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.      

Persistence of O6-guanine DNA adducts in styrene-exposed lamination workers determined by 32P-postlabelling

A modified ³²P-postlabelling method was used for the detectionof styrene-specific DNA adducts in lamination workers. The persistenceof O⁶-styrene DNA adducts was studied in DNA from lymphocytesand granulocytes of an exposed and a control group. We comparedO⁶-adduct levels obtained from a sampling prior to vacation,after 2 weeks of vacation and after an additional 1 month ofwork. In granulocytes, there was no significant difference inadduct levels between the control and the exposed groups inany individual samplings. In lymphocytes of laminators the detectedadduct levels were significantly higher (5.4 adducts/10⁸ nucleotides)than those in the controls (1.0 adduct/10⁸ nucleotides). The2 week interruption of exposure did not influence the totalO⁶-adduct level (4.9 adducts/10⁸ nucleotides in the first samplingversus 5.1 adducts/10⁸ nucleotides in the second), indicatingvery slow removal of the specific O⁶-styrene adducts from DNA.     

Characterization and evaluation by 32P-postlabelling of psoralen-type DNA adducts in HeLa cells

Samples of DNA irradiated at 405 and/or 365 nm In the presenceof 8-methoxypsoralen (8-MOP) were analysed via a modified postlabelllngassay using three hydrolysis enzymes other than those employedpreviously. These enzymes (deoxyribonucleasel, venom phosphodiesteraseand alkailne phosphatase) liberated 3'-adducted dinudeotidemonophos phate instead of the 5'-modifled dinucleotide monophosphatenormally obtained. The first separalion chromatography (Dl)of samples irradiated in the presence of 8-MOP showed a singlespot above the origin, and the next separation (D2) resolvedthis spot into two components (spots I and II). Double irradiationexperiments in which samples of DNA were first irradiated at405 am before being irradiated at 365 nm showed that spot IIcould be transformed into spot I. The use of 6,4,4'-trimethylangelicin,which induced only photo monoadducts under UVA Irradiation,gave only spot II. These two results indicated that spots Iand II were respectively due to interstrand cross-links andmonoadducts. Dose-effect experiments showed that spots I andII were dose dependent, and low-dose irradiations permittedus to measure one interstrand cross-link and two monoadductsper 10⁸ base pairs.     

32P-postlabelling with high-performance liquid chromatography for analysis of abundant DNA adducts in human tissues

Abundant complex DNA adducts can be detected in human tissues by a combined 32P-postlabelling and high-performance liquid chromatography (HPLC) method. The HPLC profiles reveal a panorama of nuclease P1-resistant human adducts, which are not among the known human DNA adducts and are suspected of being endogenous. Lipid peroxidation-induced DNA adducts and I-compounds are two possible candidates for these adducts. Therefore, we performed two experiments: one was to identify chromatographically the lipid peroxidation-induced adducts among other human adducts with two acrolein- and crotonaldehyde-derived propano adduct standards (Acr-dG3 and Cro-dG1&2) and a structurally unknown adduct (Cro-DNA) derived from crotonaldehyde-treated DNA; and the other was to analyse the adducts in breast tissue from patients with breast cancer and from controls and to compare their behaviour with that of I-compounds in cancerous tissues. In the first experiment, Acr-dG3 and Cro-dG1 were detected in three human lung tissues, at levels ranging from 3.4 to 8.9 (x 10(-8)) and from not detectable to 2.9 (x 10(-8)), respectively. Acr-dG3 and Cro-DNA were detected in three human colon tissues, at levels of 0.2-0.4 (x 10(-8)) and 1.2-3.4 (x 10(-8)), respectively. In the second experiment, adjacent and tumorous breast tissues from 15 patients with breast cancer (of an average age of 33.4 years) and normal breast tissue from 18 controls (of an average age of 57.3) were analysed for the abundant complex adducts. The total adduct levels in the adjacent and tumorous tissues were lower than in the normal tissues (with medians of 8.0, 11.8 and 13.3 (x 10(-7)), respectively). Significant differences in the adduct levels between adjacent or tumorous tissues and normal tissues were observed in three HPLC peaks, and age was significantly associated with three peaks. These results are consistent with our speculation that the abundant adducts are comprised of lipid peroxidation-induced adducts and human homologues of I-compounds.     

A comparison of 32P-postlabelling and immunological methods to examine human lung DNA for benzo[a]pyrene adducts

Human lung DNA isolated from surgical specimens has been examined for the presence of polycyclic aromatic hydrocarbon-DNA adducts using both 32P-postlabelling and immunological methods. Of 12 samples examined to date, five had detectable amounts of benzo[a]pyrene diol epoxide-DNA adducts (BPDE-DNA) as determined by the enzyme-linked immunosorbent assay (ELISA), after immunoaffinity concentration. Values ranged from 3.5 to 11.5 fmol/mg DNA. When the same group of samples was analysed using the 32P-postlabelling technique, adducts could be detected in all the samples examined. There was generally not a good correspondence between the two methods. The number of adducts measured by 32P-postlabelling ranged from 1-100 per 10(8) nucleotides, which is some two orders of magnitude higher than with the immunological method, indicating that the BPDE-DNA adduct is probably not the major adduct present in these samples.     

DNA adducts in lymphocytes and granulocytes of smokers and nonsmokers detected by the 32P-postlabelling assay

The effects of smoking and DNA adduct formation were analysed in isolated human white blood cell populations. As the white cells are composed mainly of granulocytes with a short half-life and T-lymphocytes with a half-life of several years, we isolated the lymphocytes and granulocytes of 11 smokers and 10 nonsmokers to determine any smoking-related DNA adducts by the nuclease-P1-enhanced 32P-postlabelling assay. The differences between the mean lymphocyte DNA adducts/10(8) nucleotides of 31 +/- 5.7 (SE) of smokers were significantly higher (P less than 0.05) than those in the lymphocytes 13 +/- 1.6 (SE) of nonsmokers. The total DNA adducts/10(8) nucleotides obtained from the granulocytes of smokers and nonsmokers was 9.6 +/- 1.9 and 7.6 +/- 1.9 respectively. The plasma cotinine concentrations were in good agreement with the smoking information given by the individual smokers (r = 0.847, P less than 0.001). The DNA adduct levels of the lymphocytes of the 10 smokers correlated with the plasma cotinine concentrations (r = 0.639, P less than 0.05). The variation between the results was explained by the variation among the individuals and the samples, but not by the variation in the parallel determinations. More detailed studies are needed to analyse the source of the individual variations between the smokers' adduct levels, DNA repair, and differences in the metabolism of the compounds in cigarette smoke.     

Recoveries of DNA adducts of polycyclic aromatic hydrocarbons in the 32P-postlabelling assay

The ³²P-postlabelling assay for analysis of DNA adducts of chemicalcarcinogens has been applied in a large number of experimentalanimal and human studies. Most human studies have dealt withoccupational and environmental exposures to polycyclic aromatichydrocarbons (PAHs). The postlabelling assay does not allowdirect chemical identification, and most studies with this methodhave not been performed in a quantitative way. Very little istherefore known about the identity and absolute levels of adducts,which are important contributors to the process of risk identificationand quantitation. In the present study it was, therefore, decidedto test some parameters suspected to affect recoveries of adductsin the phosphorylation step of the assay. For this purpose 12different PAHs were reacted individually and in a mixture withDNA in the presence of a rat liver S9 metabolizing system. Differentconcentrations of ATP, calcium chloride and polynucleotide kinasewere tested using the nuclease P1 enhancement. We found thateach factor contributed to adduct recovery and that optimalconditions could be defined. Diluting the modified DNA samplesup to 1000 times had little influence on the recoveries of adducts.Comparing the nuclease P1 and the butanol extraction proceduresfor adduct purification showed that both methods gave similarpaterns and levels of major adducts. The absolute recoveriesin postlabelling, based on 3H-binding of radiolabelled compounds,were for most of the tested compounds relatively low. The factthat the nuclease P1 and the butanol extraction procedures gavesimilar recoveries points towards common factor(s) involvedin the reduction of the recovered adduct levels. Based on theobserved recoveries the conclusion can be drawn that when postlabellingrelated adducts in human samples the true total adduct levelscan be considerably underestimated, even if optimal conditionsare used.     

Detection and characterization by 32P-postlabelling of DNA adducts induced by a Fenton-type oxygen radical-generating system.

Reactive oxygen species can give rise to numerous modifications of DNA. We have investigated the formation of such modifications using the nuclease P1 digestion method of the 32P-postlabelling procedure for the detection of DNA damage. Analysis of DNA that had been treated with a Fenton-type system of copper (or iron) ions and H2O2 resulted in the detection of up to ten discrete 32P-labelled spots, displaying chromatographic characteristics similar to aromatic adducts, on PEI-cellulose TLC. Maximum total levels equivalent to 28 adducts/10(8) nucleotides were achieved after 15 min of treatment with Cu2+/H2O2. The formation of adducts was 1.5 times greater if single-stranded rather than double-stranded DNA was employed, suggesting an intrastrand effect. Experiments with 3'-deoxyribonucleotides demonstrated that the adducts detected did not represent base modifications such as 8-hydroxydeoxyguanosine or thymidine glycols. However, treatment of specific dinucleotides (dApdG and dApdA) was found to produce two major adducts that were chromatographically identical by TLC and HPLC to the two major adducts formed in DNA. It is proposed that these species with aromatic adduct-like characteristics are the result of the intrastrand linking of specific adjacent bases in DNA.     

A pilot study of detection of DNA adducts in white blood cells of roofers by 32P-postlabelling

To assess the utility of DNA adducts as biomarkers of exposure to carcinogens in an industrial population, a pilot study of roofers occupationally exposed to a mixture of polycyclic aromatic hydrocarbons was conducted. DNA was isolated from peripheral white blood cells of roofers and non-occupationally exposed subjects matched for age, sex and smoking status. Occupational exposures to anthracene, fluoranthene, pyrene, benzanthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[g,h,i]perylene and benzo[k]fluoranthene were assessed by personal breathing zone air sampling and skin wipes. Exposures to benzo[a]pyrene in air of exposed subjects ranged from 0.60 microgram/m3 to 1.39 micrograms/m3, and exposures to total polycyclic aromatic hydrocarbon (the sum of eight hydrocarbons) ranged from 6.0 micrograms/m3 to 13.8 micrograms/m3 on the day before blood collection. In the biomarker studies 10 of 12 roofers, but only 2 of 12 comparison subjects, had detectable levels of aromatic DNA adducts by 32P-postlabelling assay (p less than 0.01). The two non-roofers with detectable adducts had levels at or near the detection limit of 2 adducts per 10(9) nucleotides. In two roofer samples which were studied in a mixing experiment, the major adduct spots did not co-migrate with the guanosine N2 adduct of benzo[a]pyrene diol epoxide. These results suggest that the 32P-postlabelling assay may be useful for monitoring exposures to complex mixtures of aromatic hydrocarbons in industrial populations.     

O6-substituted-2'-deoxyguanosine-3'-phosphate adducts detected by 32P past-labeling of styrene oxide treated DNA

³²P post-labeling of DNA reacted with styrene oxide resultedin the detection of six adducts. In order to determine whichof these corresponded to modification at the O⁶ position ofguanine, O⁶-substituted styrene oxide-deoxyguanosine–-3'–monophosphatederivatives were synthesized. The two synthetic isomers werepurified by HPLC and the structures were confirmed by mass spectrometryand ¹H NMR. ³²P post-labeling and co-chromatography with theDNA-styrene–7, 8–oxide reaction products resultedin the assignment of adduct number 4 as O⁶hydroxy-2-phenylethyl)-2'-deoxyguanosine-3',5'-bisphosphate and adduct number 5 as O⁶-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine-3',5'-bishosphate.     

Multiple DNA adducts in lymphocytes of smokers and nonsmokers determined by 32P-postlabeling analysis

Identification of DNA adducts in peripheral lymphocytes could serve as a means of monitoring human exposure to potential genotoxic agents. In this study, DNA from peripheral lymphocytes of smokers and nonsmokers was examined for adducts by the P1 nuclease 32P-postlabeling technique. Thin layer chromatography (TLC) maps from both groups revealed multiple DNA adducts which ranged from no adducts for one individual to six adducts for a different individual. The total DNA adduct concentrations were approximately one adduct in 10(8)-10(10) normal nucleotides. Comparison of the adduct TLC profiles revealed individual variation in both pattern and level of DNA adducts. The type and amount of adduct was not influenced by smoking history and remained unchanged in four out of six subjects who were resampled after a 1 month interval. The capacity of lymphocytes to form BaP-derived DNA adducts after a 72 h incubation with 10(-6) M [3H]BaP was measured by both high-performance liquid chromatography (HPLC) and 32P-postlabeling analysis. The in vitro adduct values detected by [3H]nucleoside concentrations on HPLC ranged from 1 to 7 fmol adduct per micrograms DNA (3.3-23.3 adducts per 10(7) nucleotides). The [3H]nucleoside values were consistent with values obtained by 32P-postlabeling of the same sample (correlation coefficient of 0.88). No relationship was apparent between the capacity of lymphocytes to form a [3H]BaP-derived adduct in vitro and the concentration of any adduct, or total adducts present in untreated lymphocytes. These results suggest that multiple DNA adducts are present in lymphocytes from nonsmokers as well as smokers, although the profile and extent of these adducts can vary among individuals. The relationship of the lymphocyte DNA adducts detected in this study to human cancer susceptibility remains to be determined.     

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.     

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).     

DNA adducts, detected by 32P-postlabelling, in the foregut of patients with familial adenomatous polyposis and in unaffected controls.

Duodenal neoplasms in patients with familial adenomatous polyposis (FAP) cluster around the ampulla; duodenal neoplasia is common but gastric neoplasia is rare. These observations suggest that bile enhances neoplasia by carrying carcinogens to target cells. A critical step in carcinogenesis is the reaction of carcinogens with DNA to form chemical adducts. Using 32P-postlabelling to measure DNA adducts we asked: (i) are there more adducts in the duodenum of patients with FAP than in the duodenum of normal patients? (ii) Are there more adducts in the duodenum than in the stomach? We measured adducts in duodenal biopsies from 51 patients with FAP and 30 age-matched controls; and paired gastric and duodenal biopsies from 31 FAP patients and six controls. The foregut of 90% of all patients studied contained DNA modifications with characteristics of aromatic non-polar DNA adducts. In duodenal biopsies, adduct labelling per 10(9) DNA nucleotides was significantly higher in FAP patients (median 15.0, range 0-162) than in normal patients (median, 7.5, range 0-40; P = 0.0002). In paired duodenal and gastric biopsies from 31 FAP patients, adduct labelling was significantly higher in duodenal DNA (median 15, range 0-109) than in stomach DNA (median 8.0, range 0-40; P = 0.0004). Age, gender or smoking did not account for these differences. Gastric adducts (median 3.5, range 0-10) were lower than duodenal adducts (median 8.5, range 0-29) in paired biopsies from six control patients, P = 0.031). These results support the hypothesis that pancreaticobiliary secretions may be involved in the pathogenesis of foregut neoplasia in FAP.     

DNA adducts, detected by 32P-postlabelling, in DNA treated in vitro with bile from patients with familial adenomatous polyposis and from unaffected controls

Patients with familial adenomatous polyposis (FAP) have a highrisk of developing duodenal adenomas and carcinomas. The distributionof these neoplasms resembles mucosal exposure to bile, suggestingthat bile may play a role in adenoma development. Our previousresults, using DNA adducts detected by ³²P-postlabelling asan index of genotoxicity, have supported this hypothesis. Wefound significantly higher adduct labelling in the duodenumof FAP patients than in the duodenum of control patients andsignificantly higher labelling in the small bowel of rats gavagedwith FAP bile than in rats given control bile. We have now investigatedthe ability of human bile to form adducts with DNA in vitro.Bile obtained from the gallbladder of 18 FAP patients immediatelybefore colectomy, and from 18 control patients, was incubatedwith salmon sperm DNA in solution at 37^°C for 1 h, afterwhich the purified DNA was analysed for DNA adducts, using thenuclease PI method of ³²P-postlabelling. Relative adduct labellingvalues (RAL, adducts per 109 nucleotides) produced by FAP bilesamples were significantly higher than RAL values produced bycontrol bile samples (medians 197 versus 86, P = 0.0016, Mann-Whitneytest). We found a consistent pattern of adduct labelling, varyingin intensity between samples. Adduct spots were eluted fromTLC plates and analyzed by reverse-phase HPLC. Each major spotgave several peaks that were consistent between bile samplesfrom different patients and were similar in FAP and controlbile. These results indicate that bile from FAP and controlpatients contains similar, directly acting genotoxic compoundsbut that levels are higher in FAP than in control patients.This suggests that bile from FAP patients is more genotoxk thanbile from control patients. Incubation of bile with free-radicalscavengers and deconjugating enzymes failed to influence adductlabelling in this system.     

Development of a 32P-postlabelling method for the analysis of 2'-deoxyguanosine-3'-monophosphate and DNA adducts of methylglyoxal

A ³²P-Postlabelling assay was developed for the analysis ofadducts arising from the reaction of 2'-deoxyguanosine-3'-monophosphatewith the 1,2-dicarbonyl compound methylglyoxal, a major mutagenin several foodstuffs, in particular, instant and brewed coffee.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 the 3'-monophosphate dephosphorylationactivity of nuclease P1 was also studied. Four isomeric reactionproducts were separated by HPLC, structurally characterizedand identified as 3-(2'-deoxy-ß-D-erythro-pentafuranosyl)-6,7-dihydro-6,7-dihydroxy-6-methylimidazo[2,3-b]purine-9(8H)one.The same adducts could be detected from calf thymus DNA thathad been reacted in vitro with methylglyoxal. DNA adducts wereisolated after enzymatic digestion to mononucleotides followedby nuclease P1 digestion of normal nucleotides. The total levelof methylglyoxal-DNA adducts obtained was 5.7 ± 1.7 (n= 15) adducts/10⁶ nucleotides. The ³²P-postlabelling methodwas further validated by the detection of adducts of methylglyoxalin DNA from freshly isolated and stimulated human lymphocytesexposed in vitro. The concentrations of the adducts detectedin these samples were 8.2 ± 0.9 (n = 3) adducts/ 10⁷nucleotides and 1.5 ± 0.1 (n = 3) adducts/10⁶ nucleotidesafter treatment with 1.5 and 3.0 mM methylglyoxal respectively.     

Metabolism and formation of DNA adducts of benzo(a)pyrene in human diploid fibroblasts.

Cultured human diploid skin fibroblasts incubated with [G-3H]benzo(a)pyrene yielded about 10 times more H2O-=soluble benzo(a)pyrene metabolites and DNA adducts of stationary growth phase than did proliferating cultures. This increased formation could be blocked by alpha-naphthoflavone. Trichloropropenoxide and cyclohexenoxide, inhibitors of the epoxide hydratase, inhibited predominantly the formation of DNA adducts. Cultures from older individuals formed significantly more benzo(a)pyrene metabolites and DNA adducts, but control cultures from patients with either lung cancer or melanoma did not. The age influence was not apparent when the ratio of DNA adducts to H2O-soluble metabolites was determined for each individual cell line. However, the proportion of DNA-bound material in the cells from patients with lung cancer was significantly increased compared to cells from melanoma patients or healthy individuals.     

Detection by 32P-postlabelling of DNA adducts induced by free radicals and unsaturated aldehydes formed during the aerobic decomposition of fecapentaene-12.

Fecapentaene-12 (fec-12), excreted in human faeces, is genotoxic to human cells and a known animal carcinogen. The mechanism of its genotoxicity is unknown but may involve direct alkylation and/or free-radical generation. The formation of reactive species during fec-12 aerobic degradation was thus investigated by electron paramagnetic resonance (EPR) and NMR spectroscopic techniques. Oxy- and alkyl-radicals were detected as the 5,5'-dimethyl-1-pyrroline-N-oxide spin-trap adducts at fec-12 concentrations of between 0.1 and 2.0 mM. Under anaerobic conditions no free-radical generation was observed. NMR spectroscopy indicated that fec-12 degraded at least initially into three unsaturated aldehydes. The co-formation of free-radicals and unsaturated aldehydes suggests that fec-12 decomposed aerobically via a process analogous to lipid peroxidation. As both types of species, thus formed, may subsequently interact with DNA to form adducts, fec-12-induced DNA damage was investigated by 32P-postlabelling techniques. Using procedures that detect alkyl-type adducts, a number of putative adducts were detected in fec-12-treated DNA; two of similar mobility were observed in fec-12-treated 2'-deoxyguanosine-3'-monophosphate. Adducts with similar mobility have been detected in acrolein-treated DNA. One adduct with similar mobility was also observed in DNA obtained from normal human fibroblasts treated with fec-12. Using a C-18 ODS column, these putative adducts were eluted in 60-85% methanol, whereas 8-hydroxydeoxyguanosine-3'-monophosphate (8OHdGp) was eluted with 1% acetonitrile. Also unlike these putative adducts, the detection of 8OHdGp required HPLC fractionation prior to 32P-postlabelling. The formation of adducts, possibly aldehyde-related, and free-radical damage suggests that fec-12 genotoxicity may be the result of several different mechanisms, the relative importance of each is as yet unknown. Hydroxyl radicals were also detected during the aerobic decomposition of deca-2,4,6,8-tetraenal, a possible degradation product of fec-12 and a less potent mutagen, suggesting that free-radical generation may have only a minor role in fec-12-induced genotoxicity.     

32P-postlabelling analysis of DNA adducts of benzo[a]pyrene formed in complex metabolic activation systems in vitro

The influence of cytochrome P-450-linked monooxygenase, epoxide hydrolase, UDP-glucuronyltransferase and glutathione S-transferases on the metabolic activation of benzo[a]pyrene (BP) was studied by incubating BP with preparations of rat-liver microsomal and cytosolic fractions in the presence of exogenous DNA. 32P-Postlabelling analysis of the DNA revealed the presence of covalently bound adducts formed by BP, which were visualised as radioactive spots on autoradiographs of thin-layer chromatograms. The effects on the adduct profile of adding different combinations of 1,2-epoxy-3,3,3-trichloropropane (TCPO), UDP-glucoronic acid (UDPGA) and glutathione (GSH) to the incubation mixture were determined. As many as 14 different DNA adducts were resolved and quantitated on the chromatograms, the numbers and quantities of which varied within a large range depending on the incubation conditions. The influence of the enzyme inhibitor and cofactors on the adduct patterns reflects the complex effects of simultaneous enzyme interactions on the metabolic activation of BP.     

DNA adducts in tumour, normal peripheral lung and bronchus, and peripheral blood lymphocytes from smoking and non-smoking lung cancer patients: correlations between tissues and detection by 32P-postlabelling and immunoassay

Smoking is a major risk factor for lung cancer. This comparative study of smoking-related carcinogen-DNA adducts in pulmonary tissues and peripheral blood lymphocytes aims to further explore the primary DNA damaging processes by cigarette smoke in target and surrogate tissues. Samples of tumour and normal peripheral lung tissue, normal bronchial tissue and peripheral blood lymphocytes were obtained from a total of 85 lung cancer patients who underwent lung resection. Bulky DNA adducts were determined by 32P-postlabelling, and polycyclic aromatic hydrocarbon (PAH)-DNA adducts were detected by (+/-)-7beta, 8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-DNA chemiluminescence immunoassay (BPDE-DNA CIA) in smaller subsets of tissue samples subject to availability of DNA. Bulky DNA adduct levels ranged between 0.3 and 27.8 adducts/10(8) nucleotides (nt) with mean adduct levels between 2.8 and 11.5 adducts/10(8) nt. Mean PAH-DNA adduct levels were 2.6-6.2 adducts/10(8) nt. Significantly higher bulky DNA adduct levels were detected in smokers' lungs as compared with non-smokers' (P < 0.02). PAH-DNA adduct levels appeared higher in the lungs of smokers compared with non-smokers but the difference was not significant. Lung tumour contained on average a 50% lower DNA adduct level compared with normal lung tissue. A statistically significant positive correlation was found between the DNA adduct levels of the corresponding tumour and normal lung tissue samples in both smokers and non-smokers using both methodologies. Bulky DNA adduct levels in normal lung and blood lymphocytes correlated significantly in non-smokers only (r = 0.55, P = 0.023). In lung tumour DNA samples there was a weak correlation between values obtained by 32P-postlabelling and by the BPDE-DNA immunoassay (r = 0.27, P = 0.054). However, with normal lung DNA samples, values obtained by the two assays did not correlate.