ICANCER RESEARCH 56, 683-688. February 15. 19961 Advances in Brief Immunoperoxidase Detection of 8-Hydroxydeoxyguanosine in Aflatoxin B1-treated Rat Liver and Human Oral Mucosal Cells' Alicja Yarborough, Yu-Jing Zhang, Ta-Ming Hsu, and Regina M. Santella2 Division of Environmental Health Sciences, Columbia School of Public Health, and Columbia Presbyterian Cancer Center, New York, New York 10932 Abstract An immunoperoxidasemethodusinga monoclonalantiserumthat recognizes8-hydroxydeoxyguanosinehas been developedfor detection and quantitationof oxidativedamagein singlecells.The methodwas Initially applied to cultured cells treated with H202 or afiatoxin B1 and thento cryostatliver sectionsof ratstreatedwithafiatoxinB1.To dem onstratethatthemethodhassufficientsensitivity fordetectionofdamage in human samples,oral mucosalcells from a total of 12 pairs of smokers andnonsmokerswereanalyzed.Mean stainingintensityof oral ceHsof smokerswas 1.6-foldhigher than in nonsmokers.The immunoperoxidase method, requiring a small number of cells and eliminating the need for isolation of DNA, will be useful for evaluation of oxidative damagein a widerangeof biological samples. Introduction 8-Hydroxy- or 8-oxo-deoxyguanosine is recognized as a useful marker for the estimation of DNA damage produced by oxygen radicals generated endogenously or exogenously. Although numerous oxidative lesions occur in DNA, oxidation of the C8 of guanine is one of the more abundant types (1); it is a major mutagenic lesion producing predominately G—*T transversion mutations (2, 3). 8-OHdG3accumulates in DNA exposed tomutagenicandcarcino genic agents, such as reducing agents, X-rays, asbestos,and polyphe nols that generate oxygen-free radicals (4). Increased levels of 8-OHdG are also observed in DNA from ‘y-irradiated mouse liver, X-irradiated HeLa cells, and H2O2-treated Salmonella typhimurium (5). A number of methods have been developed for quantitation of femtomol levels of 8-OHdG in cellular DNA, including HPLC-EC (6), GCIMS (7), 32Ppostlabeling(8), andimmunoassays (9, 10). The HPLC-EC and GC/MS methods are used most frequently, but both have some limitations that may be responsible for discrepancies in quantitative values. Two- to I I-fold greater amounts of 8-OHdG in DNA were reported with the GC/MS method than with the HPLC method (I 1). Inaccurate estimates of 8-OHdG by the GCIMS method may be due to incomplete derivatization, formation of by-products, and possible destruction or formation of oxidized bases during acid hydrolysis, whereas incomplete enzymatic hydrolysis and coelution of interfering substances may be potential problems in the HPLC-EC method.Monitoring of humantumor and nontumortissuehasdocu mented the occurrence of 8-OHdG in DNA. Higher levels of 8-OHdG were found in WBC of smokers than nonsmokers (12) and in tumor Received I 1/15/95; accepted 12/28/95. The costsof publicationof this articleweredefrayedin part by the paymentof page charges. This article must therefore be hereby marked advertisement in accordance with 18U.S.C.Section1734solely to indicatethis fact. I This work was supported by NIH Grant ESO5 I 16. A. Y. was supported by a National Cancer Institute National Research Service Award 5T32 CA09529-l0. 2 To whom requests for reprints should be addressed, at Columbia University, 701 West 168th Street, New York, NY 10032. Phone/Fax:(212) 305-1996; E-mail: rpsl @columbia.edu. 3 The abbreviations used are: 8-OHdG, 8-hydroxydeoxyguanosine; GC/MS, gas chro matography/mass spectroscopy; HPLC-EC, high-performance liquid chromatography with electrochemical detection;8-MOP,8-Methoxy-psoralen; AFB,, aflatoxin B,. tissue compared to nontumor tissues in colon, stomach, ovary, brain, breast, and lung (13, 14). Recently, we reported development of monoclonal antisera and an immunoassay for quantification of 8-OHdG in human samples (15). Antisera 1F7 was used for immunoaffinity isolation of 8-OHdG from DNA hydrolysates,followed by ELISA quantitationwith antiserum iFli. To validate the assay, DNA extracted from human placental tissue was assayedby ELISA and HPLC-EC. Values by both methods correlated well, but the levels determined by ELISA were approxi mately 6-fold higher than thosedeterminedby HPLC. This may be due to oligonucleotides detected by ELISA, but not by the HPLC method,orcross-reactionof theantiserumwith otherdamagedbases presentin the immunoaffinity-purifiedmaterial. In this study, we determined that the more selective antibody, 1F7, can be used for detection of 8-OHdG in single cells by an immu noperoxidase procedure. Initial studies were performed with cells treated in culture with two inducers of oxidative damage, H202 and AFB @. Thena small pilot studywascarriedout on humansubjectsto test the applicability of the method for detection of oxidative damage caused by smoking as a model environmental exposure. We also adapted antiserum 6A10, used previously for immunofluorescence detection of AFB1-DNA adducts (16), to the immunoperoxidase tech nique to allow side-by-side comparisons of AFB1-DNA and 8-OHdG in the same tissues. Materials and Methods Chemicals.AFB,, DNase,RNase,proteinaseK, H202,polyethylenegly col, and 3-ammnoproplytriethoxysilane werepurchased from SigmaChemical Co. (St. Louis,MO). FCSwasobtained fromSterileSystems (Logan,UT). RPM! 1640wasobtainedfrom ICN Pharmaceutical, Inc. (CostaMesa,CA). DMEM waspurchased fromGIBCO-BRLLife Technologies (Gaithersburg, MD). Permount was purchased from Fisher Scientific (Pittsburgh, PA). Eight chamber slides used here were from Miles Laboratories (Naperville, IL). Woodchuck hepatocytes (WC3) cells were obtained from Dr. C. Rogler (Albert Einstein Medical Center,New York, NY). Mouse ABC and 3,3'- dimethylaminoazobenzene kits wereobtainedfrom VectorLaboratories(Bur lingame, CA). H202 Treatment of 10T½Cells. The 101½cells were maintainedin DMEM mediumcontaining10%FCS.Exponentiallygrowingcellsin 8-cham ber slides were treated with 10—200@LM H2O2 in complete culture medium for 15 mm at 37°C.Exposure was stopped by removing the media and rinsing the cells twice with 120mistNaCI,2.7 mMKC1,1 mMEDTA, and 10mMK3P04 (pH 7.4). The cells werefixed with 75% ethanolat —20°C. AFB1 Treatment of Woodchuck Hepatocytes.WC3 cells werecultured in RPMI 1640containing10%FCS.Cellsweretreatedwith0.4—10@M AFB, in DMSO or DMSO alone for 6 h, washed with PBS twice, and fixed with 70% ethanol. AFB1 Treatment of Rats. Frozen tissuesof 1-year-oldmale Sprague Dawleyratstreatedpreviouslywith 2.5 mg/kgAFB, andsacrificedat 2, 4, 8, 24, and 48 h after treatment were available (16). Tissue samples were isolated 5 mm from the surface, sectioned (5 @m) on a cryostat (Leica, Deerfield, IL), andplacedonglassmicroscopeslidescoatedwith 3-aminopropyl-triethoxysi laneand fixed in 75% ethanolfor 10 mm at —20°C. 683 on March 4, 2016. © 1996 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from