); W.Pfau (University of Hamburg, Hamburg, Germany); A.C.Povey and D.Cooper (Paterson Institute, Manchester, UK); M.V.Reddy (Covance, Vienna, VA); P.Sagelsdorff (CIBA-GEIGY Ltd, Basel, Switzerland); B.Schoket (National Institute of Public Health, Budapest, Hungary); D.Segerback5 (CNT-NOVUM, Huddinge, Sweden); F.J.Van Schooten (University of Limburg, Maastricht, The Netherlands); R.Waters4 (University of Wales, Swansea, UK); T.Wolff and J.Topinka (GSF, Oberschleissheim, Germany).
3 To whom correspondence should be addressed. Tel: +44 181 643 8901; Fax: +44 181 770 7290; Email: davidp{at}icr.ac.uk
4 Participated in the first trial only
5 Participated in the second trial using own postlabelling procedure
Other participants in one or more workshops who did not take part directly in the postlabelling trials: P.Fu and F.A.Beland (NCTR, Jefferson, AR); N.J.Jones (University of Wales, Swansea, UK); K.Randerath and E.Randerath (Texas Medical Center, Houston, TX); L.Airoldi (Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy).
The first mini-trial (pre-trial) was conducted in the laboratories of R.A.Baan, J.Lewtas, M.Castegnaro and D.H.Phillips. The second mini-trial was carried out in the laboratories of J.Lewtas, M.Castegnaro and D.H.Phillips.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Agudo, M. Peluso, N. Sala, G. Capella, A. Munnia, S. Piro, F. Marin, R. Ibanez, P. Amiano, M.J. Tormo, et al. Aromatic DNA adducts and polymorphisms in metabolic genes in healthy adults: findings from the EPIC-Spain cohort Carcinogenesis, June 1, 2009; 30(6): 968 - 976. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Veglia, S. Loft, G. Matullo, M. Peluso, A. Munnia, F. Perera, D. H. Phillips, D. Tang, H. Autrup, O. Raaschou-Nielsen, et al. DNA adducts and cancer risk in prospective studies: a pooled analysis and a meta-analysis Carcinogenesis, May 1, 2008; 29(5): 932 - 936. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. M. Arlt, M. Stiborova, C. J. Henderson, M. Thiemann, E. Frei, D. Aimova, R. Singh, G. Gamboa da Costa, O. J. Schmitz, P. B. Farmer, et al. Metabolic activation of benzo[a]pyrene in vitro by hepatic cytochrome P450 contrasts with detoxification in vivo: experiments with hepatic cytochrome P450 reductase null mice Carcinogenesis, March 1, 2008; 29(3): 656 - 665. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Gyorffy, L. Anna, K. Kovacs, P. Rudnai, and B. Schoket Correlation between biomarkers of human exposure to genotoxins with focus on carcinogen-DNA adducts Mutagenesis, January 1, 2008; 23(1): 1 - 18. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Munnia, F. Saletta, A. Allione, S. Piro, M. Confortini, G. Matullo, and M. Peluso 32P-Post-labelling method improvements for aromatic compound-related molecular epidemiology studies Mutagenesis, November 1, 2007; 22(6): 381 - 385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J. Ritchie, C. J. Henderson, X. J. Wang, O. Vassieva, D. Carrie, P. B. Farmer, M. Gaskell, K. Park, and C. R. Wolf Glutathione Transferase {pi} Plays a Critical Role in the Development of Lung Carcinogenesis following Exposure to Tobacco-Related Carcinogens and Urethane Cancer Res., October 1, 2007; 67(19): 9248 - 9257. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Talaska, M. Al-Zoughool, C. Malaveille, L. Fiorini, B. Schumann, J. Vietas, M. Peluso, A. Munnia, M. Bianchini, G. Allegro, et al. Randomized controlled trial: effects of diet on DNA damage in heavy smokers Mutagenesis, May 1, 2006; 21(3): 179 - 183. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Bakhiya, M. Stephani, A. Bahn, B. Ugele, A. Seidel, G. Burckhardt, and H. Glatt Uptake of Chemically Reactive, DNA-Damaging Sulfuric Acid Esters into Renal Cells by Human Organic Anion Transporters J. Am. Soc. Nephrol., May 1, 2006; 17(5): 1414 - 1421. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Peluso, A. Munnia, G. Hoek, M. Krzyzanowski, F. Veglia, L. Airoldi, H. Autrup, A. Dunning, S. Garte, P. Hainaut, et al. DNA Adducts and Lung Cancer Risk: A Prospective Study Cancer Res., September 1, 2005; 65(17): 8042 - 8048. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Thapliyal, S. S. Dolas, S. S. Pakhale, and G. B. Maru Evaluation of DNA damage in mice topically exposed to total particulate matter from mainstream and sidestream smoke from cigarettes and bidis Mutagenesis, September 1, 2004; 19(5): 413 - 421. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Frederiksen, H. Frandsen, and W. Pfau Syntheses of DNA adducts of two heterocyclic amines, 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA{alpha}C) and 2-amino-9H-pyrido[2,3-b]indole (A{alpha}C) and identification of DNA adducts in organs from rats dosed with MeA{alpha}C Carcinogenesis, August 1, 2004; 25(8): 1525 - 1533. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. A. Beland, M. I. Churchwell, D. R. Doerge, D. R. Parkin, D. Malejka-Giganti, A. Hewer, D. H. Phillips, P. L. Carmichael, G. Gamboa da Costa, and M. M. Marques Electrospray Ionization-Tandem Mass Spectrometry and 32P-Postlabeling Analyses of Tamoxifen-DNA Adducts in Humans J Natl Cancer Inst, July 21, 2004; 96(14): 1099 - 1104. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Gyorffy, L. Anna, Z. Gyori, J. Segesdi, J. Minarovits, I. Soltesz, S. Kostic, A. Csekeo, M. C. Poirier, and B. Schoket DNA adducts in tumour, normal peripheral lung and bronchus, and peripheral blood lymphocytes from smoking and non-smoking lung cancer patients: correlations between tissues and detection by 32P-postlabelling and immunoassay Carcinogenesis, July 1, 2004; 25(7): 1201 - 1209. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G Castano-Vinyals, A D'Errico, N Malats, and M Kogevinas Biomarkers of exposure to polycyclic aromatic hydrocarbons from environmental air pollution Occup. Environ. Med., April 1, 2004; 61(4): e12 - 12. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. C. A. BRANDT and W. P. WATSON Monitoring Human Occupational and Environmental Exposures to Polycyclic Aromatic Compounds Ann. Hyg., July 1, 2003; 47(5): 349 - 378. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Veglia, G. Matullo, and P. Vineis Bulky DNA Adducts and Risk of Cancer: A Meta-Analysis Cancer Epidemiol. Biomarkers Prev., February 1, 2003; 12(2): 157 - 160. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Divi, F. A. Beland, P. P. Fu, L. S. Von Tungeln, B. Schoket, J. E. Camara, M. Ghei, N. Rothman, R. Sinha, and M. C. Poirier Highly sensitive chemiluminescence immunoassay for benzo[a]pyrene-DNA adducts: validation by comparison with other methods, and use in human biomonitoring Carcinogenesis, December 1, 2002; 23(12): 2043 - 2049. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. P. Perera, L. A. Mooney, M. Stampfer, D. H. Phillips, D. A. Bell, A. Rundle, S. Cho, W.-Y. Tsai, J. Ma, A. Blackwood, et al. Associations between carcinogen-DNA damage, glutathione S-transferase genotypes, and risk of lung cancer in the prospective Physicians' Health Cohort Study Carcinogenesis, October 1, 2002; 23(10): 1641 - 1646. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Tang, D. H. Phillips, M. Stampfer, L. A. Mooney, Y. Hsu, S. Cho, W.-Y. Tsai, J. Ma, K. J. Cole, M. N. She, et al. Association between Carcinogen-DNA Adducts in White Blood Cells and Lung Cancer Risk in the Physicians Health Study Cancer Res., September 1, 2001; 61(18): 6708 - 6712. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Helleberg, H. Xu, L. Ehrenberg, K. Hemminki, U. Rannug, and M. Tornqvist Studies of dose distribution, premutagenic events and mutation frequencies for benzo[a]pyrene aiming at low dose cancer risk estimation Mutagenesis, July 1, 2001; 16(4): 333 - 337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Peluso, M. Ceppi, A. Munnia, R. Puntoni, and S. Parodi Analysis of 13 32P-DNA Postlabeling Studies on Occupational Cohorts Exposed to Air Pollution Am. J. Epidemiol., March 15, 2001; 153(6): 546 - 558. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.A. Williams, F.L. Martin, G.H. Muir, A. Hewer, P.L. Grover, and D.H. Phillips Metabolic activation of carcinogens and expression of various cytochromes P450 in human prostate tissue Carcinogenesis, September 1, 2000; 21(9): 1683 - 1689. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Rundle, D. Tang, H. Hibshoosh, A. Estabrook, F. Schnabel, W. Cao, S. Grumet, and F. P. Perera The relationship between genetic damage from polycyclic aromatic hydrocarbons in breast tissue and breast cancer Carcinogenesis, July 1, 2000; 21(7): 1281 - 1289. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. P. Perera Molecular Epidemiology: On the Path to Prevention? J Natl Cancer Inst, April 19, 2000; 92(8): 602 - 612. [Abstract] [Full Text] [PDF]
|
|