Quantitation of Benzo(A)Pyrene-Deoxyguanosine Adducts by Radioimmunoassay1

[CANCER RESEARCH 40, 412-41 6, February 19801 0008-5472!80!0040-0000$02.0O Quantitation of Benzo(a)pyrene-Deoxyguanosine Adducts by Radioimmunoassay1

Miriam C. Poirier,2 Regina Santella, I. Bernard Weinstein, Dezider Grunberger, and Stuart H. Yuspa

In Vitro Pathogenesis Section, Laboratory of Experimental Pathology, National Cancer Institute, NIH, Bethesda, Maryland 20205 (M. C. P., S. H. Vi, and Division of Environmental Sciences and Cancer Center, Institute of Cancer Research, Columbia University College of Physicians and Surgeons, New York, New York 10032(R. S., I. B. W., 0. G.j

ABSTRACT is formed as a (+) and (â€”),or as a (R) and (S) enantiomer (3, 7, 15). Carcinogenicity studies in newborn and in adult mice Calf thymus DNA was modified with the benzo(a)pyrene (BP) have demonstrated strong carcinogenic potency associated derivative, (Â±)7$,8a-dihydroxy-9a,1 Oa-epoxy-7,8,9, 10-tetra with the (7R)- or the (+)BPDE I (7, 20, 24, 33), while the other hydrobenzo(a)pyrene [(Â±)BPDE I], under conditions which 3 enantiomers were much less carcinogenic. !n vitro, all 4 yielded >99% of the binding product in the form of trans-(7R)- enantiomers can form adducts with guanine and adenine resi N2- {1O[7$,8a, 9a-trihydroxy-7,8,9, 10-tetrahydrobenzo(a)py dues in nucleosides, in oligonucleotides, and in DNA (16, 18, renejyl) deoxyguanosine. Rabbits were immunized with modi 23, 26). When cells (6, 11, 15, 31), explants (1, 17), or tissues fied DNA coupled to methylated bovine serum albumin, and the (22) from either rodents or humans are exposed to the parent resulting antiserum was utilized in a competition radioimmu compound, BP, the major DNA adduct is also (7R)BPDE I-dG, noassay for the quantitation of products of BP covalently bound but there is frequently an additional minor adduct, BPDE II-dG. to DNA. The antiserum was specific for both native and dena A small portion of binding may occur as (7S)BPDE l-dG, and tured immunogen DNA's as well as for the major isolated BP deoxyadenosine adducts have also been detected in vivo (3). binding product, but it did not recognize BP, the tetrol of !n vivo exposure to a (Â±)mixture of BPDE I yields 90 to 95% (Â±)BPDEI, or unmodified deoxyguanosine. The modified DNA of DNA-bound products as trans-(7R)BPDE l-dG, the product was assayed in quantities as low as 2 pmol of adduct, a of the most carcinogenic enantiomer (6, 8). Thus, it now seems sensitivity sufficient to quantitate the extent of modification of apparent that the major pathway of BP activation leading to cellular DNA when epidermal cell cultures were exposed either DNA binding in both rodent and human cells and carcinogen to BP or to (Â±)BPDEI. High-pressure liquid chromatographic esis in rodents is through BPDE I, and that trans-(7R)BPDE I- analysis of DNA hydrolysates, obtained from epidermal cells dG is the major DNA adduct. exposed to BP or to (Â±)BPDEI, indicated that the major adduct Human exposure to BP is ubiquitous and can be as high as was the same as that on the immunogen DNA. This approach 100 ng/cu m in heavily polluted air, 23 ng/cu m in drinking should prove valuable for further studies on the mechanism of water, and 100 @sg/kgin smoked foods (2). Exposures are carcinogenesis and for monitoring human exposure to this particularly high for coke oven workers, gas works operators, ubiquitous carcinogen. and asphalters, whose occupations have been associated with increased cancer risk (14). At 20 to 50 ng/cigarette, smoking INTRODUCTION is also a major source of BP exposure (2, 14). Thus, the elucidation of the specificity of BP metabolism is of more than In recent years, considerable progress has been made in academic interest. Likewise, the development of new, specific, elucidating the specific pathways involved in BP3 metabolism and sensitive techniques to study the interaction of BP with (38), the interaction of BP metabolites with cellular macromol DNA is important both to continue studies at the molecular ecules (35), and the relationship of metabolism to BP-induced level and to detect DNA-bound BP in experimental models or cancer. In particular, the trans-7,8-dihydrodiols of BP have in exposed individuals. been identified as potent DNA-binding and carcinogenic inter Our laboratories have previously developed a RIA for detec mediates when compared to the parent compound (4, 36). tion of the C-8 guanine adduct of the carcinogen 2-acetylami These dihydrodiols undergo additional 9,10-epoxidation in nofluorene (28, 29). The antibody was specific for the adduct, mammalian (including human) cells (32) resulting in 2 diaster recognizing neither guanine nor the carcinogen independently, eomeric forms, BPDE I (anti) and BPDE II (syn), each of which and the RIA was able to detect 0.3 pmol of adduct in DNA. At this level of sensitivity, it has been possible to monitor the in 1 This research was partially supported by National Cancer Institute Grant vivo binding and the removal of adducts by DNA repair mech 2111 to Columbia University and by Contract i CPO2199 to Microbiological Associates, Inc. anisms (28, 29). Such an assay seemed appropriate for per 2Towhomrequestsforreprintsshouldbeaddressed,atBuilding37,Room forming similar studies on BP-modified DNA. This report de 3A19, NIH, Bethesda, Md. 20205. scribes the establishment of a RIA for BP-deoxyguanine ad 3 The abbreviations used are: BP, benzo(a)pyrene; (Â±)BPDE I, (Â±)-7@,8a- dihydroxy-9a, 1Oa-epoxy-7,8,9, 10-tetrahydrobenzo(a)pyrene; (7R) or (7S)BPDE ducts utilizing antiserum from rabbits immunized with BPDE I- l-dG, (7R)- or (75)-M-(i O-[7$,8a,9a-trihydroxy-7,8,9,i 0-tetrahydrobenzo modified calf thymus DNA. (a)pyrenelyl)deoxyguanosine; BPDE Il-dG, N@-{10.(7@,8a,9$-trihydroxy 7,8,9, 1O-tetrahydrobenzo(a)pyrene]yl)deoxyguanosine; RIA, radloimmunoas say; BPDE 1-tetrol, (Â±)7fi,8a,9a,10(a or fl)-tetrahydroxy-7,8,9,1 0-tetra MATERIALS AND METHODS hydrobenzo(a)pyrene; HPLC, high-pressure liquid chromatography; BPDEII, N@ (10-[7$,8a,9fl-trihydroxy-7,8,9,1 0-tetrahydrobenzo(a)pyrene]) ; BPDE I-DNA, Chemicals. (Â±)BPDEI, used for the preparation of immu DNA modified by reaction with (Â±)BPDEIand purified as described In â€œMaterials and Methodsâ€•;AAF,acetylaminofluorene. nogen DNA and nucleoside adducts and for BPDE I-[3H]dG Received September 17, 1979; accepted November 6, 1979. (267 mCi/mmol), was supplied by the National Cancer Institute,

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Standard Chemical Carcinogen Reference Repository, Division for BP adduct formation (2 to 20 @xgDNA)by RIA (28, 29). of Cancer Cause and Prevention, Bethesda, Md. [8-3H]Deoxy Equivalent amounts of DNA from unexposed cells were added guanosine (6.7 Ci/mmol) was purchased from ICN, Irvine, to the standard curve samples as controls. The procedure for Calif.; calf thymus DNA was purchased from Sigma Chemical Si nuclease hydrolysis has been described (37). DNA, isolated Co., St. Louis, Mo.; goat anti-rabbit lgG, methylated bovine and prepared in a similar manner from cells treated with radio serum albumin, and complete and incomplete Freund's adju labeled carcinogen, was incubated with RNase and proteinase vants were purchased from Miles Laboratories, Elkhart, Ind. K and extracted with chloroform-isoamyl alcohol (10) prior to BP and the BPDE I-tetrolwere obtainedfromD. W. McCourtof hydrolysis. Samples were redialyzed, reprecipitated with the Laboratory of Molecular Carcmnogenesis,National Cancer ethanol, hydrolyzed enzymatically (37), chromatographed on Institute, while repurified E3HIBP(27 Ci/mmol) was a gift from LH-20 columns, and identified by HPLC as to the types of Dr. H. Autrup of the Laboratory of Experimental Pathology, nucleoside adducts formed (see â€œImmunizationâ€•). National Cancer Institute. Immunization. Native and denatured calfthymus DNA's were RESULTS modified in vitro with (Â±)BPDE I, to a level of 1.4% (1.4 adducts/1 00 nucleotides) as described previously (18). HPLC RIA and Serum Specificity. When tested by RIA, 2 of 3 profiles (34) (Dupont 850, Waters @sBondapakcolumns,30 to rabbits given injections of denatured BPDE I-DNA, and 1 of 3 60% methanol-water gradient at 50Â°)of the hydrolyzed BP rabbits given injections of native BPDE I-DNA produced high modified DNA (37) indicated that virtually all (99%) of the titer antisera for 4 to 5 months with little increase after boosting modified nucleoside migrated as a single peak cochromato injections at 6 to 7 months. Because high-titer bleeds of all 3 graphing with the known standard, (7R)BPDE I-dG. For immu positive antisera showed superimposable competition curves nization, either native or denatured BPDE I-DNA's were com with either native or denatured BPDE I-DNA, all subsequent plexed electrostatically to equal amounts (w/w) of methylated studies were performed on a single high-titer serum obtained bovineserumalbuminin 0.9% NaCIsolution(27). The BPDE from the same bleeding. Chart 1 (â€” â€”â€”)indicates that, in a l-DNA-methylated bovine serum albumin was emulsified with RIA with this antiserum and BPDE I-[3H]dG, the greatest sen an equal volume of complete Freund's adjuvant, and an amount sitivity (50% inhibition at 5 pmol) was obtained when the equivalent to 1 mg of BP-modified DNA was injected i.m. into original immunogen, BPDE I-DNA (modified to 1.4%), was used the hindquarters of New Zealand White rabbits. Four initial as an unlabeled competitor. After denaturation and hydrolysis injections, one week apart, were followed by 5 booster injec with S1nuclease, the BPDE I-DNA was a less efficient compet tions at monthly intervals. Each rabbit received a total of 75 itor (50% inhibition at 15 pmol) when assayed by RIA. The 1sgof(7R)BPDE I-dG. Rabbitswere bled from the ear veins at authentic deoxyguanosine adducts, (7R)BPDE I-dG or BPDE regular intervals beginning 8 weeks after the first injection. II-dG, were both recognized equally, giving 50% inhibition at RIA. The radioactiveproductusedfor RIA, (7RS)BPDE I-[3H] about 40 pmol (Chart 1, 0â€”â€”â€”0,@â€”â€”â€”a).The following dG, was synthesizedby reacting[3H@deoxyguanosmne(6.7Ci/ compounds showed no competition with BPDE l-[3H]dG in the mmol) and (Â±)BPDEI in dimethylformamide (26). BPDE II-dG RIA: 5 to 22 ,sgnative or denatured unmodified DNA; 17 to 35 was synthesized similarly with a nonradioactive deoxyguano ,Lgdenatured and S1-hydrolyzed unmodified DNA; 300 to 3000 sine. [3HJDNAwas synthesized by nicked translation of calf pmol deoxyguanosine; 60 to 3000 pmol BPDE l-tetrol; and 100 thymus DNA in the presence of Escherichia CO!i,DNA polym to 1000 pmol N-(8)-deoxyguanosmnylacetylammnofluorene erase I and dCTP, dTTP, dATP, and [3H]dGTP(specific activity, (Chart 1, plus additional data not shown here). Also, when 10 33 Ci/mmol) (21). [3H]DNAwas subsequentlymodified to a @tgDNAand 5.5 ,zgBP were added simultaneously to the assay level of 1.4% with (Â±)BPDEI (see â€˜â€˜Immunization'â€˜).Proce tubes, no competition was observed. The addition of up to 22 dures for RIA were those described previously (29). The RIA used 0.1 ml (10,000 to 15,000 cpm) BPDE l-[3H]dG or BP-[3H] DNA, 0.1 ml antiserum(diluted 1:200), 0.1 ml nonradioactive 90 ,-( A BPDE I-DNA or unknownDNA, and 0.1 mlgoat anti-rabbitIgG. 80 _. â€œ/ ,I Allcomponentswere dilutedin 0.01 m Tris-HCIbuffer,pH 7.3, 70 I $ with the exception of BPDE I-[3H]dG which was in the same II @60 II buffer plus 20% ethanol to facilitate solubility. Non-equilibrium E II conditions (28, 29) were used routinely to maximize sensitivity. @50 z 1/ In the absence of cold competitor (0% inhibition), the immu @40 I, noprecipitate contained approximately 2500 cpm after sub Al traction of nonspecific background counts. Ig 70 @ C&l Culture, Carcinogen Treatment, and DNA Preparation. 114' 10 BALB/c epidermal cells were prepared from newborn mice 0'

(39) and were plated(2.5 x 10@cells)in 150-mm dishes.Cells 100 101 102 1t@1 @â€œI@ were exposed to (Â±)BPDEI (48 hr after plating) or to BP (24 PMOI hr after plating) in 0.8% dimethyl sulfoxide for 1 or 24 hr (37Â°), Chart 1. RIA standard curves in which the binding of BPDE I-(3HIdG(10,000 cpm) to specific antiserum (dIluted 1:200) was measured in the presence of respectively. After cells were harvested by scraping, DNA was increasing amounts of: the Immunogen BPDE I-DNA (â€”â€”-4); (7R)BPDE I-dG isolated and purified by CsCI buoyant density centrifugation, (0- - -0); BPDEll-dG(tx- - -is); unmodifiedDNA(Oâ€”O); deoxyguanosine and the pooled peaks were dialyzed (0.01 m Tris-HCI, pH 7.3) (L@â€”L@);BPDE1-tetrol (â€¢ â€¢);orN-8-deoxyguanosinyl-2-acetylaminofluo rene (Aâ€”A). Both native and denatured BPDE I-DNA gave identical results. and quantitated by A2se(25). Dialyzed DNA samples were Ordinate, percentage of inhibition obtained with each of the latter materials; concentrated by nitrogen evaporation, denatured, and assayed abscissa, pmol of each material added per assay.

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ftg of denatured unmodified DNA to each concentration of the 70@ BPDE I-DNA standard curve shown in Chart 1 did not change the inhibition curve, but larger amounts of unmodified DNA 60@ (>26 @sg)produced additional inhibition. These results sug gested that, although the antiserum preferentially recognized 50@ the BPDE-deoxyguanosmneadducts in DNA, there might also z 0 be some antibody specificity towards unmodified DNA. This E40 would not be surprising since intact DNA was used as the 80 immunogen and anti-DNA antibodies are readily made in rab bits (27). The presence of anti-DNA activity was apparent when BP 20 [3H]DNA rather than BPDE l-[3H]dG was utilized in the RIA. / Under these conditions, the nonradioactive BPDE I-DNA was assayed with the greatest sensitivity (50% inhibition at 1 pmol 10 adduct, 22 ng DNA). However, denatured unmodified DNA was @ also recognized, although only at a much higher concentration 5 iO 50 (40% inhibition at 350 ng DNA). Multiple absorptions of the f19 DNA Chart 2. RIA of mouse epidermal DNA obtained from cells exposed to 3.3 x antiserum with native and denatured DNA, followed by removal 1o-@m BPDE I for 1 hr. Conditions of the assay were: 10,000 cpm BPDE I-(3H1 of DNA by DEAE-cellulose, reduced the anti-DNA activity. dG; specifIc antiserum diluted 1:200; and 5 to 22 pg of either denatured However, repeated absorptions resulted in partial loss of both unmodified DNA (Oâ€”O) or mouse epidermal DNA from cells exposed to (Â±)BPDEI(Aâ€”A). [A standardcurvewithtubescontainingimmunogenDNA, activities rather than a separation of the two. similar to Fig. 1 (â€”â€”-4), and an appropriate amount of control DNA were run Assay of Modified DNA after Exposure of Cultured Cells separately to determine the levels of modification given in Table 1.@ to BPDE I or to BP. When primary mouse epidermal cells were exposed to 3.3 x 10_6 m (Â±)BPDEI for 1 hr and cellular DNA Binding of the parent hydrocarbon BP to epidermal cell DNA was isolated, purified, and assayed by RIA, binding was readily was also studied by RIA and HPLC. Mouse epidermal cells detectable (Table 1). DNA isolated under similar conditions were exposed to 4 x 10@ m BP in 0.8% dimethyl sulfoxide for from solvent-treated cultures failed to react in the RIA (using 24 hr, a time shown previously to yield maximum binding of BPDE l-[3HJdG). Increasing amounts of each modified DNA polycyclic hydrocarbons in mouse epidermal cells.4 Again, gave linear increases in percentage of inhibition in the RIA, binding to DNA was detectable by RIA, although the values while similar amounts of control DNA gave no inhibition (Chart were lower than those obtained from cultures incubated with 2). When DNA samples were obtained from cultures incubated (Â±)BPDEI. The BP binding values approached the lower limits with (Â±)BPDEI for 1 hr and then continued in culture for an of sensitivity of the RIA (Table 1). HPLC analysis was also additional 23 hr in the absence of (Â±)BPDEI, approximately performed on the DNA obtained from cultures exposed to 5 40% fewer adducts were measured at the later time point, x 10-Â°m [3H]BP for 24 hr (Chart 38). A major peak (about suggesting excision by a DNA repair process. Identification of 80% of the DNA-associated radioactivity) which cochromato the specific DNA adducts was made by HPLC analysis of graphed with the (7R)BPDE I-dG marker and a minor peak enzymatically hydrolyzed DNA from mouse epidermal cells (about 10%) which corresponded to (7S)BPDE I-dG were de treated with [3HJ(Â±)BPDEI(3.3 x 10_6 m) for 1 hr. The major tected. In addition, a minor peak (about 10%) eluting in Fraction peak detected (Chart 3A) accounted for 85% of the radioactiv 69 corresponded with the elution position of a BPDE II-dG ity bound to DNA. It chromatographed in the same position as adduct (6, 15, 18). The above HPLC analyses are consistent did marker (7R)BPDE l-dG. A minor peak, accounting for about with previous data indicating that, in vivo, there is preferential 10% of the radioactivity, eluted with authentic (7S)BPDE I-dG synthesis and DNA binding of (7R)BPDE I but that small (Chart 3A). Two very small peaks eluted later; each accounted amounts of (7S)BPDE I and BPDE II also react with DNA (6, for about 2% of the radioactivity and corresponded to deoxy 15, 16). adenosine adducts (Chart 3A). DISCUSSION Table 1 DNAMouseFormation of BPdG adducts in mouse epidermal cell We reported previously the development of specific antise!a whichtime epidermal cell cultures were exposed to (Â±)BPDEI for 1 hr at and an RIA for the detection and the quantitation of DNA solutionandcells were either harvested or washed 3 times with sterIle 0.9% NaCI adducts formed by the aromatic amine carcinogen AAF (28, incubated at 37Â°for another 23 hr. DNA's were prepared by CsCI centrifu @29).Subsequentreports have also described antisera to AAF gation,Samples(2 and the amount of DNA was determined by A2@after dialysis. werequantitatedto 20 @LgDNA)were assayedby AlA with BPDE I.{3H]dG,and adducts modified DNA (12, 30). The present study describes antisera by simultaneous assay with a standard BPDEI-DNA.fmol which detect the major deoxyguanosine adducts formed in a adduct!g@gExpo- BPdG number of cells and tissues exposed to the ubiquitous environ DNAsureCompound mental carcinogen, BP. Previous studies have demonstrated hr(Â±)BPDE Concentration(m) time (hr) i hr 24 the immunogenic properties of free polycyclic aromatic hydro I 3.3ND@'3.3 x i0' 1 335a carbons (9, 13, 19); our results demonstrate the immunogen 6BP x 10@ 1 532 Â±47 323 Â± icity of a DNA adduct formed by the anti isomer of the diol 4.OxiO' 24 ND 132Â±47 epoxide derivative of BP. Our BPDE I-dG antiserum is highly a Mean Â± range for duplicate or triplicate experiments; each value was specific, recognizing neither the free hydrocarbon nor the obtained by pooling DNA from four to five 150-mm dishes of cells. ND, not determined. 4 P. E. Dermer and S. H. Yuspa, unpublished observatIons.

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Chart 3. Cultured primary mouse epidermal cells were exposed to 3.3 X 1CT6 M [3HKÂ±)BPDE I for 1 hr (A) or 5 x 1CT6 M [3H]BP for 24 hr (B). DNA was isolated and purified on CsCI gradients and hydrolyzed, and the individual adducts were separated and identi fied by HPLC. Arrow, position of the (7FOBPDE l-dG marker; peak to the left of (7R)BPDE l-dG (about Fraction 56), (7S)BPDE l-dG; peak to the right. B (Fraction 69), BPDE ll-dG. The small peaks at LB Fraction 5 and between Fractions 75 and 85 (A and 8) have not 6 - been identified.

10 20 30 40 50 70 80 90

FRACTION NUMBERS unmodified dG. Adducts of dG from both BPDE I and II are cullures exposed to (Â±)BPDE I conlained about 4.7 adducts/ recognized by the antiserum suggesting that stereospecificities 107 dallons DNA (calculated from Table 1). This value is in the of the 7,8- and 9-hydroxyls do not play a major role as antigenic same range as that obtained in previous studies in which Ihe determinants. It is likely, therefore, that all 4 possible enantio- exlenl of covalenl binding lo DNA was measured by radioactiv meric forms of BPDE-dG adducts (18) can be detected with ity in mouse epidermal cells exposed to Ihe same concenlration this antiserum. Thus far, we have had insufficient amounts of of eilher [3H] or [14C](Â±)BPDE I (5). It is also of inlerest to noie the BPDE-deoxyadenosine and the deoxycytosine adducts to lhal Ihe HPLC profiles of DNA adducts in mouse epidermal determine if our antisera cross-react with these adducts. cultures exposed to either BP or (Â±)BPDE I were quile similar The specificity of our antiserum is of particular interest since and lhal Ihe adducls formed in epidermal cell cullure corre the immunogen was BPDE I-DNA rather than the isolated sponded wilh Ihose formed in mouse skin in vivo (22). Since deoxyguanosine adduci. This suggests that the BPDE-dG ad Ihe major adducls are BPDE-dG producÃs, il seems likely that ducts were in a configuration accessible to the immune system, our RIA detects most of Ihe covalenlly bound DNA products which might be expected if the BP moiety is situated in the resulting from in vivo exposure lo BP. minor groove of the DNA helix (35). Accessibility is also sug The data described in the present paper, together with our gested by the production of apparently identical antisera when previous studies (28, 29), demonstrate the feasibility of devel native or denatured BPDE I-DNA was used as an immunogen. oping highly sensitive and highly specific RIA's for the detection However, we cannot rule out the possibility that host nucleases and the quanlitation of specific carcinogen-DNA adducts. This digested the injected material yielding adducts or modified technique is providing a valuable tool for sludies on the inter oligonucleotides that also served as an immunogen. The fact action of carcinogens and DNA. It also provides a novel ap that intact BPDE-DNA reacted even better than free (7R)BPDE proach to monitoring a biological consequence of human ex l-dG in the RIA supports the interpretation that the adduci is posure to specific chemical carcinogens through assay of DNA accessible in intact DNA and that there were configurational from cells and from tissues of exposed individuals. elements conlribuled by the DNA (e.g., adjacent nucleotides or spatial arrangement) during the process of antibody forma ACKNOWLEDGMENTS tion. The laller resulls are in conlrasl to Ihose we oblained wilh antibodies lo A/-8-guanyl-2-acelylaminofluorene (28, 29), in Our appreciation is extended to Bob Shorr for the care and bleeding of the which nalive and denatured modified DNA's reacted less effi rabbits and to Curt Thill and Gregory Canute for their technical assistance. ciently with the antiserum than did the hydrolyzed AAF-modified DNA. Presumably, these differences can be explained REFERENCES partially by Ihe faci lhal, in the AAF sludies, Ihe immunogen 1. Autrup, H. A., Harris, C. C., Trump, B. F., and Jeffrey. A. M. Metabolism of was Ihe isolaled adduci ralher lhan carcinogen-modified DNA. benzo(a)pyrene by cultured human colon. Cancer Res., 38. 3689-3696, Additional studies with Ihese antisera may be useful for further 1978. 2. Baum, E. J. Occurrence and surveillance of polycyclic aromatic hydrocar clarification of conformational differences. bons. In: H. V. Gelboin and P. O. P. Ts'o (eds.), Polycyclic Hydrocarbons The presenl sludies indicate that RIA can be useful for and Cancer, Vol. 1, pp. 45-70. New York: Academic Press, Inc.. 1978. detecting and for quanlilaling the in vivo carcinogen-DNA 3. Beland, F. A., and Harvey, R. G. The isomerie 9,10-oxides of frans-7,8- dihydroxy-7,8-dihydrobenzo[a]pyrene. J. Chem. Soc. Chem. Commun.. 84, binding resulting from the exposure of mouse epidermal cells 1976. to (Â±)BPDE I or to BP. With Ihe RIA, we found lhal Ihe DNA of 4. Borgen, A., Darvey, H., Castagnoli, N., Crocker. T. T., Rasmussen. R. E.,

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416 CANCERRESEARCHVOL. 40

Miriam C. Poirier, Regina Santella, I. Bernard Weinstein, et al.

Cancer Res 1980;40:412-416.

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