Development of monoclonal antibodies recognizing 7-(2-hydroxyethyl)guanine and imidazole ring-opened 7-(2-hydroxyethyl)guanine

7-(2-Hydroxyethyl)guanine (7HEG) is of biological interest becauseit is formed in vivo by reaction of DNA with ethylene oxide(EO). Furthermore, the major DNA adduct of vinyl chloride, 7-(2-oxyethyl)guanine,can be converted to this adduct by reduction. Two monoclonalantibodies (9E2, 4A5) recognizing 7HEG have been developed fromBALB/c mice immunized with the adduct coupled to keyhole limpethemocyanin. In addition, another antibody (8E10) was developedagainst the imidazole ring-opened form of the adduct (ro-7HEG).ELISAs were used to determine the sensitivity and specificityof these antibodies. With antibody 9E2, 50% inhibition of antibodybinding in the competitive ELISA was at 54 pmol of the modifiedbase 7HEG/well and 67 pmol 7HEGR/well, while with antibody 4A5,the values were 3.6 pmol 7HEG/well and 6.7 pmol 7HEGR/well.Antibody 8E10 gave 50% inhibition at 48 pmol ro-7HEGR/well.Neither antibody 9E2 nor 8E10 cross-reacted with unmodifiedDNA or with the normal nudeosides at the highest concentrationtested. However, antibody 4A5 had a low affinity for deoxyguanosine(50% inhibition at 31 000 pmol). Sensitivity of adduct measurementcan be increased 3- to 10-fold using an ELISA with fluorescenceendpoint detection. These antibodies have been used to determinethe level of adducts in DNA modified in vitro with [³H]- or[¹⁴C]EO. Because of the cross-reactivity of the most sensitiveantibody, 4A5, with deoxyguanosine, a combined HPLC/immunoassaymethod was developed to quantitate 7HEG in DNA. The limit ofsensitivity of this method is dependent upon the amount of DNAavailable for analysis. Using 30 fmol as the lowest detectableamount (20% inhibition) in the fluorescent ELISA with antibody4A5 and 100 µg of DNA assayed per well, adduct levelsof 1/10⁷ nucleotide can be determined. This method was appliedto DNA adduct detection in EO-treated myeloma cells and wholeblood. Antibody 8E10 was also used in immunohistochemical studiesto visualize ring-opened adducts in cells treated with EO followedby high pH. These antibodies will be used for the detectionand quantitation of adducts in human samples.     

Preparation and characterization of antibodies against 3,2'-dimethyl-4-aminobiphenyl-modified DNA

Polyclonal antibodies for 3,2'-dimethyl-4-aminobiphenyl (DMAB)–DNAadducts were obtained from the sera of rabbits immunized withN-OH-DMAB-modified DNA. Using the enzyme linked immunosorbentassay (ELISA), these antibodies were shown to recognize DNAmodified by N-OH-DMAB and N-hydroxy-4-aminobiphenyl, but notunmodified DNA, 2-acetylaminofluorene- or 4-nitroquinoline-1-oxide-modifiedDNA, free aminobiphenyl or DMAB derivatives. Using competitiveELISA with DMAB-modified denatured DNA, a 50% inhibition ofantibody antigen binding was caused by 7 fmol of DMAB adductsapplied to each assay well as an inhibitor. Native DNA bearingadducts was associated with 50% inhibition in the range of 22–90fmol/assay well, depending on the levels of modification. Theadducts recognized by the antibody were shown to be stable againsttreatment of heat or alkali denaturation. Optimal conditionsfor the detection of adducts in DNA from the rat organs exposedto DMAB were established by means of competitive ELISA, usingalkaline-denatured DNA as an inhibitor. Thus, as little as 5fmol adducts in 1 µg of DNA could be detected. Indirectimmunofluorescence staining indicated that anti-DMAB-DNA antibodiesbound specifically to nuclear components of rat fibroblast 3Y1cells treated with N-OH-DMAB. The intensity of the fluorescencewas proportional to the dose of carcinogen administered. Theantibodies should be helpful for use in studies on the formationof adducts and their removal in cells and tissues after DMABadministration.     

Immunological quantitation of (O4-ethylthymidine in alkylated DNA: repair of minor miscoding base in human cells

Rabbit antisera specific for O⁴-ethyldeoxythymidine (O⁴-EtdThd)were developed and used in competitive and non-competitive immunoassaysfor the quantitation of the miscoding promutagenic adduct O⁴-EtdThdin DNA. High titer antiserum TB3 (affinity constant, 8.1x10⁸)gave 50% inhibition of the tracer antigen-antibody binding with0.26 pmol of O⁴-EtdThd in radioimmunoassay and negligible inhibitionwith unmodified DNA components. In competitive enzyme-linkedimmunosorbant assay (ELISA), using ENU-poly(dT) instead of hapten-proteinconjugate as antigen, inhibition of antigen-antibody bindingwas proportional to both, the concentration of ethylnitrosourea(ENU) and alkylated DNA. The sensitivity of detection of (O⁴-EtdThdin DNA was enhanced to fmol of (O⁴-EtdThd and ng amounts ofalkylated DNA by using avidin-biotin linked immuno-reagentsin non-competitive ELISA and immunoslot blot assays. An O⁴-EtdThdconcentration-dependent binding of specific antibody, detecting0.12 fmol of the modified base, was observed with DNA alkylatedin vitro with ENU at an O⁴-EtdThd/Thd molar ratio of 2.6x10^–6.The immunological quantitation of (O⁴-EtdThd in ENU-treatedhuman skin fibroblast and kidney epithelial cells indicatesa gradual removal of the modified base as a function of post-treatmenttime in culture. In both cell types about 50% of the initialdamage was repaired during a 72-h period. Earlier studies usinghuman organ extracts ruled out the participation of an alkyltransferaseor glycosylase enzyme for the repair of O⁴ -alkylthymine. Nonethe less, the kinetics data presented indicate that normal humancells in culture are proficient for the repair of critical pyrimidinealkylation adduct that may be associated with cellular oncogenictransformation of susceptible mammalian cells.     

Quantification of methoxsalen-DNA adducts with specific antibodies

Methods are now available for the quantification of carcinogen-DNA adducts in human tissues and can be used to screen populations for exposure to environmental carcinogens. One approach utilizes highly specific antibodies in sensitive immunoassays for quantifying adduct levels in DNA from various tissues. We have recently developed a panel of monoclonal antibodies that specifically recognize DNA modified by methoxsalen and ultraviolet A light (320-400 nm) (UVA). These antibodies have been characterized as to sensitivity and specificity by an enzyme-linked immunosorbent assay (ELISA). In a competitive ELISA, 50% inhibition of antibody binding occurred with 17 fmol methoxsalen-DNA photo adducts. There was also some antibody cross-reactivity with DNA modified by 4'-aminomethyl-4,5,8-trimethylpsoralen and 4',5-dimethylangelicin but not with free methoxsalen. A more sensitive ELISA has also been developed using fluorescence detection of enzyme activity. With this assay, one adduct per 10(8) bases can now be detected reliably. Adduct levels have been quantified in myeloma cells and lymphocytes treated in vitro with methoxsalen and UVA. In addition, in preliminary studies, adducts have been measured in lymphocytes isolated from patients undergoing extracorporeal photophoresis for cutaneous T-cell lymphoma. Quantification of methoxsalen adducts in patients should provide a basis for estimating risk resulting from psoralen plus UVA (PUVA) treatment.     

Quantitation of O6-ethyldeoxyguanosine in ENU alkylated DNA by polyclonal and monoclonal antibodies

Rabbit polyclonal and mouse monoclonal antibodies were developedagainst O⁶-ethylguanosine conjugated with keyhole limpet hemocyanin.Radioimmunoassay (RIA) and a modified enzyme-linked immunosorbantassay (ELISA) were established for the determination of antigen-antibodybinding and quantitation of potentially mutagenic O⁶-ethyldeoxy-guanosine(O⁶-EtdGuo) in DNA treated with ethylnitrosourea (ENU) in vitroand in vivo. Optimum as well as reproducible antibody bindingcould be observed with conjugate concentrations at 0.1 ng/wellimmobilized by overnight drying at 37°C. RIA was several-foldmore sensitive than ELISA in detecting inhibition with O⁶-EtdGuorequiring 0.1 pmol for the 50% inhibition of tracer-antibodybinding. In competitive inhibition assays with polyclonal andmonoclonal antibodies, a linear dose resonse relation was obtainedwith DNA hydrolyzates alkylated in vitro with increasing concentrationsof ENU. Significantly lower modification levels, e.g., 16.0fmol O⁶-EtdGuo, at an O⁶-EtdGuo/dGuo molar ratio of 2.6 x 10^–7in a hydrolyzate of 80 µg rat liver DNA ethylated in vivowith 10 µg ENU/g body weight was determined immunologically.The rate of elimination of O⁶-EtdGuo determined in human fetalkidney epithelial cells treated with 0.65 mM ENU showed that50% of initial O⁶-EtdGuo was removed within 1 h followed bya slow phase of repair with 23% remaining at 8 h post-treatment.     

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.     

Sensitive detection of DNA modifications induced by cisplatin and carboplatin in vitro and in vivo using a monoclonal antibody.

An assay that is based upon a monoclonal antibody (ICR4) is described that enables the quantitation of cisplatin-induced adducts on DNA down to 3 nmol Pt/g DNA (i.e., 1 Pt adduct/10(6) bases), the level necessary to produce toxic effects in cells in vitro and in vivo, using just a few micrograms of DNA. Detection is possible below this level (although probably not necessary for in vivo studies) but the cross-reactivity of unmodified DNA sequences complicates absolute quantitation of adducts. Therefore, it will be possible to investigate the distribution of clinically useful platinum drugs in patients undergoing chemotherapy. Rats of strain F344 appeared to be the best, among several tested, for the production of antibodies to modified DNA, and they were used for the production of hybridomas. Fifteen hybridomas which secreted antibodies that bound to DNA that was highly modified with cisplatin but not to normal DNA were obtained. One (ICR4) was chosen for further characterization because of its relatively strong binding to DNA modified to a moderate level with cisplatin. The characterization included the development of a sensitive competitive enzyme-linked immunoabsorbent assay and the use of DNA that had been reacted with cisplatin both in vitro and in vivo. The levels of platination of both types of DNA samples were determined by atomic absorbance spectroscopy. For DNA that had been exposed to cisplatin in vitro, 50% inhibition of antibody binding was caused by about 15 fmol of total DNA-bound Pt/assay well. At moderate levels of platination, heating of the DNA solution at 100 degrees C for 5 min increased its immunoreactivity such that 50% inhibition was caused by 2.5 fmol Pt adducts/well. Pt adducts on DNA extracted from cells that had been treated with cisplatin were less immunoreactive than DNA treated with cisplatin in vitro, but after heating the immunoreactivity increased such that 50% inhibition in the assay was caused by 2 fmol Pt adduct/well. This sensitivity was invariant over a wide range of levels of platinum adduct frequency. DNA adducts formed by the second generation anticancer drug carboplatin were recognized similarly to the adducts formed by cisplatin, but those formed by the clinically inactive trans-diamminedichloroplatinum(II) or chloro(diethylenetriamine)-platinum(II)-chloride were not significantly immunoreactive. Control DNA cross-reacted in the competitive assay but the immunoreactivity per mol base was 10(7) times lower than the immunoreactivity of cisplatin adducts.     

A 32P-postlabeling method for simultaneous detection and quantification of exocyclic etheno and propano adducts in DNA

A ³²P-postlabeling method is described that specifically detectsand quantifies the 1,N² adducts derived from acrolein (AdG)and crotonaldehyde (CdG) and 1,N²-ethenodexoxyguanosine (EdG)in DNA. These exocyclic adducts are potential DNA lesions causedby exposure to enals as environmental pollutants and as endogenouscompounds. This method was developed with the use of the syntheticadduct standards of these exocyclic adducts. The assay relieson HPLC for adduct enrichment prior to labeling and for quantitationand identification after labeling. The labeling efficienciesof adducts at the 1 fmol level ranged from 74 to 96%, whereasthey were only 49–60% at the 100 fmol level. This methodcan detect as low as 0.2 fmol of adduct and allows the detectionand quantitative determination of stereolsomers of AdG and CdG.The method was validated by using a sample of enzyme digestsof 180 µg calf thymus DNA spiked with 25 or 75 fmol ofadducts, which is equivalent to 5 or 15 adducts in 10⁸ nucleotides.The recovery rates of these adducts in DNA ranged from 30 to90% at the 25 fmol level and 21 to 55% at the 75 fmol level.Similar to the labeling efficiency, a greater recovery was observedwith a lower amount of adduct in DNA. Overall, this method allowsthe simultaneous identification and quantification of exocycicadducts AdG, CdG and EdG in DNA. Therefore, it provides a potentialtool for studies of the in vivo formation of exocyclic adducts.     

Quantitation by electron microscopy of the binding of highly specific antibodies to benzo[a]pyrene-DNA adducts

Highly specific antibodies bound to carcinogen adducts in DNAmodified with (+ / - )7ß, 8-dihydroxy-9, 10-epoxy-7,8, 9, 10-tetrahydrobenzo[a]pyrene (BPDE I) were quantitatedby electron microscopy (EM) visualization and these observationswere compared with quantitation of adducts by enzyme-linkedimmunosorbent assay (ELISA). The antiserum, elicited in rabbitsfollowing inoculation with BPDE I-modified DNA, has been foundto be highly specific in its recognition of BPDE I-deoxyguanosinemoieties. Parallel DNA samples prepared for analysis by ELISAand EM quantitation were randomized, encoded, and analyzed todetermine extents of carcinogen modification in double-blindstudies. After levels of modification were determined by immunoassays,DNA samples were prepared for EM analysis by incubation withamounts of anti-BPdG-DNA serum in excess of that necessary forcomplete binding of antibody to antigenic sites. At equilibrium,samples were enzymatically digested with papain in order tocleave anti-BPdG-DNA IgG molecules into Fab fragments in situ.Following column exclusion chromatography, BPdG-DNA-Fab complexeswere incubated with ferritin-labeled Fab‘ fragments ofgoat [anti-rabbit F(ab’)2] IgG in amounts in excess ofthose necessary for complete binding. When DNA samples weremodified to between 0 and 40 fmol adduct/ µg DNA, excellentagreement was obtained between ELISA quantitation and visualizationby EM of antibodies bound to adducts.     

Immunological Detection and Quantitation of DNA Adducts Formed by 4-Aminoazobenzene Species in vivo

Antibodies to 3-methoxy-4-aminoazobenzene (3-MeO-AAB) and 2-methoxy-4-aminoazobenzene (2-MeO-AAB) DNA adducts were raised in rabbits against in vitro-adducted DNA samples. The enzyme-linked immunosorbent assay (ELISA) was used to determine the sensitivity and specificity of these antibodies. They proved highly specific for the modified DNA used as the immunogen, but cross-reacted with each other. Moreover, they showed cross reactivity with DNA modified by 4-( o -tolylazo)- o -toluidine, but not by other carcinogens, such as 4-aminobiphenyl or 4-nitroquinoline 1-oxide. The 50% inhibition level of antibody binding in the competitive ELISA was at 10–20 fmol of modified base per assay (equivalent to 1–2 adducts per 10⁶ bases). Immunohistochemical staining indicated that these antibodies bind specifically to nuclear components of the liver in rats given either 3-MeO-AAB or 2-MeO-AAB at the dose of 50 mg/kg body weight.     

Immunological detection and quantitation of DNA adducts formed by 4-aminoazobenzene species in vivo.

Antibodies to 3-methoxy-4-aminoazobenzene (3-MeO-AAB) and 2-methoxy-4-aminoazobenzene (2-MeO-AAB) DNA adducts were raised in rabbits against in vitro-adducted DNA samples. The enzyme-linked immunosorbent assay (ELISA) was used to determine the sensitivity and specificity of these antibodies. They proved highly specific for the modified DNA used as the immunogen, but cross-reacted with each other. Moreover, they showed cross reactivity with DNA modified by 4-(o-tolylazo)-o-toluidine, but not by other carcinogens, such as 4-aminobiphenyl or 4-nitroquinoline 1-oxide. The 50% inhibition level of antibody binding in the competitive ELISA was at 10-20 fmol of modified base per assay (equivalent to 1-2 adducts per 10(6) bases). Immunohistochemical staining indicated that these antibodies bind specifically to nuclear components of the liver in rats given either 3-MeO-AAB or 2-MeO-AAB at the dose of 50 mg/kg body weight.     

Effects of dietary 1,4-phenylenbis(methylene)selenocyanate on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced DNA adduct formation in lung and liver of A/J mice and F344 rats

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) was testedfor its ability to inhibit DNA adduct formation induced by thetobacco-specific N-nitrosamine 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone(NNK) in the liver and lung of A/L mice and F344 rats. Dietaryp-XSC, providing a dose of 5 p.p.m. selenium, significantlyinhibited the formation of 7-methylguanine (7-mGua) inducedby a single i.p. injection of 10 µmol of NNK (12.8% inhibitionat 4 h and 19.9% at 96 h) and O⁶-methylguanine (O⁶-mGua) (16.5%at 4 h and 34.8% at 96 h) in the liver of A/J mice. Dietarysupplements of p-XSC providing 15 p.p.m. of selenium reducedthe levels of 7-mGua by 17.3% (4 h) and 33.6% (96 h). The formationof O⁶-mGua was inhibited by 69.5% (4 h) and 73.8% (96 h). InA/J mouse lung DNA the most significant reduction was observedin levels of O⁶-mGua. Dietary p-XSC at 5 p.p.m. as seleniuminhibited the formation of this adduct by 73.1% (4 h). Ninety-sixhours after NNK injection, and at both time points with p-XSCproviding 15 p.p.m. selenium,O⁶-mGua was not detected. Althoughlevels of 7-mGua in mouse lung DNA were also reduced, this wassignificant only 4 h after carcinogen administration. In general,selenite at 5 p.p.m. as selenium had no significant effect onthe levels of these lesions; however, it inhibited O⁶-mGua inthe liver only4 h after NNK administration. These effects mayexplain why there is chemopreventive activity for p-XSC, butnot for selenite, in NNK-induced lung carcinogenesis in A/Jmice. Moreover, these findings raised our interest in determiningthe potential chemopreventive activity of p-XSC against NNK-inducedlung adenocarcinomas in male F344 rats by first determiningits effects on NNK-induced DNA methylation in the lungs of rats.Diet supplemented with 10 p.p.m. selenium as p-XSC did indeedinhibit the formation of adducts in pulmonary DNA of F344 ratstreatedwith four consecutive injections of 81 mg/kg of NNK. Statisticallysignificant inhibition of O⁶-mGua formation was observed 4 hafter carcinogen treatment in both pulmonary (49.1% inhibition)and hepatic (39.8%) DNA. Statistically significant inhibitionof 7-mGua formation was also measured in lung DNA isolated 24h after the last NNK injection (45.0%) and in liver DNA 4 hafter carcinogen treatment (31.8%). Theseresults suggest thatp-XSC would also inhibit induction of lung adenocarcinoma inmale F344 rats by NNK.     

DNA adduct formation and removal in specific liver cell populations during chronic dietary administration of 2-acetylaminofluorene

The concentration of DNA adducts in specific hepatic cell typeshas been determined in F344 rats fed 0.02% 2-acetyl-aminofluorene(AAF) for 28 days followed by control diet for an additional28 days. In animals killed at 28 days of AAF feeding, the majorDNA adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene, was presentin each cell type in the order: hepatocytes (282? 28 fmol/µgDNA) > whole liver (232 ? 33 fmol/µg DNA) > nonparenchymalcells (128 ? 30 fmol/µg DNA) > bile duct fraction (60?12 fmol/µg DNA). After an additional 28 days on controldiet, the adduct level in each cell fraction was 30–40fmol/µg DNA. Adduct removal was biphasic in whole liver,hepatocytes and nonparenchymal cells, with a fast phase apparentuntil the adduct concentration reached 60 fmol/µg DNA.In whole liver and hepatocytes this level was obtained in approximatelyseven days, and in nonparenchymal cells the fast phase was completein about two days. Adduct removal in the bile duct fractionexhibited only a single slow phase. At the end of the AAF feeding,hepatocytes accounted for 86% of the total liver DNA adducts.After an additional 28 days on control diet, hepatocyte adductsstill contributed a major fraction (67%) of the total persistentadduct population. Thus, hepatocytes, the target cell for AAF-inducedhepatic tumors, dominate the adduct formation and removal profileobserved in whole liver.     

Monoclonal antibodies to 1-aminopyrene-DNA

Monoclonal antibodies were obtained after fusion of mouse P3X63-AG.8.653myeloma cells with spleen cells isolated from BALB/cCr miceimmunized with denatured DNA modified by 1-nitrosopyrene reducedwith sodium ascorbate (AP-d-DNA) and complexed electrostaticallyto methylated bovine serum albumin. Ten stable hybridoma lineshave been isolated and characterized by enzyme-linked immunosorbentassay (ELISA). They all recognize 1-aminopyrene (1-AP)modifiedDNA, but not free 1-nitropyrene or 1-aminopyrene. Antibody 11H2is the most specific for AP-DNA showing no cross-reactivitywith unmodified native DNA. It also recognizes 8-nitro-1-APand 6-nitro-1-AP modified DNA. There was some low cross-reactivitywith DNA modified by a benzo[a]pyrene diol epoxide and N-acetoxy-N-2-acetylaminofluorene.Competitive ELISA with antibody 11H2 reliably detected AP-DNAadducts formed when 1-nitropyrene was incubated with Salmonellatyphimurium TA1538. By immunological methods, AP-DNA adductswere shown to be unstable to heat denaturation. This suggeststhat specific monoclonal antibodies to carcinogen-DNA adductswill be useful not only for detecting and quantitating carcinogen-DNAdamage but also for probing adduct stability.     

Immunological detection of DNA damage caused by melphalan using monoclonal antibodies

Immunological detection of melphalan adducts on DNA should permit new types of clinical and experimental investigations. Five cloned rat hybridoma cell lines were derived, each producing an antibody that bound to DNA alkylated with melphalan (phenylalanine mustard) but not to normal DNA. Further characterization of one melphalan specific antibody (MP5/73) used a competitive fluorogenic enzyme-linked immunoabsorbent assay. Using denatured DNA, 50% inhibition of antibody binding was caused by 30 fmol of total melphalan adducts (determined using radioactive melphalan) per assay well. Denatured control DNA caused 16 to 24% inhibition at 45 micrograms (130 nmol)/well, the maximum concentration tested. Adducts on RNA behaved similarly to those on denatured DNA. Adducts on native DNA caused 50% inhibition at 272 to 1335 fmol/well dependent upon alkylation frequency and sonication treatment. Native control DNA caused no detectable inhibition at 45 micrograms/well. The adducts recognized by the antibody were thermo- and alkali labile. Denaturation of the alkylated DNA by moderate heating in the presence of 75% formamide gave 50% inhibition at 50 fmol/well, indicating that only 5% of the recognized adducts could bind antibody in native DNA.     

High avidity monoclonal antibody to imidazole ring-opened 7-methylguanine

A hybridoma (K1A8) secreting a high affinity antibody to imidazole ring-opened 7-methylguanine (N5-methyl-N5-formyl-2,5,6-triamino-4-hydroxypyrimidine) was obtained from spleen cells of a mouse immunized with a conjugate of keyhole-limpet hemocyanin and imidazole ring-opened 7-methylguanylic acid (iro-7mGMP). The antibody recognizes the iro-7-methylguanine (iro-7mG) determinant in the BSA-iro-7mGMP conjugate, in chemically methylated, denatured DNA, and in the ring-opened 7-methylguanosine, 7-methyldeoxyguanosine and 7mGMP haptens. In the competitive ELISA of DNA-iro-7mG, 50% inhibition (I50) was observed at 4 fmol determinant per well (8 x 10(-11) M) using BSA-iro-7mGMP as the immobilized antigen. The lower limit of 7-methylguanine (7mG) detection in DNA is determined by the binding of unmodified DNA per se to the antibody. The intrinsic reaction of DNA with antibody is low; in the competitive ELISA I50 was obtained with 330 micrograms calf thymus DNA per 50 microliters well, equivalent to 4 nmol iro-7mG per mol nucleotide. The 7mG content of calf thymus DNA is 7 nmol per mol nucleotide (approximately 20 amol per micrograms DNA). The limit of detection of 7mG by competitive ELISA is quoted provisionally as 7 nmol iro-7mG per mol nucleotide, where 30% inhibition of antibody binding is obtained in the presence of 105 micrograms DNA per 50 microliters well. Nuclear DNAs of tissue culture cells treated with 0, 0.01 and 0.1 mM N-methyl-N'-nitro-N-nitrosoguanidine contained 0.18, 31 and 320 mumol, respectively, of 7-methylguanine adducts per mol of nucleotides. This report indicates that the K1A8 antibody will serve to quantify DNA alkylation in human populations exposed to low levels of methylating carcinogens.     

Quantitation of protein adducts as a marker of genotoxic exposure: immunologic detection of benzo(a)pyrene -- globin adducts in mice

Immunologic methods have been developed for the determinationof benzo(a)pyrene (BP)-protein adducts and validated in animalstreated with (³H)BP. A previously developed antibody, 8E11,which recongnizes 7ß, 8-dihydroxy-9, 10-epoxy-7, 8,9, 10tetrahydrobenzo(a)pyrene (BPDE-I)-modified DNA or proteinas well as BPDE-I- tetraols, was used. The sensitivity of theassay was increased by enzymatic digestion of the modified proteinwith insoluble protease into peptides and amino acids beforeanalysis. In a competitive enzymelinked immunosorbent assay(ELISA) with digested BPDE-I-modified bovine serum albumin,50% inhibition occured at 400 fmol of adduct compared to 1450fmol for the nondigested albumin. Analysis of globin (Gb) isolatedfrom animals treated in vivo with 0.3–3 mg (³H)BP indicatedthat the ELISA could detect 90–100% of the adducts determinedby radioactivity. Levels of adducts in lung and liver DNA andserum albumin were correlated with the levels of Gb adducts.Of the total radioactivity associated with hemoglobin, only10% was from Gb while {small tilde}80% was from the heme fractionand the remainder from free BP metabolites. Significant cross-reactivityof antibody 8E11 was found with several BP-diols and phenols,suggesting that the immunoassay will not only be specific forBPDE-I adducts but will also detect adducts of other BP metabolitesas well as other aromatic hydrocarbon diol epoxides. An immunoaffinitycolumn of antibody 8E11 coupled to Sepharose 4B was used toisolate modified peptides from the digested Gb. About 65% ofthe applied radioactivity was retained on the column. Between1 and 2 mg of non-modified digested Gb could be added to thesample without interfering with binding of adducts. Proteindigestion and immunoaffinity chromatography should be usefulfor the measurement of protein adducts in biomonitoring studies.     

Preparation of compound-specific and group-specific antibodies to 7-methylguanine and related 7-alkylguanines and their use in immunoaffinity purification

The preparation and characteristics of compound-specific andgroup-specific antibodies against 7-alkylguanines (7-alkGua)are described. A compound-specific antibody against 7-methylguaninewas prepared using a hapten bound to carrier protein throughthe N² position. In a competitive enzyme-linked immunosorbantassay (ELISA) 7-methyl-guanine (7-MeGua) showed 50% inhibition(I_50% at 10 pmol/ well at room temperature, but the inhibitionwas found to be 40 times better at 4°C (I_50% at 250 fmol/well).When the antibody was bound to protein A-Sepharose CL4B 7-MeGuawas retained in immunoaffinity columns. A group-specific antibodyto 7-alkGua was prepared using 7-(2-carboxyethyl)guanine (7-CEGua)bound to carrier protein via the carboxyl group. In a competitiveELISA, this antibody cross- reacted well with 7-CEGua, 7-ethylguanine(7-EtGua), 7-(2-hydroxyethyl)guanine (7-HOEtGua) and 7-(2',3'-dihydroxy)-propylguanine(7-DHPGua) and some inhibition was seen with 7-MeGua. Immunoaffinitycolumns prepared from this antibody retained a number of 7-alkGuaof diverse structure. 7-EtGua in calf thymus DNA treated withdiethyl sulphate and ethylnitrosourea was isolated by immunoaffinitypurification and quantified by HPLC-fluorescence. These resultsillustrate the potential of immunoaffinity purification forboth individual DNA adducts and groups of adducts.     

High selectivity of polyclonal antibodies against DNA modified by diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxides.

Polyclonal antibodies were developed in New Zealand White rabbits against DNA modified with diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxide (B[c]PhDE)-1 (4-hydroxyl and epoxide cis) and B[c]PhDE-2 (4-hydroxyl and epoxide trans). Antiserum developed against B[c]PhDE-2-DNA was stereoselective. In competitive ELISA assays using wells coated with 160 fmol B[c]PhDE-2-DNA adducts, B[c]PhDE-2-DNA gave 50% inhibition at 200 fmol adducts/well. B[c]PhDE-1-DNA required a 10-fold higher amount of adducts/well to give 50% inhibition. Benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA and 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA caused only a 30% inhibition even at the highest doses tested (greater than 4000 fmol adducts/well). For antiserum developed against B[c]PhDE-1-DNA, 50% inhibition required 570 fmol B[c]PhDE-1-DNA adducts in wells coated with 100 fmol B[c]PhDE-1-DNA adducts. 7,12-Dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA and B[c]PhDE-2-DNA were also effective competitors: they caused 50% inhibition at 1900 and 1800 fmol adducts/well respectively. In contrast, benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA gave no inhibition at the highest dose of competitor tested (4050 fmol adducts/well). Antisera from three rabbits immunized with B[c]PhDE-2-DNA demonstrated similar antigen specificities. The properties of these antisera differ from those reported previously for antibodies developed against benzo[a]pyrene-DNA in that they show selectivity for DNA modified by specific hydrocarbon diolepoxides, in one case for B[c]PhDE-2-DNA and in the other for B[c]PhDE-DNA or 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA. The specificity of these antisera will facilitate analysis of the modification of DNA by different polycyclic aromatic hydrocarbon diolepoxides.     

Antibodies to the food mutagens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline: useful for immunoassay and immunoaffinity chromatography of biological samples

Monoclonal mouse IgG₁ and IgG₃ antibodies were developed tothe food mutagens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline(4,8-DiMeIQx) in order to make specific and sensitive detectionand purification systems suitable for biological samples. Theantibodies were developed with the strategy that cross-reactionwith analogues modified in the N²-position was desirable. Competitiveenzyme-linked immunosorbent assays (ELISA) with 50% inhibitionby 0.4–6 pmol food mutagen were developed. The epitopesrecognized by the antibodies have been characterized by ELISAusing 52 synthetic analogues and metabolites of PhIP, 4,8-DiMeIQx,and other food mutagens. One of the anti-PhIP antibodies onlyrecognizes PhIP and those PhIP-analogues which have minor modificationsin the N²-amino group, whereas the other, 7B7-1, is less stringentand also recognizes several other modified metabolites, includingbulky adducts at the N²-amino group e.g. the major guanine anddeoxyguanosine adducts isolated from PhIP-modified DNA. Theantibodies to DiMeIQx also recognize the food mutagens 2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline(4-MeIQx), 2-amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline (8-MeIQx),and the corresponding quinolines (4-MeIQ and 8-MeIQ). Two ofthese antibodies only bind analogues with minor modificationsin the free amino group, whereas analogues with major modificationsin this position, including a deoxyguanosine adduct, react withthe third antibody. Urine samples and faecal extracts from ³H-PhIPor 2-¹⁴C-DiMeIQx dosed rats were analysed by these ELISA assays,and high correlations between radioactivity and response inthe ELISA assays were observed. Urine samples and faecal extractsfrom ³H-PhIP-dosed rats were purified on an affinity columncontaining the less stringent anti-PhIP antibody, 7B7-1. Theaffinity column was found by high performance liquid chromatography(HPLC) analysis to concentrate exclusively labelled material.This affinity column also bound PhIP-related materials fromdilute samples of acid hydrolysed PhIP-DNA with high efficiency.Only