Identification Of DNA Adducts Research Articles

Abstract 2257: DNA adductome analysis in human colorectal tissues

Abstract Due to recent advancement of DNA sequencing technologies, cancer genome analysis has shown that somatic mutations have different trends in various tissues and individuals. Some of trends has been associated with exposure of chemically reactive compounds derived from exogenous or endogenous sources and genetic deficiencies in DNA repair and DNA replication as mutational signatures. Accumulation of these information will ultimately expect to identify carcinogenic etiologies in an individual case. However, what types of DNA damage caused the mutational signatures and how extent they contribute to human carcinogenesis has not yet fully explained.To directly evaluate the chemical types of DNA damage in human colorectal tissues, we performed comprehensive identification of DNA adducts by a liquid chromatography coupled with mass spectrometry. In this DNA adductome methodology, genome DNA was enzymatically hydrolyzed into mononucleoside and separated using a reversed-phase chromatography. Chemical structure of DNA adducts including products of epigenetic modification in genome DNA was identified using column retention time and m/z of chemical standard library of DNA adducts. Peak areas of DNA adducts were normalized by that of naturally-occurring isotopologues of canonical DNA nucleoside and compared with clinicopathological information. We identified several DNA adducts in human colorectal tissue. These included DNA adducts of alkylation, oxidation, and lipid peroxidation. C5-methyl-2’-deoxycytidine was most abundant atypical DNA in human colorectal tissue and prevalent in all cases. C5-hydroxymethyl-2’-deoxycytdine was decreased in colorectal cancer cases compared with non-colorectal cancer cases. Other DNA adducts like 1,N6-etheno-2’-deoxyadenosine were present with group- or individual-specificity. Compared with our previous results of DNA adductome analysis in human stomach mucosae and kidney tissues, DNA adducts observed in human colorectal tissue showed some tissue specificity. We will discuss whether profiles of DNA adducts are based on similar contexts of chemical exposure among individuals. DNA adducts observed in our study potentially indicated chemical causes of DNA mutation in human colorectal tissue. We propose that the integration of DNA adductomics using mass spectrometric profiling with other genetic analysis such as mutational signature analysis leverage the exploration of chemical and genetic etiology in an individual carcinogenic context and evaluate gene-environment interaction in human carcinogenesis. Citation Format: Yuji Iwashita, Shunsuke Ohtsuka, Ippei Ohnishi, Yuto Matsushita, Takashi Yamashita, Keisuke Inaba, Atsuko Fukazawa, Hideto Ochiai, Keigo Matsumoto, Nobuhito Kurono, Yoshitaka Matsushima, Hiroki Mori, Shioto Suzuki, Shohachi Suzuki, Fumihiko Tanioka, Haruhiko Sugimura. DNA adductome analysis in human colorectal tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2257.

Data-Independent Mass Spectrometry Approach for Screening and Identification of DNA Adducts.

Long-term exposures to environmental toxicants and endogenous electrophiles are causative factors for human diseases including cancer. DNA adducts reflect the internal exposure to genotoxicants and can serve as biomarkers for risk assessment. Liquid chromatography-multistage mass spectrometry (LC-MSn) is the most common method for biomonitoring DNA adducts, generally targeting single exposures and measuring up to several adducts. However, the data often provide limited evidence for a role of a chemical in the etiology of cancer. An "untargeted" method is required that captures global exposures to chemicals, by simultaneously detecting their DNA adducts in the genome; some of which may induce cancer-causing mutations. We established a wide selected ion monitoring tandem mass spectrometry (wide-SIM/MS2) screening method utilizing ultraperformance-LC nanoelectrospray ionization Orbitrap MSn with online trapping to enrich bulky, nonpolar adducts. Wide-SIM scan events are followed by MS2 scans to screen for modified nucleosides by coeluting peaks containing precursor and fragment ions differing by -116.0473 Da, attributed to the neutral loss of deoxyribose. Wide-SIM/MS2 was shown to be superior in sensitivity, specificity, and breadth of adduct coverage to other tested adductomic methods with detection possible at adduct levels as low as 4 per 109 nucleotides. Wide-SIM/MS2 data can be analyzed in a "targeted" fashion by generation of extracted ion chromatograms or in an "untargeted" fashion where a chromatographic peak-picking algorithm can be used to detect putative DNA adducts. Wide-SIM/MS2 successfully detected DNA adducts, derived from chemicals in the diet and traditional medicines and from lipid peroxidation products, in human prostate and renal specimens.

Human Biomonitoring of DNA Adducts by Ion Trap Multistage Mass Spectrometry.

Humans are continuously exposed to hazardous chemicals in the environment. These chemicals or their electrophilic metabolites can form adducts with genomic DNA, which can lead to mutations and the initiation of cancer. The identification of DNA adducts is required for understanding exposure and the etiological role of a genotoxic chemical in cancer risk. The analytical chemist is confronted with a great challenge because the levels of DNA adducts generally occur at <1 adduct per 10(7) nucleotides, and the amount of tissue available for measurement is limited. Ion trap mass spectrometry has emerged as an important technique to screen for DNA adducts because of the high level sensitivity and selectivity, particularly when employing multi-stage scanning (MS(n) ). The product ion spectra provide rich structural information and corroborate the adduct identities even at trace levels in human tissues. Ion trap technology represents a significant advance in measuring DNA adducts in humans. © 2016 by John Wiley & Sons, Inc.

DNA adduct identification using gold-aptamer nanoprobes.

The optical and physico-chemical properties of gold nanoparticles (AuNPs) have prompted new and improved approaches which have greatly evolved the fields of biosensing and molecular detection. In this study, the authors took advantage of AuNPs' ease of modification and functionalised it with selected DNA aptamers using a salt aging method to produce gold-aptamer nanoprobes. After characterisation, these nanoprobes were subsequently used for biomolecular detection of glycidamide (GA)-guanine (Gua) adducts generated in vitro. The results are based on differences in nanoprobe stabilisation against salt-induced aggregation, similar to the non-cross-linking method developed by Baptista for discrimination of specific sequences. Alkylated Guas were efficiently discriminated from deoxyguanosine and GA in solution. Despite this, a clear identification of DNA adducts derived from genomic DNA alkylation has proven to be a more challenging task.

Abstract 4791: Development of a high resolution mass spectrometry method for the observation and characterization of unknown adducts in human DNA

Abstract The development of analytical approaches for identification of DNA adducts may provide important tools to help clarify their role as biomarkers of exposure and as potential markers of cancer risk. New sensitive mass spectrometric methods are needed to screen for previously unidentified modified bases to allow for identification of adducts of multiple types, potentially leading to the identification of base modification patterns in DNA from human tissues. Liquid chromatography-mass spectrometry (LC-MS) is now the method of choice for characterization and quantitation of DNA adducts in tissue samples. We have developed a unique LC-High Resolution-MSn-based DNA adductomics approach which has the potential to identify previously uncharacterized DNA adducts in human tissues because it possesses the required combination of sensitivity, specificity, and generation of chemical information that is absent from previous MS techniques. DNA hydrolysis produces deoxynucleosides (dR) which upon ionization can undergo fragmentation resulting in loss of deoxyribose, resulting in an overall [M+H - 116.0474]+ ion. This universally observed fragmentation can be used to identify the analyte as a dR. The methodology described here uses the appearance of this fragment ion at high mass tolerance to trigger a second fragmentation step (MS3) leading to production of fragment ion spectra which provide additional structural information. An additional fragmentation step (MS4) is triggered for those MS3 spectra, which contain ions corresponding to one of the four nucleobases to provide confirmation of base identity. The final output is an LC chromatogram of DNA adducts in which each peak provides an accurate mass sufficient for unambiguous molecular formula determination and extensive fragmentation data potentially leading to its structural identification. We performed preliminary experiments to test the performance of our approach on mixtures of 18 DNA adducts selected to represent modifications of all four nucleobases and modifications at various reactive sites and with differing polarity. All 18 DNA adducts showed clear, sharp and symmetric peaks in the full scan and [M+H - 116.0474]+ ions upon MS2 fragmentation which then triggered the MS3 event on the product ions. The fragmentation spectrum of each adduct contained the ion corresponding to the mass of the base it was derived from and triggered the fragmentation of the base ion in the MS4 event. To test the effect of a real sample matrix on DNA adduct detection, the mixture of 18 DNA adducts was added to a human leukocyte DNA sample which had previously been passed through the purification and enrichment steps used for our LC-MS/MS analysis of N2-ethyl-dGuo. All 18 DNA adduct standards were detected as clear and sharp peaks. These results clearly demonstrate the potential of this method for DNA adduct detection and identification in human tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4791. doi:1538-7445.AM2012-4791

Abstract 1701: Elevated DNA adducts detected in human breast tumors than normal tissues from mammoplasty

Abstract Breast cancer is the most common cancer among women worldwide and the second leading cause of cancer-related death in women in the United States. The etiology of the majority of human breast cancers is unknown. Elevated levels of estrogen and exposure to environmental carcinogens have been suggested to be involved in human breast cancer development. Several studies have also provided evidence of the potential role of oxidative stress and lipid peroxidation in breast cancer etiology. Identification of DNA adducts and monitoring change in DNA adduct profiles/burden in normal and cancer tissue may provide clues for risk of breast cancer. In this study, we detected and compared the levels of a wide range of polar to lipophilic adducts in tumor (n= 23) and adjacent normal tissue (n= 30) from breast cancer patients with normal tissue (n=124) from mammoplasty using a 32P-postlabeling adductomics. Adducts were classified into polar (P-1, P-2a and P2b or 8-oxodG) and lipophilic (L-1) in descending order of their polarities. Our results indicated significantly elevated total DNA adduct levels in tumor and adjacent normal tissue of breast cancer patients compared to normal tissues from mammoplasty. The levels of most polar adducts (P-1) were not significantly different among the tumor, adjacent and normal breast tissue. However, relatively lesser polar adducts, i.e., P-2a, P-2b (8-oxodG) and L-1 adducts were significantly higher in tumor and adjacent normal tissues of breast cancer patients compared with normal tissues from mammoplasty. Thus, there is a clear trend of adduct accumulation in the breast tissues of cancer patients that could have acted as a precursor event in carcinogenesis process. Therefore, elevated oxidative DNA adducts such as 8-oxodG can be a possible biomarker for early detection of breast cancer risk and subjects with high DNA adduct load may potentially be at higher risk for cancer development, and could be stratified for chemopreventive intervention. (Supported from CA-90892 and Agnes Brown Duggan Endowment) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1701.

Reaction of Glyoxal with 2‘-Deoxyguanosine, 2‘-Deoxyadenosine, 2‘-Deoxycytidine, Cytidine, Thymidine, and Calf Thymus DNA: Identification of DNA Adducts

Glyoxal (ethanedial) is an increasingly used industrial chemical that has been found to be mutagenic in bacteria and mammalian cells. In this study, the reactions of glyoxal with 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, cytidine, thymidine, and calf thymus DNA have been studied in aqueous buffered solutions. The nucleoside adducts were isolated by reversed-phase liquid chromatography and characterized by their UV absorbance and 1H and 13C NMR spectroscopic and mass spectrometric features. The reaction with 2'-deoxyguanosine gave one adduct, the previously known 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one adduct. The reaction of 2'-deoxyadenosine with glyoxal resulted in the formation of a previously not reported N6-(hydroxyacetyl)-2'-deoxyadenosine adduct. In the reaction of glyoxal with 2'-deoxycytidine and cytidine at neutral conditions and 37 degrees C, 5-hydroxyacetyl pyrimidine derivatives were obtained. When the cytidine reaction was performed at pH 4.5 and 50 degrees C, the 5-hydroxyacetyl derivative of uridine was formed through deamination of cytidine-glyoxal. Adducts in the thymidine reaction could not be detected. In the reaction of glyoxal with calf thymus DNA, the 2'-deoxyguanosine-glyoxal and 2'-deoxyadenosine-glyoxal adducts were obtained, the former being the major adduct.

Measuring DNA Nucleobase Adducts Using Neutral Hydrolysis and Liquid Chromatography-Mass Spectrometry

The detection and identification of DNA adducts is important for predicting human cancer risk posed by chemicals and for uncovering potential genotoxicity of new drug and agricultural chemical candidates. For compounds that react with DNA to form N7-guanine and/or N3-adenine adducts, neutral thermal hydrolysis provides a simple procedure for sample preparation. The N7-guanine and N3-adenine adducts are selectively ejected from the DNA chain, resulting in a clean sample matrix enriched in nucleobase adducts. Coupling neutral thermal hydrolysis with liquid chromatography-mass spectrometry (LC-MS) provides sensitive methods to detect and quantitate DNA adducts, and structural information is provided by MS. Combining these technologies with capillary liquid chromatography sample preconcentration systems can provide exquisitely sensitive detection. In this review, we first summarize the chemistry of nucleobase adduct formation, briefly summarize modern methods to detect DNA adducts, and then describe neutral thermal hydrolysis coupled to LC-MS/MS and some of its applications to DNA damage studies. Finally, we review recent applications of neutral thermal hydrolysis and LC-MS to toxicity screening of chemicals.

Syntheses of DNA adducts of two heterocyclic amines, 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) and 2-amino-9H-pyrido[2,3-b]indole (AalphaC) and identification of DNA adducts in organs from rats dosed with MeAalphaC.

2-Amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (AalphaC) are mutagenic and carcinogenic heterocyclic amines formed during ordinary cooking. MeAalphaC and AalphaC are activated to mutagenic metabolites by cytochrome P450-mediated N-oxidation to the corresponding N2-OH derivatives. The proximate mutagenic N2-OH derivatives of MeAalphaC and AalphaC did not react with deoxynucleosides or DNA. However, upon acetylation with acetic anhydride both reacted with 2'-deoxyguannosine and 3'-phospho-2'-deoxyguanosine, resulting in one adduct each, but not with other nucleosides or nucleotides. The adducts were identified as N2-(2'-deoxyguanosin-8-yl)-MeAalphaC, N2-(2'-deoxyguanosin-8-yl)-AalphaC, N2-(3'-phospho-2'-deoxyguanosin-8-yl)-MeAalphaC and N2-(3'-phospho-2'-deoxyguanosin-8-yl)-AalphaC by comparison with adducts of known structure obtained by reaction of the parent amines with acetylated guanine N3-oxide. N2-OH-MeAalphaC and N2-OH-AalphaC reacted with calf thymus DNA after addition of acetic anhydride. 32P-postlabelling analysis of modified DNA showed one major adduct co-migrating with N2-(3',5'-diphospho-2'-deoxyguanosin-8-yl)-MeAalphaC and N2-(3',5'-diphospho-2'-deoxyguanosin-8-yl)-AalphaC, respectively. Some minor adducts presumed to be undigested oligomers were also detected. 32P-postlabelling analysis of DNA from several organs of rats dosed orally with MeAalphaC showed that in vivo N2-(2'-deoxyguanosin-8-yl)-MeAalphaC also was the major adduct formed. Relative adduct level in DNA isolated from the liver of the rats was about 50.40 adducts/10(9) nt. The adduct levels were approximately 4-fold lower in the colon and the heart and approximately 12-fold lower in the kidney of the rats.

Identification of DNA Adducts Using HPLC/MS/MS Following In Vitro and In Vivo Experiments with Arylamines and Nitroarenes

Arylamines and nitroarenes are suspected of playing a key role in chemical carcinogenesis. Therefore, the study of DNA adduct formation is an important step to determine the genotoxic potential of these compounds. Calf thymus DNA was modified in vitro by reaction with activated N-hydroxyarylamines: 2-chloroaniline (2CA), 4-chloroaniline (4CA), 2-methylaniline (2MA), 4-methylaniline (4MA), 2,4-dimethylaniline (24DMA), 2,6-dimethylaniline (26DMA), 2-aminobiphenyl (2ABP), 3-aminobiphenyl (3ABP), and 4-aminobiphenyl (4ABP). Female Wistar rats (n = 2) were given a single dose of the above arylamines and their analogous nitro derivatives by oral gavage and sacrificed after 24 h. Hepatic DNA and in vitro modified DNA were hydrolyzed enzymatically to individual 2'-deoxyribonucleosides. Adducts were determined using HPLC/MS/MS by comparison to synthesized standards. The hydrolysis efficiency was monitored by HPLC with UV detection. Each arylamine described above formed adducts to 2'-deoxyguanosine and 2'-deoxyadenosine after in vitro reaction with DNA. DNA adducts were found in rats dosed with 4ABP or with 4-nitrobiphenyl. DNA adducts were not detected in rats dosed with 2CA, 4CA, 2MA, 4MA, 24DMA, 26DMA, 2ABP, 3ABP, 2-chloronitrobenzene, 4-chloronitrobenzene, 2-nitrotoluene, and 4-nitrotoluene. All compounds formed hydrolyzable hemoglobin adducts. Therefore, biologically available N-hydroxyarylamines yielded hemoglobin adducts but not hepatic DNA adducts, except for 4ABP.

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Ochratoxin A (OTA) is an important food and feed contaminant with potential adverse effects in humans and animals. In view of present discussions on limit values for OTA in foods, essential elements of a toxicological risk assessment are outlined. The exposure situation in Europe is now well documented. The data base, with respect to a characterization of hazard and dose-response relationships, allowed to calculate a provisional tolerable daily intake for OTA suited to protect the consumer against undesirable toxic effects. Nonetheless, further research on OTA is indicated in view of unresolved issues regarding the following points: 1. mechanisms of action (mode of genotoxicity, role of bioactivation/metabolism, identification of DNA-adducts and dose-dependency); 2. combinations of OTA and other mycotoxins (studies of relevant mixtures/conditions); 3. individual susceptibility and/or situation-based vulnerability. Better information on mechanistic aspects of mycotoxin-induced toxicities will further improve our knowledge on the "margin of safety" between a given exposure and a potential impairment of human health.

Microsome-mediated bioactivation of dibenzo[a,l]pyrene and identification of DNA adducts by 32P-postlabeling.

Dibenzo[a,l]pyrene (DBP) is one of the most potent bacterial mutagen and mammary carcinogens. When DBP (50 microM) was incubated with calf thymus DNA (300 microg/ml) in the presence of liver microsomes from beta-naphthoflavone (beta-NF)- or Aroclor 1254-treated rats, at least eight adduct spots were detected as analyzed by nuclease P1-enhanced 32P-postlabeling assay. DNA adduction was enhanced by nearly 20- and 60-fold with beta-NF- and Aroclor 1254-induced microsomes, respectively, as compared with uninduced microsomes, suggesting a possible involvement of CYP1A family in DBP activation. Inclusion of the selective P4501A1 inhibitor, alpha-naphthoflavone (50 microM) in the activation reaction almost completely (>98%) abolished adduct formation further supporting involvement of P4501A in DBP activation. Analysis of DNA and 2'-deoxynucleosides 3'-mononucleotide reacted with anti- and syn-DBP-11,12-diol-13,14-epoxides (DBPDEs) and co-chromatography analyses in multiple solvents showed that the microsomal DBP-DNA adducts were derived by interaction of both anti- and syn-DBPDEs with adenine and guanine in DNA in the following order: anti-DBPDE-dA approximately syn-DBPDE-dG >> anti-DBPDE-dG approximately syn-DBPDE-dA. It is concluded that (i) most or all DBP adducts were P4501A-mediated; (ii) both the anti- and syn-stereoisomers were involved in the DNA adduct formation; and (iii) both adenine and guanine in the DNA contributed equally to the formation of the major and minor adducts.

Development of a nanoscale liquid chromatography/electrospray mass spectrometry methodology for the detection and identification of DNA adducts.

In this work, the coupling of liquid nanochromatography to NanoFlow electrospray mass spectrometry was evaluated for the detection of DNA adducts. The NanoFlow ES LC/MS system was compared with the capillary and conventional ES LC/MS system by analyzing an in vitro reaction mixture resulting from the interaction of 2'-deoxyguanosine 5'-monophosphate with bisphenol A diglycidyl ether and by injecting 2'-deoxyadenosine. By using NanoFlow ES LC/MS, the mass sensitivity could be improved by a factor of 3300. Three different injection methods used in liquid nanochromatography, i.e., split, large-volume, and column-switching injections were compared in terms of sensitivity. Furthermore, NanoFlow ES LC/MS was used to detect 2'-deoxynucleotide adducts isolated from an in vitro mixture of calf thymus DNA and bisphenol A diglycidyl ether. Different 2'-deoxynucleotide adducts could be identified by monitoring typical product ions, diagnostic for the adducts.

Immunocytochemical identification of DNA adducts, O6-methylguanine and 7-methylguanine, in respiratory and other tissues of rat, mouse and Syrian hamster exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The present paper reports about an immunocytochemical inventory of the cell types involved in the metabolic activation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to a DNA methylating metabolite. The formation and distribution of the methylated DNA bases O6-methylguanine (O6-meGua) and 7-methylguanine (7-MeGua) were studied in respiratory tissues, oesophagus, liver, kidneys, pancreas, small intestine, colon and prostate of rat, mouse and hamster 6 h after treatment with a single dose of 30 mg NNK/kg. The tissue- and cell-specific distribution of O6-meGua- and 7-meGua-specific nuclear staining showed the same patterns and were remarkably similar in rat, mouse and hamster in spite of the diverging spectra of NNK-induced tumours in these species. In nasal tissue, a target for NNK-induced tumourigenesis in rat and hamster, but not in mouse, adduct-specific nuclear staining was observed in all three species in sustentacular cells, Bowman glands, respiratory epithelial cells and serous glands. Both methylated DNA bases were also observed in basal cells of the olfactory epithelium of rat and (occasionally) hamster, but not in those of the mouse. In the trachea, a target for NNK-induced tumourigenesis in hamster only, substantial adduct-specific nuclear staining was found in basal epithelial and glandular cells of the hamster; in the same cells of rat and mouse only a weak nuclear staining was found. In the lung, a common target for NNK-induced tumourigenesis, the formation of O6-meGua and 7-meGua was restricted predominantly to bronchial and proximal bronchiolar epithelium. Nuclear staining in the rat was occasionally found in alveolar cells and was also observed in hepatocytes. In the three species investigated, O6-meGua- and 7-MeGua-specific nuclear staining was found in target and non-target tissues. Apparently, and in analogy with results obtained in other studies, the species-specific organotropy for tumour formation of NNK is not exclusively determined by DNA methylation. Expanding methylation data with literature data on factors considered to be involved in tumour formation, namely proliferation, toxicity and DNA repair among others, still did not lead to a satisfactory explanation for the species-specific organotropy observed. Additional factors (yet to be identified), need to be taken into account in order to explain (and predict) tumourigenic effects induced by monofunctional methylating agents.

Identification of dna adducts at specific locations by sequencing techniques

1. 1. A simple procedure for the accurate identification of the positions of adduct formation when viral M13mpl8 single stranded DNA is treated with chloroacetaldehyde (CAA) is presented. 2. 2. The normal dideoxy sequencing reaction protocol was employed except no dideoxy nucleotides were used to cause chain termination. 3. 3. Instead, the CAA adducted bases of the template DNA act as points of polymerase fall off causing the appearance of bands of DNA sequencing gel autoradiographs marking locations where adduct formation may have occurred.

DNA adducts, mutations and cancer.

Anyone having lectured on DNA adducts is likely to be familiar with the first question after the presentation: 'What is the relationship of DNA adducts and cancer?'. Although there are a number of reviews on the topic (1-5), my personal response, instead of going into lengthy circumstantial argumentation and hand-waving, has been a quotation from Mortimer Mendelson: 'I wouldn't like to have my DNA messed up'. In this commentary I will indulge in this still circumstantial argumentation. The reason being that never before has it appeared to be so easy, or less difficult, to entertain the question. Hopefully this effort will spare colleagues confronting the same question some time. At the same time I will take the opportunity to describe developments in the identification of DNA adducts. A vivid history of the development of ideas on chemical causes of cancer has been published by Lawley.

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.