32P-Postlabelling of diastereomeric 7-alkylguanine adducts of butadiene monoepoxide

The reaction of 3, 4-epoxy-1-butene (BMO) with deoxygu-anosine-3'-monophosphate(3'-dGMP) resulted in the formation of two pairs of diastereomeric7-alkyl-3'-dGMP derivatives corresponding to two isomers C'-1and C'-2. The T4 polynucleotide kinase-mediated phosphorylationwith (     

32P-Postlabelling analysis of the DNA adducts formed by aristolochic acid I and II

We report the quantitation of DNA adducts in target and non-targetorgans of male Wistar rats treated orally with five daily doses(10 mg/kg body wt) aristolochic acid I (AAI) or aristolochicacid II (AAII), the major components of the herbal drug aristolochicacid, a forestomach carcinogen In the rat. DNA adducts weredetected and analysed using the nuclease P1-enhanced variationof the Randerath 32 postlabeiling assay. The highest level ofDNA adducts formed was by AAI inthe target organ, forestomach(330 ± 30 adducts/10⁸ nucleotides), but high levels werealso observed in a non-target tissue, the glandular stomach(180 ± 15). Lower amounts of adducts were detected inliver, kidney and urinary bladder epltheliuin. With AAII thebinding Levels were generally lower than the AAII, the highestLevel of adducts being detected in kidney (80 ± 20 adducts/10⁸nucleotides) and lower levels in liver, stomach and urinarybladder epithelia. Adduct patterns similar to those in vivowere observed in two new in vitro assays. Rat faecal bacteriawere shown to be able to activate AM and AAII to reactive species,which were trapped with exogenous calf thymus DNA and analysedby postlabelling. llncuhatlon of AM and AAII in explanted ratstomach held in short-term organ culture resulted In DNA adductformation in the epithelia of both forestomach and glandularstomach. To assign the recently characterized in vitro nucleosideadducts of AII to the bisphosphate derivatives, a new ion-pairH]PLC procedure on a reversed-phase column was developed. Bymonitoring Cerenkov radiation on-line, a good separation ofAII adducts was observed, demonstrating that adducts formedin vivo were chromatographically indistinguishable with thoseformed in vitro, and previously characterized as an aristolactammoiety bound covalently to the exocydlic amino groups of deoxyadenosineand deoxyguanosine.     

32P-Postlabelling of DNA adducts formed by allyl glycidyl ether in vitro and in vivo

32P-Postlabelling analysis of allyl glycidyl ether-treated DNA after adduct enrichment on anion-exchange cartridges revealed two major and one minor DNA adducts. The major adducts were shown to originate from alkylation at N-7-guanine and N-1-adenine, respectively, while the minor adduct was at N-3-cytosine. In addition, rearrangement products of the 1-adenine and 3-cytosine adducts to N6-adenine and 3-uracil were indicated. The relative amounts of adenine, cytosine and uracil products appeared to be dependent upon conditions (in particular pH) during sample processing and analysis. When nuclease P1 was used for adduct enrichment the adenine, cytosine and uracil adducts, but not the 7-guanine adduct, were detected. The labelling efficiency of the 7- guanine adduct standard was 40-45%. Total recovery of this adduct from allyl glycidyl ether-modified DNA was 9-12%. The labelling efficiency of the 1-adenine adduct standard was 78-82%. Total recovery of this adduct from DNA was approximately 20% when using anion-exchange chromatography for adduct enrichment and 30-34% when using nuclease P1. Preliminary analysis of DNA from mice treated with allyl glycidyl ether indicated 57 times higher level of the 7-guanine adduct, per unit dose, in skin DNA (120 per 10(8) normal nucleotides) after topical application when compared to liver DNA after i.p. administration. The 1- adenine adduct could not be quantified in liver DNA (due to an interfering background product present in untreated animals) and the level of the 3-cytosine adduct was below the detection limit of the method. After topical application the level of the 1 adenine adduct in skin DNA was approximately 30 per 10(8), using either column or nuclease P1 enrichment. The 3-cytosine adduct was detected in skin, but was not quantified.      

32P-Postlabelling analysis of aromatic DNA adducts in human oral mucosal cells

Exfoliated mucosal cells were collected from the oral cavityof three groups at high risk for oral cancer: Indian betel nutchewers, Filipino inverted smokers (burning end of cigar inmouth) and Indian Khaini tobacco chewers. DNA was extractedfrom these samples, as well as from samples of exfoliated cellsof Canadian non-smoking controls. DNA was analyzed for the presenceof aromatic DNA adducts using ³²P-postlabel-ling analysis. Fivechromatographically distinct adducts were found in samples fromboth the high risk groups and the nonsmoking controls. Individualadducts were detectable in 30–95% of samples, dependingon the adduct and population group. Estimated levels of specificadducts ranged from non-detectable (prevalence relative to normalnucleotides < 1 x 10^-9) to occasionally > 1x 10^-7. Noadducts were found in high risk groups which did not also appearin control subjects.     

32P-postlabelling analysis of isomeric 7-alkylguanine adducts of styrene oxide

Styrene 7,8-oxide, which reacts preferentially at the N-7 position of guanine, yielded two pairs of diastereomers 7-(1'-hydroxy-2'- phenylethyl)-dGMP (the alpha-isomer) and 7-(2-hydroxy-2-phenylethyl)- dGMP (the beta-isomer) on reaction with deoxyguanosine-3'-monophosphate (3'-dGMP). The alpha- and beta-isomers were formed in the ratio 32:68. T4 polynucleotide kinase preferentially mediated labelling of diastereomers corresponding to the beta-isomer. The beta-diastereomers showed a labelling efficiency of 52%, whereas the alpha-isomers showed a labelling efficiency of 4%. Molecular modelling experiments showed intrinsic differences between the two isomers. The torsion angles of C8- N7-2'-Ar and C8-N7-2'-1' for the alpha-isomers were 149.9 degrees and - 26.4 degrees, whereas the torsion angles of C-8-N7-1'-2' and N7-1'-2'- Ar for the beta-isomers were 105.3 degrees and 179.4 degrees. The consequent interatomic distance between one of the hydrogens on the alpha-carbon and the 3'-phosphate group on the sugar residue was 5.3 A in the alpha-isomer whereas the closest distance between the hydrogens attached to the alpha-carbon and 3'-phosphate group in the beta-isomer was 6.2 A. This arrangement probably leads to steric overcrowding at the 3'-phosphate group in alpha-isomers and these are less efficiently phosphorylated than beta-isomers. In in vitro styrene oxide-modified salmon testis DNA alpha- and beta-isomers of 7-alkylguanines were formed in the ratio 37:63. The recovery of two diastereomeric beta- isomers in a 32P-postlabelling assay was 14%, but one of the diastereomers was obtained in 3-fold greater yield than the second isomer.      

Identification of the major adducts formed by reaction of 5-methylchrysene anti-dihydrodiol-epoxides with DNA in vitro

5-Methylchrysene is metabolically converted to the bay-region dihydrodiol-epoxides, trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (DE-I), in which the methyl group and the epoxide ring are in the same bay region, and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-5-methylchrysene (DE-II). Previous studies have indicated that DE-I is more important in 5-methylchrysene carcinogenesis than is DE-II. Both DE-I and DE-II were individually reacted with calf thymus DNA in vitro. The DNA was enzymatically hydrolyzed to deoxyribonucleosides, and the modified deoxyribonucleosides were separated by chromatography on Sephadex LH-20 and analyzed by high-performance liquid chromatography. One major adduct and seven minor adducts were formed from each dihydrodiol-epoxide. The major adduct was, in each case, characterized by its pH-dependent partition coefficient, stability to base, mass spectrum, ultraviolet spectrum, and nuclear magnetic resonance spectrum as a deoxyguanosine derivative resulting from addition of the exocyclic amino group of deoxyguanosine to the benzylic carbon of the epoxide ring of the dihydrodiol-epoxide. The results of this study show that the major DNA adducts formed from 5-methylchrysene via DE-I and DE-II are structurally similar.     

32P-Postlabelling determination of DNA adducts of malonadlehyde in humans: total white blood cells and breast tissue

DNA adducts of malonaldehyde (MA) were measured in total whiteblood cells (TWBC) fo healthy individuals from both sexes anddifferent ages, as well as in breast tissue (BT) samples obtainedfrom, healthy females undergoing reduction mammaplasty. A largeinterindividual variation in adduct levels was observed. Theaverage adduct level found in TWBC, considering both sexes andall ages was 2.6 ± 1.2 adducts/10⁷ nucleotides (n = 26).A similar average DNA adduct concetratioon was found in BT andamounted to 3.0 ± 1.3 (n = 7) adducts/10⁷ nucleotides.Our results whow that DNA adducts of MA can be measured in humansusing ³²P-postlabelling in combination with nucleas P1 and reversed-phaseHPLC as adduct enrichment procedures, and furher validate theseadducts as suitable biomarkers for the measurement of DNA damageinflicted by endogenously induced oxidative processes such aslipd peroxidation.     

32P-HPLC suitable for characterization of DNA adducts formed in vitro by polycyclic aromatic hydrocarbons and derivatives

Analysis of DNA adducts demands both high sensitivity and goodresolution. A high-performance liquid chromato-graphy methodfor ³²P-postlabeled DNA adducts (³²P-HPLC) was used to investigateDNA adduct formation from 38 polycyclic hydrocarbons and biphenylsin vitro. The ³²P-HPLC method proved to be useful for separation,detection and characterization of DNA adducts from most of thesubstances. The in vitro method used to form the DNA adducts,with calf thymus DNA, nucleotide 3'-phosphates and metabolicactivation through S-9 liver homo-genate, gave poor quantitativereproducibility. However, the results showed that the ³²P-HPLCmethod was suitable for characterizing DNA adducts from manysubstances. From 35 of the tested substances 365 DNA and nucleotide3'-phospate adducts were detected and characterized concerningretention times. Of the adducts, 171 were detected in DNA and39 of them from five substances were characterized concerningtarget nucleotides. The retention time library built can beused in future analyses of DNA with complex patterns of DNAadducts.     

32P-post-labelling analysis of DNA adducts formed by aristolochic acid in tissues from patients with Chinese herbs nephropathy

Recently, we reported that aristolochic acid (AA) a naturally occurring nephrotoxin and carcinogen is implicated in a unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN). Indeed, we identified the principal aristolochic acid-DNA adduct in the kidney of five such patients. We now extend these observations and demonstrate the presence of additional AA-DNA adducts by the 32P-post-labelling method not only in the kidneys, but also in a ureter obtained after renal transplantation. Using the nuclease P1 version of the assay not only the major DNA adduct of aristolochic acid, 7-(deoxyadenosin-N6-yl)- aristolactam I (dA-AAI), but also the minor adducts, 7-(deoxyguanosin- N2-yl)-aristolactam I (dG-AAI) and 7-(deoxyadenosin-N6-yl)-aristolactam II (dA-AAII) were detected, and identified by cochromatographic analyses with TLC and HPLC. Quantitative analyses of six kidneys revealed relative adduct levels from 0.7 to 5.3/10(7) for dA-AAI, from 0.02 to 0.12/10(7) for dG-AAI and 0.06 to 0.24/ 10(7) nucleotides for dA-AAII. The detection of the dA-AAII adduct is consistent with the occurrence of aristolochic acid II (AAII) in the herb powder imported under the name of Stephania tetrandra and confirms that the patients had indeed ingested the natural mixture of AAI and AAII. 32P-post- labelling analyses of further biopsy samples of one patient showed the known adduct pattern of AA exposure not only in the kidney, but also in the ureter, whereas in skin and muscle tissue no adduct spots were detectable. In an attempt to explain the higher level of the dA-AAI adduct compared to the dG-AAI adduct level in renal tissue even 44 months after the end of regimen, the persistence of these two purine adducts was investigated in the kidney of rats given a single oral dose of pure AAI. In contrast to the dG-AAI adduct, the dA-AAI adduct exhibited a lifelong persistence in the kidney of rats. Our data demonstrate that AA forms DNA adducts in human tissue by the same activation mechanism(s) reported from animal studies. Thus, the carcinogenic/mutagenic activity of AA observed in animals could also be responsible for the urothelial cancers observed in two of the CHN patients.      

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.     

Optimization of an HPLC method for analyses of 32P-postlabeled DNA adducts

A further development of an HPLC method to analyze ³²P-postlabeledDNA adducts is presented. The method is based on on-line detectionof ³²P radioactivity after separation by reversed-phase chromatography.The method has an advantage in that the postlabeling mixturecan be injected directly into the HPLC system without any priorpurification, with the background radioactivity on a low level.The analysis includes the whole range of substances from orthophosphateto non-polar DNA adducts, which makes it possible to analyzenormal nucleotides and ATP together with DNA adducts. The analyticalsystem has a high reproducibility and separates complex mixturesof DNA adducts. The slightly lower sensitivity compared to theTLC method is compensated for by the possibility of injectinglarge amounts of DNA into the system without affecting the analyticalproperties. The system can be applied to different DNA adductsas well as complex mixtures of DNA adducts.     

Structural characterization of the major adducts formed by reaction of 4,5-epoxy-4,5-dihydro-1-nitropyrene with DNA.

The only available marker of DNA adducts formed from 1-nitropyrene (1-NP) and DNA, N-(deoxyguanosin-8-yl)-1-aminopyrene, is derived from the nitroreduction pathway. Our studies, as well as those of others, have indicated that multiple DNA adducts are formed from 1-NP in vivo and in vitro. Thus the need for additional DNA adduct markers was apparent. Therefore, it was our goal to characterize the DNA adducts formed from 4,5-epoxy-4,5-dihydro-1-nitropyrene, a metabolite of 1-NP. The epoxide was incubated with calf thymus DNA (pH 5.4). The DNA was enzymatically hydrolyzed to deoxyribonucleosides which were analyzed by reverse phase HPLC. Three major peaks were obtained in yields less than 5%. The structural assignment of these adducts was made by comparison of their proton nuclear magnetic resonance spectra with those of cis- and trans-4,5-dihydro-4.5-dihydroxy-1-nitropyrene, and by long range coupling constants, decoupling experiments, D2O exchange, partitions and acid hydrolysis. Two adducts result from trans and one from cis addition of the N2-exocyclic amino group of deoxyguanosine to the C5-benzylic carbon of the epoxide ring. This is the first report that describes the structure of the DNA adducts formed with a ring-oxidized metabolite of 1-NP. On the basis of this finding we suggest that K-region oxides of 1-NP may be responsible for the formation of the putative 1-NP-DNA adducts in vivo.     

Characterization of DNA adducts formed by aristolochic acids in the target organ (forestomach) of rats by 32P-postlabelling analysis using different chromatographic procedures

We report the analysis of DNA adducts in the target organ (forestomach)of male Sprague–Dawley rats treated orally with two doses(10 mg/kg body wt) per week for 2 weeks of either aristolochicacid I (AAI), aristolochic acid II (AAII) or the plant extractaristolochic acid (AA). DNA adducts were detected and quantitatedusing the nuclease P1-enhanced version of the ³²P-postlabellingassay. For identification of adducts, reference compounds wereprepared by reaction of enzymatically activated AAI and AAIIwith 3'-purine phosphonucleosides and analysed by the ⁿ-butanolenrichment procedure. These reference compounds were assignedto the previously characterized DNA adducts of AAI [7-(deoxy-guanosin-N²-yl)-aristolactamI = dG-AAI, 7-(deoxyadenosin-N⁶-yl) I = dA-AAI] and AAII [7-(deoxyadenosin-N⁶-yl)-aristolactamII = dA-AAII]. Cross referencing of the carcinogen-modifiednucleoside bisphosphates obtained from forestomach DNA withthe synthetic standard compounds by ion-exchange chromatographyand reversed-phase HPLC demonstrated that the major DNA adductsformed by AAI and AA were identical to dG-AAI and dA-AAI. Likewise,forestomach DNA isolated from AAII-treated rats showed two purinederived adduct spots, the major one being dA-AAII, the minorone being tentatively identified as 7-(deoxyguanosin-N²-yl)-aristolactamII. A minor adduct detected in forestomach DNA of rats treatedwith AAI was found to be chromatographically indistinguishablefrom the adduct identified as dA-AAII, indicating a possibledemethoxylation reaction of AAI. Quantitation of DNA adductsrevealed that in in vitro reactions with 3'-phosphonucleosidesthe adduct levels were approximately one order higher for bothAAI and AAII-derived adducts than in forestomach DNA modifiedwith AAI or AAII in vivo. In vitro as well as in vivo adductionby AAI was more efficient than adduction by AAII. The patternof adduct spots obtained from forestomach DNA of rats treatedwith the plant extract AA reflected the composition of the extractdetermined by HPLC analysis. Irrespective of the aristolochicacid used to induce DNA adducts, deoxyadenosine is the majortarget of modification, pointing to the general importance ofdeoxyadenosine adducts for chemical carcinogenesis of thesenaturally occurring products. This study shows that the combinationof two independent chromatographic systems considerably enhancesthe fidelity of identification of DNA adducts with the ³²P-posthbellingassay.     

Analysis of 7-methylbenz[a]anthracene-DNA adducts formed in SENCAR mouse epidermis by 32P-postlabeling

The present study has analysed the DNA adducts formed in SENCAR mouse epidermis following topical application of 7-methylbenz[a]anthracene (7- MBA). Mice were treated with 400 nmol of 7-MBA, which represents an initiating dose of this hydrocarbon for SENCAR mice. DNA adducts were analysed 24 h after topical application of the hydrocarbon by 32P- postlabeling coupled with either HPLC analysis or an improved TLC procedure giving better resolution of DNA adducts through the use of a D6 solvent [isopropanol:4N NH4OH (1:1)] following D5. Twenty-four hours after topical application of 400 nmol 7-MBA, the level of total covalent binding was 0.37 +/- 0.07 pmol/mg DNA as determined by 32P- postlabeling. This level of binding correlated well with the relative tumor initiating activity of this hydrocarbon compared to 7,12- dimethylbenz[a]anthracene (6.4 +/- 0.01 pmol/mg DNA) and dibenz[a,j]anthracene (0.03 +/- 0.01 pmol/mg DNA). Analysis of the 32P- labeled 3',5'-diphosphodeoxyribonucleosides by HPLC and TLC revealed the presence of deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts formed from both the anti- and syn-bay-region diol-epoxides of 7-MBA (anti- and syn-7-MBADEs). The major DNA adduct derived from 7-MBA in mouse epidermis was tentatively identified as (+) anti-7-MBADE-trans-N2- dGuo. In addition, a minor dGuo adduct derived from the bay-region syn- diol-epoxide of 7-MBA was detected as well as a minor dAdo adduct from this diol-epoxide. Another minor dAdo adduct was also detectably present which arose from either the anti- or syn-diol epoxide. Furthermore, several unidentified DNA adducts were present in both HPLC and TLC chromatograms of DNA samples from 7-MBA-treated mice. These results are discussed in terms of the role of specific 7-MBA-DNA adducts in tumor initiation by this hydrocarbon.      

Development of a 32P-postlabelling method for the analysis of adducts arising through the reaction of acetaldehyde with 2'-deoxyguanosine-3'-monophosphate and DNA

A ³²P-postlabelling assay was developed for the analysis ofadducts arising from the reaction of 2'-deoxyguanosine-3'-monophosphatewith acetaldehyde, the primary oxidative metabolite of ethanol.The ³²P-postlabelling reaction was optimized by testing variousparameters such as the kinetics of phosphorylation by T₄ polynucleotidekinase, substrate-concentration-dependent labelling efficiencyand the concentration of the various ingredients of the phosphorylationreaction. The sensitivity to 3'-monophosphate dephosphorylationactivity of nuclease P1 was also studied. Three stable adductswere separated by reversed-phase HPLC. The major stable adductwas structurally characterized and identified as N²-ethyl-2'-deoxyguanosineand could be detected, after reduction with NaBH₄ or a mixtureof ascorbic acid and GSH, in calf thymus DNA samples that hadbeen reacted in vitro with acetaldehyde. DNA adducts were isolatedafter enzymatic digestion to mononucleotides followed by nucleaseP1 digestion of normal nucleotides. The average levels of acetaldehyde-DNAadducts detected in these samples were 12.1 2.3 (n = 17) and4.9 0.9 (n = 9) adducts/10⁷ nucleotides after reduction withNaBH₄, or ascorbic acid and GSH respectively. The ³²P-postlabellingmethod was further validated by the detection of acetaldehydeadducts in liver DNA from mice treated with ethanol. The averageconcentration of the adducts detected in these animals was 1.5 0.8 (n = 7) adducts/10⁸ nucleotides, as analyzed by reversed-phaseHPLC with online detection of radioactivity.     

Storage phosphor imaging technique for detection and quantitation of DNA adducts measured by the 32P-postlabeling assay.

The 32P-postlabeling method has found wide application as a sensitive technique for detecting the presence of a broad range of bulky aromatic compounds covalently bound to DNA. In this method, the modified DNA is enzymatically degraded to 3'-mononucleotides and labeled with [32P]-phosphate at the 5'-position using [gamma-32P]ATP and T4 polynucleotide kinase. The 32P-labeled DNA digest is then chromatographed in two dimensions on polyethyleneimine - cellulose thin-layer plates. Screen-enhanced autoradiography is used to locate the presence of the radiolabeled adducts on the chromatogram, and the radioactive areas are generally excised and quantitated by liquid scintillation spectrometry. However, on a chromatogram with multiple adducts, it can be difficult to quantitative partially resolved adducts and evaluated background radioactivity levels. We have evaluated the use of storage phosphor imaging techniques to quantitate and map the radioactivity on chromatograms generated by the 32P-postlabeling method. The results showed that storage phosphor imaging was approximately 10 times more sensitive than screen-enhanced autoradiography at -80 degrees C for the detection of 32P, exhibits a greater linear range of response, has a resolution that compares favorably to film and has a lower background than does liquid scintillation spectrometry. Further, the generation of a digitized record of the distribution and intensity of radioactivity allows for computer-assisted assessment of adduct profiles and can facilitate quantitation of individual adducts and radioactive zones comprised of multiple overlapping adducts in complex chromatograms. Additionally, the permanent record created by the imaging technology permits facile retrospective analysis of samples, whereas with autoradiography and liquid scintillation spectrometry reanalysis of a replicate sample is required.     

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.     

Detection and identification of mutagens by the adducts formed upon reaction with guanosine derivatives

Mutagens present in crude samples such as heated glucose can be detected or identified by means of the adducts formed upon reaction with a fluorescent guanosine derivative (FG) or isopropylideneguanosine (IPG). After the reaction of IPG with heated glucose, two adducts were isolated by high-performance liquid chromatography. One of the adducts was identified as the cyclic adduct formed between IPG and glyoxal. Mesoxaldialdehyde, which is structurally related to glyoxal, also produced a cyclic IPG-adduct and showed mutagenic activity in Salmonella typhimurium strain TA100. The other adduct isolated from the reaction mixture of IPG and heated glucose was 8-hydroxy-IPG. Various reagents which generate oxygen radicals were effective in the hydroxylation of guanosine derivatives at the C-8 position. These reagents also cause hydroxylation of guanine residues in DNA.     

Three-dimensional fluorometry for the detection of DNA adducts

We describe the interfacing of a fluorometer to a desk-top computer by means of a commercially available interface box, for the purpose of generating three-dimensional fluorescence spectra. The important features of a self-designed program in BASIC are discussed in detail.     

Smoking-related DNA adducts: 32P-postlabeling analysis of 7-methylguanine in human bronchial and lymphocyte DNA

7-methylguanine DNA adducts were determined in macroscopicallynormal bronchial specimens and peripheral blood lymphocytesof 20 patients undergoing pulmonary surgery. A recently developed³²P-postlabeling assay was applied with anion exchange chromatographyas an adduct enrichment method. The material consisted of 13smokers and 7 non-smokers. The mean bronchial 7-methylguaninelevels of 11 smokers and 6 non-smokers were 17.3 and 4.7 adducts/10⁷nucleotides. In lymphocyte DNA, the respective mean levels were11.5 and 2.3 adducts/10⁷ nucleotides. The bronchial DNA adductlevels in smokers were statistically higher than those in non-smokers.Among 5 smokers, for whom both bronchial and lymphocyte DNAwas available, 7-methylguanine levels correlated in the twotissues (r = 0.77).