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.     

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.     

Genotoxicity of the food mutagen 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) and analogs

The food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)is an analogue of quinoline, a hepatocarcinogen. 2-Aminofluorene,benzldine and 3,2'-dimethyl-4-aminobiphenyl (DMAB) are potentinducers of unscheduled DNA repair in primary culture rat liverhepatocytes, as was IQ (151 grains/ nucleus at 1 x 10^–6M). Quinoline, on the other hand, is only weakly positive inthis assay (15 grains/nucleus at 1 x 10^–3 M). IQ, quinolineand DMAB were applied topically to shaved skin of Sencar micewith promotion by 12-O-tetradecanoylphorbol 13-acetate (TPA)for 20 weeks, when 14 of 20 mice in the quinoline group had25 tumors, but only one of 30 animals in the IQ group and fiveof 30 in the DMAB group were tumor-bearing. Analogs of IQ synthesizedby substitution at the 2- or 3-position with amino or methylgroups were assayed with the Ames Salmonella typhimurium testerstrains TA98 and TA100. Mutagenicity for TA98 is reduced inthe absence of the 3-methyl group and is completely abolishedwith removal of the 2-amino moiety. None of these analogs arestrong mutagens for TA100. Exocyclic N-oxidation is a likelyobligatory step in the activation of IQ to a mutagen.     

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.     

Development of techniques to monitor for exposure to vinyl chloride: monoclonal antibodies to ethenoadenosine and ethenocytidine

Monoclonal antibodies which specifically recognize etheno-adenosineand ethenocytidine, two of the adducts resulting from exposureto vinyl chloride, have been developed. The sensitivity andspecificity of these antibodies have been determined by enzyme-linkedimmunosorbent assay (ELISA). The antibody to ethenoadenosine(1G4) reacts with both the ribose (50% inhibition at 600 fmol)and deoxyribose (50% inhibition at 980 fmol) form of the adduct.The antibody to ethenocytidine (6F5) also reacts with both theribose (50% inhibition at 800 fmol) and deoxyribose (50% inhibitionat 1000 fmol) form of the adduct. Neither antibody cross-reactswith non-modified DNA or the normal nucleotides. A more sensitivefluorescence ELISA was developed for antibody 1G4 with 50% inhibitionat 212 fmol of ethenoadenosine and for antibody 6F5 with 50%inhibition at 192 fmol ethenocytidine. These antibodies havebeen used to determine the level of etheno derivatives in DNAmodified in vitro with chloroacet-aldehyde and in the DNA andRNA of cells treated in culture.     

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.     

Comparison of various immunochemical assays for the detection of ethylene oxide-DNA adducts with monoclonal antibodies against imidazole ring-opened N7-(2-hydroxyethyl)guanosine: application in a biological monitoring study

Monoclonal antibodies have been developed for the analysis ofthe predominant lesion in DNA induced by ethylene oxide (EtOx),namely N7-(2-hydroxyethyl)guanine (N7-EtOHGua). Two monoclonalantibodies raised against imidazole ring-opened N7-(2-hydroxyethyl)guanine(RON7-EtOHGua), N7EO-E and N7EO-11, and the previously isolatedantibody N7E-102 were characterized by competitive ELISA withvarious inhibitors. N7EO-E and N7EO-11 recognize 2-hydroxyethyllesions better than ethyl or methyl lesions, while N7E-102 recognizes2-hydroxyethyl and ethyl modifications equally well. All antibodiesshow a preference for imidazole ring-opened adducts, bind betterto adducts in DNA compared to alkylated nucleosides or basesand bind 10⁶- to 3x10⁶-fold less well to unmodified DNA. Thesensitivity of detection of RON7-EtOHGua in DNA and in the nucleiof cells in situ by antibody N7EO-E was investigated in severalassays. The immunoslot blot assay was the most sensitive method(0.34 RON7-EtOHGua per 10⁶ nucleotides was detectable), followedby competitive ELISA, direct ELISA and in situ detection byimmuno-fluorescence microscopy. The immunoslot blot assay wasused to analyse N7-EtOHGua levels in white blood cell DNA fromindividuals exposed to EtOx (2–5 p.p.m.) and of controls.This exposure did not result in a statistically significantincrease in the N7-EtOHGua level.     

Polycyclic aromatic hydrocarbon-DNA adducts in smokers and their relationship to micronutrient levels and the glutathione-S-transferase M1 genotype

Sixty-three male cigarette smokers were entered into a cross-sectionalstudy to determine whether inverse associations existed betweenpolycydlic aromatic hydrocarbon (PAH)—DNA adduct levelsand intake/serum levels of vitamin A, vitamin C and vitaminE. Associations between PAH–DNA adducts and intakes ofcarotene, as well as serum levels of ß-carotene, werealso determined. Fasting blood samples were collected for assaysof PAH—DNA adducts in circulating mononuclear cells, plasmacotinine and serum levels of vitamin A, ß-carotene,vitamin C and vitamin E. Since genetic deficiency in the detoxifyingenzyme glutathione S-traasferase M1 (GSTM1) has been associatedwith increased risk of lung cancer, GSTM1 genotype was alsodetermined. Analysis of PAH—DNA adducts by competitiveenzyme-linked immunosorbent assay (ELISA) indicated that 70%of the subjects had detectable adducts, with a mean of 4.38adducts/10⁸ nucleotides (range 1.00–24.1/10⁸ Pearson'smethod was utilized to determine whether any associations existedbetween the various host variables and PAH—DNA adducts.Previously, no significant associations were found between PAH—DNAadducts and cigarettes smoked/day, pack-years, daily/life timetar exposures or plasma cotinine levels (Santella et al., Carcinogenesis,13, 2041–2045, 1992). PAH—DNA adducts were inverselyassociated with serum cholesterol-adjusted vitamin E levels(r = –0.25, P     

Determination of stereospecificity of benzo[a]pyrene diolepoxide-DNA antisera with site-specifically modified oligonucleotides

Antisera developed against benzo[a]pyrene diolepoxide (BPDE)—DNAadducts are sensitive tools for detection of DNA adducts inhuman samples. All antisera currently used for biomonitoringstudies were produced against DNA or guanosine modified withracemic anti-BPDE. Using a non-competitive enzyme-linked immunosorbentassay (ELISA), Venkatachalam and Wani (Carcinogenesis, 15, 565–572,1994) recently tested polyclonal and monoclonal (5D2) antiserafor cross-reactivity against oligonucleotides containing (+)-and (–)-trans-anti-BPDE-N²-guanine or N⁶-adenine adductsand showed different stereospecificity for the two antisera.Because of the importance of antiserum specificity in humanbiomonitoring studies, we have tested several monoclonal (Mab5D11 and 5D2) and polyclonal (Pab #29) antisera developed againstracemic anti-BPDE-DNA adducts, and Mab 8E11 developed againstanti-BPDE-guanosine adducts. Stereoisomeric anti-BPDE-modifiedoligonucleotide adducts in the sequence 5'-d(CCAT-CG^*CTACC)-3'where G^* = anti-BPDE-N²-dG with (+ )-trans, (–)-trans,(+ )-cis and (–)-cis adduct stereochemistry at the C10position of anti-BPDE were tested by competitive ELISA. Twostructurally related 5-methylchrysene diolepoxide adducts withG^* = (+)- and (–)-trans-anti-5-MeCDE-N²-dG in the sameoligonucleotide were also tested. While Mab5D2 had the highestaffinity for the (–)-trans-anti-BPDE-modified oligomer,Mab 5D11 and 8E11 and Pab #29 recognized the (+ )-trans-anti-BPDE-modifiedoligomer better than the (–)-trans-anti-BPDE modifiedoligomer. Mab 5D11 and Pab #29 recognized racemic anti-BPDE-modifiedDNA adducts better than trans-anti-BPDE-modified oligonucleotides;however, Mab 8E11 showed similar sensitivity to racemic anti-BPDE-DNAadducts and (+ )-and (–)-trans-anti-BPDE-modified oligomers.All antisera exhibited lower reactivities with both 5-MeCDEmodified oligomers. Because of their sensitive detection of(+)-trans-anti-BPDE-dG adducts, the primary adduct producedin vivo, Mab 8E11 and 5D11 and Pab #29 are appropriate for measurementof most adducts formed in humans.     

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.     

Consequences of 3-methylcholanthrene-type induction for the metabolism of 4-aminobiphenyl in isolated rat hepatocytes

Carcinogenic aromatic amines such as 4-aminobiphenyl (4-ABP)are extensively metabolized by both oxidative and conjugationreactions. Thus the burden of genotoxic metabolites of 4-ABPin a target organ is probably influenced by the balance of N-hydroxylationand alternative metabolic pathways in the hepatocyte. In freshlyisolated rat hepatocytes, 4-ABP (at a substrate concentrationof 10 µM) was mainly N-acetylated (54% of total metabolites),while 2% Ar-hydroxy-4-ABP-N-glucuronide and 21% of unconjugatedN-hydroxylated metabolites were detectable. Ring-hydroxylatedmetabolites and the primary N-glucuronide of 4-ABP accountedfor 8% and 4%, respectively. Pretreatment of rats with 3-methylcholanthrene(MC), a dioxin-type inducer of CYP1A isozymes and phenol UDP-glucuronosyltransferase(UGT1A1), led to a dramatic decrease of W-acetylated (2% oftotal metabolites) and an increase of N-hydroxylated (54% asfree and glucuronidated compound) and ring-hydroxylated (35%)metabolites. Essentially similar effects were seen at a substrateconcentration of 50 µM. Consistently, MC-type inductionwith ß-naphthoflavone resulted in a significant increasein the formation of DNA adducts of 4-ABP, detected by ³²P-postlabellingof hepatocellular DNA. The results suggest that, similar toa previous study with 2-naphthylamine (2-NA), MC treatment leadsto a marked shift from conjugation to N-oxidation. However,N-hydroxy-4-ABP (in contrast to N-hydroxy-2-NA) is mostly releasedfrom hepatocytes in the unconjugated form.     

DNA adduct formation by tamoxifen with rat and human liver microsomal activation systems

Using microsomal preparations from rat and human liver, we investigatedthe activation of the anti-estrogen compound tamoxifen (TMX)to form DNA adducts. Pretreatment of rats with phenobarbitalincreased DNA adduct formation by microsomal activation of TMX3- to 6-fold, depending on the cofactors used. When reducednicotinamide-adenine dinucleotide phosphate (NADPH) was usedas a cofactor in human and rat microsomal activation systems,the relative DNA adduct levels were 2.9 and 5.2 x 10^–8respectively and 1-3 TMX-DNA adducts were detected by ²³P-postlabeling;DNA adduct 1 was the same in both microsomal systems. When cumenehydroperoxide (CuOOH) was used as a cofactor, activation ofTMX produced four major DNA adducts and several minor DNA adductsin both rat and human liver microsomes; the relative adductlevels were 11.1 and 23.1 xlO^–8 respectively. TMX-DNAadducts 1, 4, 5 and 6 were similar in both human and rat microsomalsystems with CuOOH as the cofactor. The TMX-DNA adducts formedwith NADPH as the cofactor were clearly different from thoseformed with CuOOH as the cofactor, which implies that the metabolitesleading to the individual DNA adducts were different. Additionof a P450 inhibitor, either n-octylamine or     

Production of monoclonal antibodies to guanine imidazole ring-opened aflatoxin B1DNA, the persistent DNA adduct in vivo

Monoclonal antibodies were produced following immunisation ofmice with guanine imidazole ring-opened aflatoxin B₁ DNA (iroAFB₁ DNA), coupled electrostatically to methylated keyhole limpethaemocyanin. Three monoclonal hybridoma lines producing antibodiesspecific for iro AFB₁ DNA were grown as ascites tumours andsuitable dilutions of the ascitic fluid (1:8000–1:50 000)used in a competitive enzyme linked immunosorbent assay (ELISA)to measure reactivity of the antibodies to a variety of aflatoxinand nucleic acid-related compounds. These antibodies recogniseAFB₁ bound to DNA at levels 10⁴–10⁵ times lower concentrationthan unmodified calf thymus DNA or 8,9-dihydro-8,9-dihydroxy-aflatoxinB₁; and show 2–5 times the affinity to iro AFB₁ DNA comparedto AFB₁ DNA. The concentration of AFB₁ in iro AFB₁ DNA producing50% inhibition in a competitive ELISA was 1.8 x 10^–7 molar.Using the most sensitive hybridoma line, levels of 1 adductin 300 000 nudeotides would be detectable, which is the levelof binding found in the rat and hamster in vivo. These monoclonalantibodies should therefore prove useful in detecting theselesions in animal and human tissue samples exposed to aflatoxins.     

Genotoxic effects of cyclopenta-fused polycyclic aromatic hydrocarbons in isolated rat hepatocytes and rabbit lung cells

Benz[j]aceanthrylene (B[j]A) and benz[l)aceanthrylene (B[l]A),two isomeric cyclopenta polycyclic aromatic hydrocarbons (CP-PAH)structurally related to 3-methylcholanthrene, were studied withrespect to their genotoxic effects in isolated liver and lungcells. Both compounds were found to cause DNA adducts measuredby the ³²P-postlabelling technique. The level of DNA-adductsin rat hepatocytes exposed to 30 µg/ml B[l]A and B[j]Afor 4 h were 46.5 ± 22.0 and 8.3 ± 5.1 fmol/µgDNA respectively. Using butanol extractions, the major and oneof the minor B[l] A adducts co-chromatographed with B[j]A-l,2-oxide adducts of 2'-deoxyadenosine and 2'-deoxyguanosine.Thus, oxidation at the cyclopenta-ring of B[j]A appears to bean important activation pathway. In hepatocytes, 3-30 µg/mlof B[j]A and B[j]A induced DNA damage and repair measured bothas increased alkaline elution of DNA and as increased incorporationof [³H]TdR in the DNA. B[l]A was somewhat more potent than B[j]Ain inducing DNA repair. Reactive CP-PAH intermediates formedin the hepatocytes caused mutations in Salmonella typhimuriumTA98 upon co-incubation. DNA adducts were also observed in isolatedrabbit lung cells exposed to 30 µg/ml B[l]A or B[j]A for2 h. A total of 14.5 ± 6.9, 2.9 ± 2.1 and 0.2± 0.6 fmol B[l] A adducts/µg DNA were observedin Clara cells, type II pneumocytes and alveolar macrophagesrespectively. The main B[l]A adduct observed in the liver cellswas not found in the lung cells. On the other hand, the levelsof B[j]A adducts in the lung cells were in the range 4-14% ofthat found in liver cells, and no major differences betweenthe various lung cells were observed. Neither B[l]A nor B[j]Ainduced DNA damage measured by alkaline elution in the lungcells, indicating that these adducts are not alkali labile.     

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.     

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.     

Covalent DNA adducts formed in mouse epidermis by benzo(g)chrysene

The metabolic activation in mouse skin of benzo(g)chrysene (B(g)C),a moderately carcinogenic polycyclic aromatic hydrocarbon (PAH)present in coal tar, was investigated. Male Parkes mice weretreated topically with 0.5 µmol B(g)C and DNA was isolatedfrom the treated areas of skin at various times after treatmentand analysed by 32P-post-labelling. Seven major adduct spotswere detected, at a maximum level of 6.55 fmol adducts/µgDNA. Mouse skin treated with the PAH benzo(c)phenanthrene (B(c)Ph)gave a total of 0.24 fmol adducts/µg DNA. B(g)C-DNA adductspersisted in skin for at least 3 weeks. Treatment of mice with0.5 µmol of the optically pure putative proximate carcinogens,the (+)- and (–)-trans benzo(g)chrysene-11, 12-dihydrodiols,led to the formation of adducts which co-migrated on TLC andHPLC with those formed in B(g)C-treatedmice, which suggestedthat the detected adducts were formed by the fjord region B(g)C-11,12-dihydrodiol-13, 14-epoxides (B(g)CDEs). To test this, thefour optically pure synthetic B(g)CDEs were reacted in vitrowith DNA and the heteroco-polymers poly(dA.dT) and poly(dG-dC)and these samples 32P-postlabelled. Co-chromatography, onbothTLC and HPLC, of in vitro and in vivo adducts indicated thatB(g)Cis activated in mouse skin through formation of the (–)-anti-(11R,12S,13S,14R) and (+)-syn-(11S, 12R,13S,14R) B(g)CDEs. (–)-anti-B(g)CDEformed five adducts with DNA, two of them with adenine and threewith guanine bases. (+)-syn-B(g)CDE formed one adduct with eachof these bases in DNA. The adenine adducts accounted for 64%of the total major adducts formed in B(g)C-treated mouse skin.The route of metabolic activation of B(g)C is similar to thatreported for B(c)Ph, but the extent of activation to the fjordregion diol-epoxides is significantly greater in the case ofB(g)C, as demonstrated by the higher levels of adduct formationin vivo.     

On the maternal transfer of 4-aminobiphenyl in rats

The potential for 4-aminobiphenyl (4-ABP) to be transferredfrom circulating blood into the milk of lactating Sprague—Dawleyrats was determined. The distribution of ¹⁴C-labeled 4-ABP intomilk was examined at time intervals of < 1, 20, 60, 120,240 and 480 min after i.v. dose administration. Eliminationof radioactivity from blood and milk was determined to be biphasic.The levels of 4-ABP and/or metabolites were lower in milk thanin blood at all time points examined. The levels of radioactivitydetected in blood declined less rapidly than in milk. That is,the percent of the dose per ml of blood declined from 0.81–0.45,while the percent of the dose per ml of milk declined from 0.38–0.06during the 8 h time period. The radioactivity present in milkwas partially extractable with ethyl acetate with 43% of theradioactivity being extractable at the earliest time point whileonly 16% was extractable after 8 h. The level of radioactivityassociated with the protein precipitate of the milk samplesincreased from 4–21% within 4 h after treatment. The potentialof 4-ABP or its metabolites to exert a genotoxic effect on newbornpups via maternal transfer was also examined. Dams were treatedon day 1 post partum and then daily with 4-ABP (10 mg/kg) incorn oil or corn oil alone for 2 weeks. Each experimental grouphad four litters of pups each containing 5 pups. Pups were sacrificedat 15 days of age, separated by sex and the levels of 4-ABP:DNA adducts in liver determined using ³²P-postlabeling. DNAadduct profiles were similar between male and female pups withtotal adduct levels of 332 and 338 fmol of adducts/mg of DNA,respectively. These results indicate that the genotoxic effectsof 4-ABP can be transmitted from exposed dams to the nursingoffspring.     

Association between DNA strand breaks and specific DNA adducts in murine hepatocytes following in vivo and in vitro exposure to N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene

N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene(N-OAc-AAF) have previously been shown to induce dose-dependentDNA strand breaks in primary hepatocytes from mice and rats.In an attempt to determine the relationship between the extentof DNA strand breaks and the formation of specific DNA-carcinogenbound adducts in murine liver, the capability of N-OH-AAF andN-OAc-AAF to induce both DNA single strand breaks and adductformation in in vivo and in primary hepatocytes was measured.N-OH-AAF induced a low level of DNA damage in F344 rats (10mg/kg, i.p.) and in B6 mice (40 mg/kg, i.p.) 4 h after treatment.The DNA adducts identified in vivo were N-(guanin-8-yl)-2-acetylaminofluorene(Gua-C8-AAF) 55% versus 11%, N-(guanin-8-yl)-2-aminofluorene(Gua-C8-AF) 34% versus 67% and Mguanin-N²-yl)-2-acetylaminofluorene(Gua-N²-AAF) 11% versus 10%, respectively, for rat and mouseliver. An additional unknown adduct (12%) was detected in mouseliver. Dose dependent DNA binding and formation of individualDNA adducts were observed in rat and mouse primary hepatocytesfollowing 1 h exposure to [ring-³H]-N-OH-AAF (0.1-20 µM)and [ring-³-N-OAc-AAF (5–20 /M). The patterns of DNA adductsin mouse and rat primary hepatocytes exposed to N-OH-AAF andN-OAc-AF were similar to those obtained in liver following invivo treatment with N-OH-AAF. The deacetylase inhibitor, paraoxon(10^–4M) completely inhibited DNA damage induced by N-OH-AAFin mouse and partially in rat hepatocytes while DNA damage causedby N-OAc-AAF was only partially inhibited by paraoxon (10^–4M) in both species. Parallel experiments showed that paraoxon,at low concentration (10^– M), did not alter either thelevel of DNA binding or the pattern of adduct formation in rathepatocytes treated with N-OH-AAF (20 µM). However, at10^–4 M paraoxon partially blocked DNA binding (60%) andthe formation of Gua-C8-AAF (95%) and Gua-N²-AAF (80%) whileGua-C8-AF was increased twofold. In mouse hepatocytes paraoxonpretreatment (10^–4M) inhibited the formation of Gua-C8-AFby 70% following exposure to N-OH-AAF (20 µM). Gua-C8-AAFand Gua-N²-AAF were also inhibited but only at 10^–4M paraoxon.Paraoxon (10^–6 and 10^–4 M) pretreatment induceddosedependent partial inhibition of the covalent binding ofN-OAc-AAF to rat DNA and the formation of all guanine adducts.In the mouse, paraoxon (10^–6 and 10^–4 M) inhibitedthe formation of Gua-C8-AF while it increased Gua-C8-AAF. Theseresults indicate that a positive correlation exists betweenthe extent of DNA strand breaks and the formation of eitherGua-C8-AAF or Gua-C8-AF.     

Persistence, gestation stage-dependent formation and interrelationship of benzo[a]pyrene-induced DNA adducts in mothers, placentae and fetuses of Erythrocebus patas monkeys

Since DNA adducts have been detected in the placentae of pregnantwomen who smoke cigarettes, the importance of these adductsas biomarkers of fetal exposure and risk has been evaluatedusing a non-human primate as a model. Pregnant Erythrocebuspatas monkeys on days 50, 100 or 150 of gestation (term = 160± 5 days) were treated once with 5–50 mg/kg benzo[a]pyrene(B[a]P), p.o. Fetuses were removed by Cesarean section 1–50days after treatment and analyzed for DNA adducts by the nucleaseP₁ version of the ³²P-postlabeling method. B[a]P induced highlevels of DNA adducts in all fetal organs, placentae and maternallivers in all three trimesters of gestation. DNA adduct levelswere higher in mid-gestation compared to early and late gestation.The major adduct detected was 10ß-(deoxyguanosin)-N²-yl-7ß,8