Correlation between DNA or protein adducts and benzo[a]pyrene diol epoxide I-triglyceride adduct detected in vitro and in vivo

In this study, we demonstrated the in vitro and in vivo formation of carcinogen-lipid adduct and its correlation with DNA or protein adducts. The lipids from serum or hepatocyte membranes of Sprague-Dawley rats, human serum and standard major lipids were in vitro reacted with benzo[a]pyrene (B[a]P) and B[a]P metabolites. 7, 8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE-I), an ultimate carcinogenic form of B[a]P, was covalently bound to triglyceride (TG). BPDE-I-TG adducts isolated by thin-layer chromatography (TLC) were further detected by high-performance liquid chromatography. TGs, including triolein, tripalmitin and tristearin, showed positive reactions with BPDE-I. However, cholesterol, phospholipids (phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidyl-inositol and sphingomyelin) and non-esterified fatty acids (palmitic acid, oleic acid, linoleic acid and stearic acid) did not react with BPDE-I. In addition, other B[a]P metabolites (B[a]P-phenols and -diols) did not react with TG. TG appeared to be the most reactive lipid yet studied with respect to its ability to form an adduct with BPDE-I. There was a clear-cut dose-related formation of [1,3-(3)H]BPDE-I-lipid adducts in vitro between TG and [1,3-(3)H]BPDE-I. In an animal study, BPDE-I-TG was also formed in the serum of rats orally treated with B[a]P (25 mg/rat). Also, obvious correlations between [(3)H]B[a]P related-biomolecule adducts (DNA or protein) or lipid damage and the BPDE-I-TG adducts were obtained in various tissues of mice i.p. treated with [(3)H]B[a]P. These data suggest that TG can form an adduct with BPDE-I, as do other macromolecules (DNA, RNA and protein). Therefore, a carcinogen-lipid adduct would be a useful biomarker for chemical carcinogenesis research and cancer risk assessment.     

Monoclonal antibodies to DNA modified by a benzo[a]pyrene diol epoxide

Monoclonal antibodies were obtained after fusion of mouse P3x63Ag8.653 myeloma cells with spleen cells isolated from BALB/cCrmice immunized with either DNA modified by 7ß, 8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (BPDE-I-DNA) complexedelectrostatically to methylated bovine serum albumin or withBPDE-I modified guanosine conjugated with bovine serum albumin,BPDE-I-G-BSA. One monoclonal hybridoma line from each type ofimmunization was grown as ascites tumors or in defined mediaand characterized in an enzyme linked immunosorbent assay (ELISA).The antibody produced from the spleen cells of a BPDE-I-DNAimmunized mouse, designated 5D11, recognizes BPDE-I-DNA andDNA modified by 7ß, 8-dihydroxy-9ß, 10ß-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE II) but not unmodified DNA,N-2-acetylamino-fluorene (AAF) or 1-nitropyrene (NP) modifiedDNA, BPDE-II-dG or BPDE-I tetraol. It does recognize BPDE-I-dGbut with a much lower affinity than when the adduct is presentin DNA. In contrast, antibody 8E11 produced from the spleencells of a BPDE-I-G-BSA immunized mouse recognizes the monoadductBPDE-I-dG better than BPDE-I-DNA. It also recognizes BPDE-Itetraol but not BPDE-II-DNA, unmodified DNA, AAF- or NP-DNAor BPDE-II-dG. In a noncompetitive ELISA as little as 3 fmolof BPDE-I-DNA adduct can be detected with either antibody 5D11or 8E11. The combination of the highly sensitive ELISA and highlyspecific monoclonal antibodies should be valuable in the detectionand quantitation of human exposure to benzo-[a]pyrene.     

Synthesis and characterization of covalent adducts derived from the binding of benzo[a]pyrene diol epoxide to a -GGG- sequence in a deoxyoligonucleotide

Direct synthesis and purification procedures are described forthe preparation of adducts derived from the covalent bindingof 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene[(+)-ante-BPDE or (+)-BPDE 2] to each of the three guanine residues(trans-N² lesions) in the oligodeoxyribonucleotide d(CTATG¹G²G³TATC)The positions of the modified Gs are defined by Maxam-Gilbertsequencing techniques. Six different oligonucleotides with oneor two precisely positioned (+)-anti-BPDE residues are identified.The absorbance, circular dichroism and fluorescence characteristicsare changed upon formation of duplexes with the complementarystrands d(GATACCCATAG). In the doubly-modified oligonucleotides,a broad, excimer-like long wavelength fluorescence emissionband is observed with a maximum near 455 nm only if the two(+)-anti-BPDE-modified Gs are adjacent to one another. The covalentlyattached (+)-anti-BPDE residues decrease the thermodynamic stabilitiesof the duplexes; their melting points are markedly dependenton the position of the lesions, being highest with the (+)-anti-BPDEresidue at G¹ (T^m=40°C, only 2°C lower than in the caseof the unmodified oligonucleotide) and lowest when it is situatedat G³ (T^m=29°C). The implications of these and other physicalcharacteristics are discussed. The facile synthesis of theseor similar site-specific and stereochemically defined (+)-trans-anti-BPDE-N²-dGlesions in runs of contiguous guanines in oligodeoxyribonucleotidesof specified base sequence should be useful for the design ofsite-directed mutagenesis studies in vitro and in vivo.     

Concentration dependent alterations of DNA replication initiation and elongation by benzo[a]pyrene diol epoxide

Vero cells treated with various concentrations of (±)7ß,8dihydroxy-9, 10-epoxy, 7,8,9,10-dihydrobenzo[a]pyrene (BP-diolepoxide I) exhibited dose-dependent inhibition in both the rateof DNA synthesis and in the size of nascent DNA. The maximuminhibition was seen 2–3 h after addition of BP-diol epoxideI. A recovery in both the rate of synthesis and size of nascentDNA was observed 5–10 h after treatment. The pH step alkalineelution assay which separates different nascent DNA replicationintermediates was used to investigate whether the inhibitionand recovery noted above could be accounted for by alterationsin DNA replication initiation (DNA synthesis within a replicon)or elongation (rejoining of replicons). At lowest dose studied(0.166 µM BPdiol epoxide I) a reversible inhibition inDNA initiation was observed. At the higher dose levels (0.66µM and 1.66 µM BPdiol epoxide I) inhibition of bothDNA initiation and elongation were observed and inhibition ofelongation predominated. The inhibition in elongation was detectedby an increase in the relative amount of low molecular weightnascent DNA associated with DNA synthesis within a repliconand a relative decrease in the higher molecular weight elongatedDNA. A reversal in the inhibition of both initiation and elongationwas noted.     

Presence of benzo[a]pyrene diol epoxide adducts in target DNA leads to an increase in UV-induced DNA single strand breaks and supF gene mutations

Exposure to DNA damaging agents and mutagens often occurs as combinations of agents, or as complex mixtures of chemicals. We found that plasmid DNA adducted with benzo[a]pyrene diol epoxide (BPDE) was more susceptible to UV-induced single strand breaks than was control DNA. To determine whether the increase in DNA damage also applied to mutagenic lesions, the supF gene forward mutation assay was used to compare mutations induced by BPDE alone, UVB, UVC, BPDE followed by UVB and BPDE followed by UVC. It was found that the mutation frequency for BPDE + UVB (1167 in 10(4) transformants) was higher than BPDE alone (12 in 10(4) transformants) or UVB alone (446 in 10(4) transformants), and the mutation frequency for BPDE + UVC (197 in 10(4) transformants) was higher than BPDE alone or UVC alone (26 in 10(4) transformants). For BPDE + UVB and BPDE + UVC there was a significant increase in plasmids with multiple mutations. Whilst these indicate error prone repair due to the single strand breaks, the different mutation frequencies in plasmids treated to give similar levels of strand breaks suggest other mechanisms for the mutations in plasmids with single mutation events. The spectrum of non-multiple mutations in the two combined treatments included both UV signature mutations (GC-->AT as the most common mutation) and BPDE signature mutations (GC-->TA and GC-->CG as the most common mutations). However, the increase in absolute mutation frequency of BPDE signature mutations between BPDE treatment and BPDE + UV treatment was greater than the increase in absolute mutation frequency of UV signature mutations, even though the level of BPDE adducts was identical in each case. These results suggest two possibilities: (i) the BPDE adducts are photoactivated to a more mutagenic lesion, or (ii) the presence of UV lesions lead to the BPDE adducts becoming more mutagenic.     

Myeloperoxidase--463A variant reduces benzo[a]pyrene diol epoxide DNA adducts in skin of coal tar treated patients.

The skin of atopic dermatitis patients provides an excellent model to study the role of inflammation in benzo[a]pyrene (BaP) activation, since these individuals are often topically treated with ointments containing high concentrations of BaP. In this study we have determined, by HPLC with fluorescence detection, the BaP diol epoxide (BPDE)-DNA adduct levels in human skin after topical treatment with coal tar and their modulation by the -463G-->A myeloperoxidase (MPO) polymorphism, which reduces MPO mRNA expression. BPDE-DNA adduct levels were 2.2 and 14.2 adducts/10(8) nt for MPO-463AA/AG and -463GG, respectively. The predominant BaP tetrol observed was tetrol I-1, which is derived after hydrolysis of the anti-BPDE-DNA adduct. The tetrol I-1/II-2 ratio, corresponding to the anti/syn ratio, was 6.7. The (32)P-post-labeling assay was also performed and thin layer chromatograms showed a major spot with a chromatographic location corresponding to BPDE-DNA. The mean values of the BPDE-DNA adduct spots were 3.8 +/- 2.4 per 10(8) nt for MPO-463AA/AG (n = 3) and 18.4 +/- 11.0 per 10(8) nt for MPO-463GG (n = 7), respectively (P = 0.03). One individual with the homozygous mutant genotype (-463AA) even had a 13-fold lower adduct level (1.4 per 10(8) nt) as compared to MPO-463GG subjects. In conclusion, these data show for the first time: (i) the in vivo formation of BPDE-DNA adducts in human skin treated with coal tar; (ii) that the MPO-463AA/AG genotype reduced BPDE-DNA adduct levels in human skin.     

Properties of covalent benzo[a] pyrene diol eporide-DNA adducts investigated by fluorescence techniques

The spectroscopic absorption and fluorescence properties ofadducts derived from the covalent binding of (±)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,l0-tetrahydrobenzo[a]pymne(BPDE) to DNA are re-examined in view of conflicting interpretationsregarding the Coaformations of these adducts which currentlyexist in the literature. The fluorescence decay profiles wereaccurately determined utilizing synchrotron-pulsed light sourceexcitation and the time-correlated single photon counting technique.The coaformational properties of the adducts were probed bydetermining their accessibilities to acrylamide, a known fluorescencequencher, and by comparing the accessibilities of the BPDE-DNAadducts with those of known model systems with intercalative,partially intercalative and minor groove binding conformations.In contrast toanyofthesemodelsystems,tkfhmesmmofthearomahicpyrenyl residues in the covalent BPDE - DNA ad- exhibit significantsensitivity to acrylamide, suggesting that these residues arelocated at binding sites with significant solvent exposure.A quantitative analysis of the acrylamide fluorescence quenchingaccording to a dynamic Stem—Volmer quenching model suggeststhe following characteristics: the major (65%) component (1.4ns lifetime) is characterized by significant exposure to thesotvent environment; the second component (6–7 ns lifetime)can be subdivided into a solvent-accessible and a solvent-inaccessiblecomponent, the inaccessible fraction being attributed to minoradducts, possibly with a quasi-intercalative conformation. Theamplitude of the third, long-lived (200-ns) component is variable;it arises from the photochemical decomposition of the adductswhich gives rise to tetraols (7,8,9,1O-tetrahydro-tetrahydroxybenzo[a]pyrene).The variable content of these degradation products accountsfor most discrepancies in the fluorescence properties of thecovalent BPDE-DNA adducts previously reported.     

Enhancement of benzo[a]pyrene diol epoxide mutagenicity by sulfite in a mammalian test system

Sulfur dioxide, a ubiquitous air pollutant, is a co-carcinogenfor benzo[a]pyrene (BP). We have demonstrated previously thatthe interaction between sulfite, the physiological form of sulfurdioxide, and (±)-7r, 8t-dihydroxy-9t, 10t-epoxy-7, 8,9, 10-tetrahydrobenzo[a]pyrene (anti-BPDE), the ultimate carcinogenicform of BP, results in an enhanced mutagenic effect in Salmonellatyphimurium strains TA98 and TA100. We report here that thissame co-mutagenic effect of sulfite occurs in a mammalian cellline. Treatment of Chinese hamster V79 cells with 50 nM anti-BPDE,a concentration on the linear portion of the dose-response,resulted in a four-fold increase in mutations at the hprt locusrelative to the spontaneous rate. When V79 cells were exposedto 1 or 10 mM sulfite immediately prior to the addition of anti-BPDE,the mutation rate increased by 73% and 210%, respectively, overthat elicited by anti-BPDE alone. Sulfite itself was moderatelycytotoxic, but caused no increase in mutation over the spontaneousrate. Characterization of the dose- and time-dependance of thisenhancement of diol epoxide mutagenicity by sulfite closelyresembled the effects seen previously in the bacterial system.In particular, enhancement by sulfite was evident when sulfitewas added to the cells between 60 min and 1 min prior to theaddition of the diol epoxide. Concurrent addition of sulfiteand the diol epoxide attenuated the enhancement, and the effectwas lost altogether when sulfite was added 10 min after thediol epoxide. The specificity of this effect of sulfite wasshown by comparison with sulfate, which at concentrations ofeither 1 or 10 mM exhibited modest cytotoxicity, but neitherwas directly mutagenic nor able to enhance the mutagenic effectof anti-BPDE. Binding studies with labeled anti-BPDE showedthat the addition of 10 mM sulfite increased binding of anti-BPDEto DNA by over 43%, corresponding to the observed increase inmutant frequency. Interestingly, this difference in level ofDNA modification was not apparent after 30 min to 2 h exposures,but only emerged at the 4 h time point. The 4 h point was routinelyused for all mutagenicity studies. Binding of anti-BPDE-derivedmaterials to cellular RNA was not altered by 10 mM sulfite.The emergence of increased DNA modification at the latest timepoint suggests either a more prolonged period of active DNAbinding than would occur with diol epoxide, or a differencein the ability to recognize and clear specific DNA adducts.Both possibilities are discussed in regard to the observed formationof 7r,8t,9t-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene- lOc-sulfonate(BPT-10-sulfonate) in those incubations. BPT-10-sulfonate isa relatively stable BP derivative which retains the abilityto covalently modify DNA. The role of this derivative in theenhancement of diol epoxide mutagenicity by sulfite is stronglysuggested by these data.     

In vitro benzo[a]pyrene diol epoxide-induced DNA adducts and risk of squamous cell carcinoma of head and neck

In this large confirmatory study of 803 patients with squamous cell carcinoma of head and neck (SCCHN) and 839 controls frequency matched by age, sex, and ethnicity, we further examined potential predictors of benzo[a]pyrene diol epoxide (BPDE)-induced adduct levels and their associations with SCCHN risk. BPDE-DNA adduct levels were determined by the (32)P-postlabeling method in peripheral lymphocytes after in vitro challenged by BPDE. We also genotyped for GSTM1 null, GSTT1 null, GSTP1 Ile(105)Val, and GSTP1 Ala(114)Val. Potential predictors of BPDE-DNA adducts were evaluated by stratification and multivariate linear regression analyses and the association between adduct levels and SCCHN risk by multivariate logistic regression analyses. We found that mean BPDE-DNA adduct levels (the relative adduct labeling x 10(7) +/- SD) were significantly higher in cases (77.6 +/- 111.8) than in controls (57.3 +/- 98.3; P < 0.001). Using the median control value (29.22) as a cutoff, 63% of the cases were distributed above this level (adjusted odds ratio, 1.71; 95% confidence interval, 1.39-2.10). A significant dose-response relationship was observed between adduct quartiles and SCCHN risk (P(trend) < 0.001). Multivariate linear regression analysis revealed that ethnicity and smoking were significant predictors of BPDE-DNA adduct levels in controls. In conclusion, we confirmed the previously reported association between in vitro BPDE-induced DNA adduct levels and SCCHN risk, and the assay may help identify individuals at high risk of developing smoking-related cancers.     

Inhibition of DNA synthesis, independent of DNA adduct formation, by benzo[a]pyrene diol epoxide in mammalian cells

Treatment of SV40-infected CV-1 cells with the ultimate carcinogenanti-benzo[a]pyrene diol epoxide (BPDE) at 1 x 10 ^–4 mg/mlor higher reduced the rate of viral DNA synthesis to an extentdependent on the BPDE concentration; similar reductions in cellularDNA synthesis were produced in infected and uninfected cells.Treatment of cells with BPDE, followed by removal of BPDE, atvarious times before infection with SV40 gave the same results.Recovery or partial recovery of DNA synthesis occurred whenthe BPDE concentration was below 6 x 10 ^–4 mg/ml; at higherconcentrations the cells were killed. Simultaneously replicatingviral DNA's from viruses infecting the same cells before andafter BPDE treatment of the cells exhibited the same reducedrate of synthesis. The evidence indicates that covalent adductsin viral DNA are not responsible for its reduced replicationrate; moreover, it is probable that an insignificant numberof adducts is produced in intracellular viral DNA at BPDE concentrationsthat do not kill CV-1 cells. Rather, it appears likely thatBPDE inhibits viral DNA synthesis by attacking cellular DNAor non-DNA targets. Caution is therefore required in relatingthe effects of BPDE and other carcinogens to DNA adduct formation.     

High mobility group 1 and 2 proteins bind preferentially to DNA that contains bulky adducts induced by benzo[a]pyrene diol epoxide and N-acetoxy-acetylaminofluorene

High mobility group (HMG) proteins 1 and 2 are abundant non-histone chromosomal proteins that bind preferentially DNA that is bent or underwound. Previous studies have shown that these proteins preferentially bind to DNA damaged by the crosslinking agents cis-diammine-dichloro-platinum(II), chromium(III) and UV-C radiation. Here we have studied the binding of HMG-1/2 proteins to a duplex oligonucleotide damaged by benzo(a)pyrene diol epoxide or N-acetoxy-acetylaminofluorene using an electrophoretic mobility shift assay. Both chemicals induce monoadducts that are known to distort DNA structure. The affinities of HMG-1/2 for DNA damaged by benzo[a]pyrene diol epoxide or N-acetoxy-acetylaminofluorene were similar to that for UV-irradiated DNA, which were an order of magnitude higher than for undamaged DNA. In contrast, DNA modified by dimethyl sulfate was not preferentially recognised by HMG-1/2.     

Base specificity in the binding of benzo[a]pyrene diol epoxide to simian virus 40 DNA.

[14C]Simian virus 40 (SV40) DNA was reacted with [3H]7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to give 0.60 adducts per genome. The modified DNA was digested to completion with Hind III restriction endonuclease and the 6 fragments isolated by polyacrylamide gel electrophoresis. Hydrocarbon binding to the fragments was proportional to their guanine--cytosine (G--C) content, reflecting selective reaction of the hydrocarbon with deoxyguanosine residues. No sites unusually susceptible to alkylation were detected.     

Induction of cyclooxygenase-2 by benzo[a]pyrene diol epoxide through inhibition of retinoic acid receptor-beta 2 expression

Benzo[a]pyrene diol epoxide (BPDE, a carcinogen present in tobacco smoke and environmental pollution) has been shown to suppress retinoic acid receptor-beta2 (RAR-beta(2)) and induce cyclooxygenase-2 (COX-2) expression. Restoration of RAR-beta(2) inhibited growth and colony formation of esophageal cancer cells, which was correlated with COX-2 suppression. In this study, we investigated the molecular mechanisms for RAR-beta(2)-mediated suppression of COX-2 expression using BPDE as a tool. We found that BPDE-induced COX-2 expression was through inhibition of RAR-beta(2) and consequently, induction of epidermal growth factor receptor (EGFR), extracellular signal-regulated protein kinases 1/2 (Erk1/2) phosphorylation, and c-Jun expression. Esophageal cancer cells that do not express RAR-beta(2) did not respond to BPDE for induction of COX-2. BPDE was also unable to induce COX-2 expression after RAR-beta(2) expression was manipulated in these esophageal cancer cells. Furthermore, BPDE induced time-dependent methylation of RAR-beta(2) gene promoter in esophageal cancer cells. Transfection of RAR-beta(2) expression vector into esophageal cancer cells suppressed expression of EGFR, Erk1/2 phosphorylation, c-Jun, and COX-2. In addition, co-treatment of RAR-beta(2)-positive cells with BPDE and the MEK1/2 inhibitor U0126 caused little change in c-Jun and COX-2 expression. This study demonstrated that BPDE-suppressed expression of RAR-beta(2) results in COX-2 induction and restoration of RAR-beta(2) expression reduces COX-2 protein in esophageal cancer cells, thereby further supporting our previous finding that RAR-beta(2) plays an important role in suppressing esophageal carcinogenesis.     

Synchronous fluorescence spectroscopic, immunoaffinity chromatographic and 32P-postlabeling analysis of human placental DNA known to contain benzo[a]pyrene diol epoxide adducts

Human placenta readily catalyzes the biotransformation of polycyclic aromatic hydrocarbons (PAHs) and other carcinogens to reactive metabolites that can damage DNA through formation of covalent adducts. Placenta is widely available for epidemiologic studies and may be a useful dosimeter for carcinogen exposures in humans. However, previous studies of human placental DNA have yielded discrepant results with respect to PAH-DNA adducts. In order to resolve some of the issues surrounding these discrepancies, placental DNA samples known to contain benzo[a]pyrene diol epoxide adducts were also analyzed by 32P-postlabeling and immunoaffinity chromatography. Results indicate that previous discrepancies can be accounted for by methodologic factors affecting the specificities of adduct assays in biological samples and suggest that human placental DNA contains adducts derived from multiple PAHs.     

Reduced DNA repair of benzo[a]pyrene diol epoxide-induced adducts and common XPD polymorphisms in breast cancer patients

Environmental chemicals are thought to play a role in the etiology of breast cancer, because polycyclic acromatic hydrocarbon (PAH)-DNA adducts are detectable in normal and malignant breast tissues. Peripheral blood lymphocytes (PBLs) from female breast cancer patients were more sensitive to in vitro exposure to benzo[a]pyrene diol epoxide (BPDE) than those from healthy controls. Therefore, we hypothesized that reduced DNA repair is associated with risk of breast cancer in women and the risk may be modulated by polymorphisms of DNA repair genes. In a case-control pilot study, we included 69 previously untreated female breast cancer patients and 79 controls frequency matched to the cases on age and ethnicity. The PBLs were used to measure DNA repair capacity (DRC) by using the host-cell reactivation (HCR) assay with a reporter gene damaged by exposure to 60 micro M BPDE prior to transfection. We also genotyped for two common XPD polymorphisms Lys751Gln and Asp312Asn. We found that the mean DRC level was significantly lower in breast cancer patients (10.1%) than in controls (11.1%) (P = 0.008). Subjects with DRC lower than the median level of controls (11.0%) had >3-fold increased risk (OR = 3.36, 95% CI = 1.15-9.80) for breast cancer than did those with higher DRC after adjustment for age, smoking status and assay-related variables. None of the genotypes was statistically significantly associated with an increased risk of breast cancer, which may be due to the small number of observations in each subgroup. The XPD variant genotypes in general predicted the DRC better in the controls than in the cases, suggesting genetic variants of other DNA repair genes may be involved in these breast cancer patients. These findings suggest that women with reduced DRC may be at an increased risk of developing breast cancer. Large studies are warranted to confirm these preliminary findings.     

The binding efficiency of polyclonal and monoclonal antibodies to DNA modified with benzo[a]pyrene diol epoxide is dependent on the level of modification. Implications for quantitation of benzo[a]pyrene-DNA adducts in vivo

A number of polyclonal antibodies specific for DNA modifiedwith (±)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE) were obtained from the sera of New Zealand white rabbitsimmunized with BPDE-DNA, complexed with methylated bovine serumalbumin (mBSA). Monoclonal antibodies were developed by fusionof mouse myeloma cells with spleen cells isolated from BALB/cmice immunized with the same complex of BPDE-DNA and mBSA. Theseantibodies have been characterized for specificity in a highlysensitive, enzyme-linked immunosorbent assay (ELISA). All antibodiesshowed a very high affinity for single-stranded BPDE-DNA, buthad lower affinity towards native BPDE-DNA. The affinity forthe free mononucleoside BPDE-dG was at least 100-fold lowerthan that for BPDE-DNA, and no affinity was detected for BPtetrols or DNA modified with N-acetoxy-N-acetyl-2-aminofluorene.A high cross reactivity was observed with DNA modified with(±)-trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene.Using five different antibodies, monoclonal or polyclonal, weobserved that the antibody affinity for BPDE-DNA was dependenton the level of modification; in the competitive ELISA as littleas 4 fmol BPDE-DNA (50 pmol/µg) was sufficient for 50%inhibition with our best antisera, but 17 fmol of the adductwas required when [³H]BPDE-DNA of low modification (1–10fmol/µg) was used as inhibitor. When samples of [³H]BP-DNAisolated from the livers of mice, treated i.p. with differentdoses of [³H]BP were examined by competitive ELISA and calibratedwith [³H]BPDE-DNA of low modification (1–10 fmol/µg),binding values calculated from the immunoassay were in goodagreement with those obtained from radioactivity measurements.In contrast, when this DNA was quantitated in competitive ELISAusing highly modified BPDE-DNA as standards, values by ELISAwere 20–40% of those obtained by radioactivity. Theseresults indicate that the use of serially diluted BPDE-DNA ofhigh modification as standard competitor in the ELISA will leadto erroneous results in the measurement of adducts in DNAs modifiedto a low extent (biological samples). The property of antiseraspecific for BP-DNA, recognizing highly modified DNA more efficientlythan DNA modified to a low extent, may be common to all antiseraelicited against highly modified DNA immunogens. Therefore weconclude that antibody affinity must be tested also with DNAsamples of low modification, obtained either in vitro or invivo.