Purification and recovery of bulky hydrophobic DNA adducts.

For many years (32)P postlabeling has detected DNA adducts at very low levels and yet has not been able to identify unknown adducts. Mass spectrometry offers substantially improved identification powers, albeit at some loss in detection limits. With this ultimate utilization of mass spectrometry in mind, the current research presents a new method to quantitatively purify bulky hydrophobic DNA adducts at levels that are pertinent to ongoing DNA adduct research in human health and environmental fields. This method was demonstrated with benzo[a]pyrene adducts. Purification was accomplished with the use of small columns (7.5-mm frits) with an 11 mg bed of polystyrene-divinlybenzene beads which retained the adducts while permitting the nonadducted nucleotides to be washed out with water. Subsequently, the adducts were eluted with 50% MeOH and the sample was reduced in volume in an evacuated centrifuge. Purification was demonstrated at adduct levels ranging from 4 adducts in 10(6) nonadducted nucleotides to 4 in 10(8). For these levels, analyses by capillary electrophoresis with sample stacking and UV detection determined that recoveries ranged from 91 to 54%, respectively. The adduct quantities isolated should be sufficient to allow the use of current MS capabilities that are linked on-line to separation methodologies such as capillary electrophoresis, capillary electrochromatography, and high-pressure liquid chromatography.     

Analysis of DNA and protein adducts of benzo[a]pyrene in human tissues using structure-specific methods

We review studies which investigate the presence, using structure-specific analytical methods, of DNA or protein adducts of the carcinogen benzo[a]pyrene (BaP) in human tissues. The analytical methods include high performance liquid chromatography with fluorescence detection and gas chromatography-mass spectrometry. Although, for DNA detection these methods are somewhat less sensitive than non-specific techniques such as 32P-postlabeling and immunoassay, they have the distinct advantage of providing reliable structural information. In order to achieve adequate sensitivity, these methods often require the use of fairly large amounts of DNA (>100 microg) or protein (50-100mg). Most studies reviewed here measured tetraols released from DNA or protein by hydrolysis of adducts derived from (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), a major ultimate carcinogen of BaP. BPDE-DNA adducts were detected in 39% of 705 samples analyzed. BPDE-protein adducts were found in 59% of 772 samples. There was no single exposure situation that led to an overwhelming presence of detectable adducts. For example, BPDE-DNA adducts were detected in 45% of smokers, 33% of former smokers, 52% of non-smokers, 39% of occupationally exposed individuals, and 34% of environmentally exposed people. Adduct levels were influenced by polymorphisms in carcinogen metabolizing genes such as GSTM1, the presence of which was frequently protective. The relatively high occurrence of non-detectable adducts may result from low levels of BaP exposure and host factors such as genetic polymorphisms. Our analysis demonstrates that the presence of BaP adducts in human tissues cannot be assumed, even in situations where exposure to BaP is relatively high.     

Speciation of oxaliplatin adducts with DNA nucleotides

This paper describes a set of fast and selective high performance liquid chromatography (HPLC) methods coupled to electro-spray ionisation linear ion trap mass spectrometry (ESI-MS), sector-field inductively coupled plasma mass spectrometry (SF-ICP-MS) and UV detection for in vitro studies of the bifunctional adducts of oxaliplatin with mono-nucleotides, di-nucleotides and cellular DNA. The stationary phases and the optimised conditions used for each separation are discussed. Interaction of oxaliplatin with A and G mono-nucleotides resulted in the formation of five bifunctional platinum diaminocyclohexane (DACHPt) adducts. These were two isomers of the A-DACHPt-A and A-DACHPt-G adducts, and one G-DACHPt-G adduct, as confirmed by MS/MS spectra obtained by collision induced dissociation. These adducts were also characterised by UV absorption data and SF-ICP-MS elemental (195)Pt and (31)P signals. Further, interaction of oxaliplatin with AG and GG di-nucleotides resulted in the formation of three adducts: DACHPt-GG and two isomers of the DACHPt-AG adduct, as confirmed by ESI-MS and the complementary data obtained by UV and SF-ICP-MS. Finally, a very sensitive LC-ICP-MS method for the quantification of oxaliplatin GG intra-strand adducts (DACHPt-GG) was developed and used for monitoring the in vitro formation and repair of these adducts in human colorectal cancer cells. The method detection limit was 0.14 ppb Pt which was equivalent to 0.22 Pt adduct per 10(6) nucleotides based on a 10 μg DNA sample. This detection limit makes this method suitable for in vivo assessment of DACHPt-GG adducts in patients undergoing oxaliplatin chemotherapy.     

A solid-phase extraction/high-performance liquid chromatography-based (32)P-postlabeling method for detection of cyclic 1,N(2)-propanodeoxyguanosine adducts derived from enals

The cyclic 1,N(2)-propanodeoxyguanosine (PdG) adducts are Michael addition products from reactions of deoxyguanosine (dG) with enals, including acrolein (Acr), crotonaldehyde (Cro), pentenal (Pen), heptenal (Hep), and 4-hydroxy-2-nonenal (HNE). Although this is a general reaction, only the PdG adducts derived from Acr, Cro, and HNE have been detected in vivo as endogenous DNA lesions. Our previous in vitro study demonstrated that PdG adducts of Acr, Cro, and Pen are predominantly derived from oxidation of omega-3 polyunsaturated fatty acids (PUFAs), whereas the long-chain Hep and HNE adducts are from omega-6 PUFAs. PdG adducts are important because they represent a new class of endogenous promutagenic DNA lesions with potential roles in carcinogenesis. Earlier, we developed a (32)P-postlabeling method for detecting PdG adducts from Acr and Cro and a modified method for the long-chain HNE adducts. Both methods require multiple high-performance liquid chromatography steps and, in some cases, time-consuming thin-layer chromatography for purification. There is a lack of a single, versatile, and efficient method for simultaneous detection of all five enal-derived PdG adducts. In this paper, we report an improved (32)P-postlabeling method which permits detection of Acr, Cro, Pen, Hep, and HNE adducts in a single DNA sample. This method relies on solid-phase extraction for adduct enrichment before and after (32)P-labeling; all five PdG adducts were converted to the ring-opened derivatives for confirmation of identities and quantification. The method was validated using the synthetic adducts and enal-modified DNA and was finally applied to rat liver DNA and rat liver DNA samples spiked with different amount of standards. The detection limit was determined to be as low as 0.5 fmol in 80 microg DNA, corresponding to 9 adducts/10(9) dG.     

Postlabelling HPLC analysis of lipophilic DNA adducts from human lung

A new modification of the 32P postlabelling method was described for the analysis of lipophilic DNA in human tissues. To isolate these DNA adducts the method applied nuclease P1 enrichment before labelling and butanol extraction after labelling, followed by high performance liquid chromatography HPLC separation and flow through radioactivity detection. These enrichment methods are known to work for many adducts of polycyclic aromatic hydrocarbons PAHs . In human peripheral lung tissue fro m smokers the apparent level of the DNA adducts observed was 25-244 adducts per 108 nucelotides; in two alleged non smokers the level of adducts was 17 and 109 adducts per 108 nucleotides. When the same samples were analysed by thin layer chromatography TLC , as in the conventional postlabelling assay, the recovery was 5 of that of the HPLC method. Nevertheless, the results from the two methods correlated. In TLC the adducts were lost because they did not remain in the origin in D1 of the TLC development. There was no large difference in recovery between the two techniques for the PAH-DNA adduct standards used. The present results are underestimates of the true adduct levels because there is no way to correct for labelling efficiency and recovery of unknown adducts. Yet they give a lower estimate of the level of lipophilic DNA adducts in human lung tissue.     

Postlabelling HPLC analysis of lipophilic DNA adducts from human lung

A new modification of the 32P postlabelling method was described for the analysis of lipophilic DNA in human tissues. To isolate these DNA adducts the method applied nuclease P1 enrichment before labelling and butanol extraction after labelling, followed by high performance liquid chromatography HPLC separation and flow through radioactivity detection. These enrichment methods are known to work for many adducts of polycyclic aromatic hydrocarbons PAHs. In human peripheral lung tissue fro m smokers the apparent level of the DNA adducts observed was 25-244 adducts per 108 nucelotides; in two alleged non smokers the level of adducts was 17 and 109 adducts per 108 nucleotides. When the same samples were analysed by thin layer chromatography TLC, as in the conventional postlabelling assay, the recovery was 5 of that of the HPLC method. Nevertheless, the results from the two methods correlated. In TLC the adducts were lost because they did not remain in the origin in D1 of the TLC development. There was no large difference in recovery between the two techniques for the PAH-DNA adduct standards used. The present results are underestimates of the true adduct levels because there is no way to correct for labelling efficiency and recovery of unknown adducts. Yet they give a lower estimate of the level of lipophilic DNA adducts in human lung tissue.     

Applications of mass spectrometry for quantitation of DNA adducts

DNA adducts are formed when electrophilic molecules or free radicals attack DNA. 32P-postlabeling has been the most commonly used assay for quantitation of DNA adducts due mainly to its excellent sensitivity that allows quantitation at concentrations as low as approximately 1 adduct per 10(9) normal bases. Such methods, however, do not have the specificity desired for accurate and reliable quantitation, and are prone to produce false positives and artifacts. In the last decade, mass spectrometry in combination with liquid and gas chromatography has presented itself as a good alternative to these techniques since it can satisfy the need for specificity and reliability through the use of stable isotope-labeled internal standards and highly specific detection modes such as selected reaction monitoring and high-resolution mass spectrometry. In this article, the contribution of mass spectrometry to the quantitation of DNA adducts is reviewed with special emphasis on unique applications of mass spectrometry in the area of DNA adduct quantitation and recent applications with improvements in sensitivity.     

The organ-specific induction of DNA adducts in 2-acetylaminofluorene-treated rats, studied by means of a sensitive immunochemical method

Exposure of cells to chemical carcinogens and mutagens may result in the formation of DNA adducts, which can give rise to mutations in the genome and to cellular transformation. Methods to measure DNA-adduct formation may be useful for ‘biomonitoring’, to establish exposure of laboratory animals or humans to DNA-damaging agents. For such purposes, immunochemical methods appear to be suitable, because they allow sensitive detection and quantification of DNA adducts in small amounts of sample in a non-radiolabelled form. We have worked out optimal conditions for the detection of DNA adducts by means of competitive enzyme-linked immunosorbent assay (ELISA). This technique involves interaction of soluble antigen, immobilized antigen and antibody. It appeared that the sensitivity of the competitive assay can be improved by lowering the amount of immobilized antigen, adsorbed to the wall of the plastic reaction vessel. On the basis of these observations, suitable conditions were selected for a sensitive quantitative assay of adducts in DNA isolated from various organs of rats, treated (p.o.) with the liver carcinogen 2-acetylaminofluorene (2-AAF). Under the conditions of these experiments, the available rabbit antiserum recognizes the guanosine-AAF adduct with high specificity. A time- and dose-dependent induction of AAF adducts could be measured in liver DNA from exposed rats, whereas the amount of adducts in DNA from spleen and nucleated blood cells remained below the detection limit (1 adduct/10⁸ nucleotides). The implications of these findings with respect to the relevance of blood cell biomonitoring for target cell exposure are discussed.     

Methodologies for bulky DNA adduct analysis and biomonitoring of environmental and occupational exposures

It is undisputed that DNA adduct formation is one of the key processes in early carcinogenesis. Therefore, analysis of DNA adduct levels may be one of the best tools available to characterize exposure to complex mixtures of genotoxic chemicals as occurring in different environmental and occupational exposure settings. However, from an analytical point of view the detection and quantification of DNA adducts is a challenging enterprise as extremely high sensitivity and selectivity are required. The entire spectrum of chromatographic techniques, including thin-layer chromatography (TLC), gas and liquid chromatography as well as capillary electrophoresis has been used in combination with different detection systems, all with their own specific characteristics. Among the various combinations of techniques, the TLC-(32)P-postlabeling combination appears to meet best with criteria of sensitivity and requirements of minimal amounts of material. Recent developments in the application of capillary electrophoresis in combination with either immunochemical or mass spectrometric detection techniques may offer new and promising approaches, with higher selectivity as compared to TLC-(32)P postlabeling. The applicability of these new techniques in biomonitoring studies aiming at the exposure and risk assessment of low and chronic exposures remains to be determined. In this paper we compare and discuss the advantages and limitations of different techniques used in DNA adduct analysis, with specific emphasis on those adducts formed by the polycyclic aromatic hydrocarbons and heterocyclic aromatic amines.     

DNA adducts: effects of low exposure to ethylene oxide, vinyl chloride and butadiene

Dose-response relationships of genotoxic agents differ greatly depending on the agent and the endpoint being evaluated. Simple conclusions that genotoxic effects are linear cannot be applied universally. The shape of the molecular dose of DNA adducts varies from linear, to supralinear, to sublinear depending on metabolic activation and detoxication, and repair of individual types of DNA adducts. For mutagenesis and other genotoxicity endpoints, the dose-response reflects the molecular dose of each type of DNA adduct, cell proliferation, as well as endogenous factors that lead to mutagenesis such as the formation and repair of endogenous DNA adducts. These same factors are important when interpreting the shape of dose-response data for carcinogenesis of genotoxic agents, however, tumor background variability adds additional complexity. Endogenously formed DNA adducts may be identical to those formed by chemicals, as in the case of vinyl chloride and ethylene oxide, or they may be those associated with oxidative stress. Data presented in this paper demonstrate that the exogenous number of adducts induced by 5 days of exposure to 10 ppm vinyl chloride is only 2. 2-fold greater than that present as a steady-state amount in unexposed control rats. Similar data are shown for ethylene oxide. Extremely sensitive methods have been developed for measuring the molecular dose of genotoxins. These methods can detect DNA adducts as low as 1 per 10(9) to 10(10). However, in view of the high number of endogenous DNA adducts that are present in all cells, it is unlikely that causal relationships can be attributed to very low numbers of such DNA adducts. Effects of both exogenous and endogenous DNA adducts need to be factored into the interpretation of chemical exposures.     

Solid phase immunoenzyme analysis of the immunospecificity of DNA from calf spleen alkylated by thiophosphamide

DNA binding activity of rabbit antiserum against calf spleen DNA's modified by thiophosphamide (DNA-T) was studied by means of solid enzyme immunoassays (ELISA). The studies demonstrated the preferential binding of the immobilized DNA-T compared to immobilized single-stranded DNA (ss-DNA) and only small preference compared to native DNA. Two antisera against DNA-T were purified by affinity chromatography on a ss-DNA-CNBr agarose from antibodies to calf spleen ss-DNA. They interacted only with the immobilized DNA-T, but not with ss-DNA or native DNA. These results demonstrated that DNA modification by thiophosphamide, decreases the immunogenicity of usual nitrogen-containing DNA bases, but detected new immunogenic specificity for adducts. Detection of new immunogenic specificity in DNA's alkylated by thiophosphamide, resulted in the development of a sensitive enzyme immunoassay for the detection of these adducts in nucleic acids, in monitoring their formation, persistence and repair damages in DNA.     

High-performance liquid chromatographic analysis of 32P-postlabeled DNA-aromatic carcinogen adducts

The technique of 32P postlabeling of DNA-carcinogen adducts is a useful and extremely sensitive method of detecting and quantitating DNA damage by carcinogens. We have adapted the 32P method to analysis by high-pressure liquid chromatography, making the procedure more rapid and convenient than when thin-layer chromatography is used. Following DNA isolation and hydrolysis, nucleotide-carcinogen adducts are enhanced relative to normal nucleotides by solvent extraction and then labeled with high-specific-activity [gamma-32P]ATP. The resulting 32P-postlabeled nucleotides are resolved by reverse-phase ion-pair HPLC. After as little as 3 h of exposure to carcinogens, DNA adducts can be demonstrated from 1 microgram or less of mouse hepatic DNA. Acetylated and nonacetylated adducts can be resolved from hepatic DNA of mice treated with 2-aminofluorene. Differences in DNA damage as measured by adduct formation were demonstrated between "rapid" and "slow" acetylator mouse strains. Rapid-acetylator C57BL/6J mice had three times the amount of hepatic DNA adducts as slow-acetylator A/J mice 3 h after a 60 mg/kg dose of 2-aminofluorene. 4-Aminobiphenyl and 2-naphthylamine each showed an adduct peak with retention time similar to that of the nonacetylated 2-aminofluorene adduct, while benzidine gave a major adduct that eluted somewhat earlier as would be expected for an acetylated adduct. The alkenylbenzenes, safrole and methyleugenol, also formed DNA adducts detectable by this method. DNA prepared from skin of mice painted with benzo[a]pyrene also contained carcinogen-DNA adducts detectable and resolvable by HPLC analysis following 32P postlabeling. The combination of HPLC with 32P postlabeling appears to be a useful technique for the rapid detection and quantitation of DNA damage caused by several classes of aromatic carcinogens.     

Analysis of tamoxifen-DNA adducts in endometrial explants by MS and 32P-postlabeling

The nonsteroidal antiestrogen tamoxifen increases the risk of endometrial cancer; however, the mechanism for the induction of these tumors is not known. Recently, Sharma et al. [Biochem. Biophys. Res. Commun. 307 (2003) 157], using high performance liquid chromatography (HPLC) with online postcolumn photochemical activation and fluorescence detection, reported the presence of (E)-alpha-(deoxyguanosin- N2-yl)tamoxifen in DNA from human endometrial explants incubated with tamoxifen. Inasmuch as the methodology used by these investigators does not allow unambiguous characterization of tamoxifen-DNA adducts, we have used two additional techniques (HPLC coupled with electrospray ionization tandem mass spectrometry and 32P-postlabeling analyses) to assay for the presence of tamoxifen-DNA adducts in the human endometrial explant DNA. Tamoxifen-DNA adducts were not detected by either method.     

Deoxyribonuclease I footprinting reveals different DNA binding modes of bifunctional platinum complexes

Deoxyribonuclease I (DNase I) footprinting methodology was used to analyze oligodeoxyribonucleotide duplexes containing unique and single, site-specific adducts of trinuclear bifunctional platinum compound, [{trans-PtCl(NH3)2}2 mu-trans-Pt(NH3)2{H2N(CH2)6NH2}2]4+ (BBR3464) and the results were compared with DNase I footprints of some adducts of conventional mononuclear cis-diamminedichloroplatinum(II) (cisplatin). These examinations took into account the fact that the local conformation of the DNA at the sites of the contacts of DNase I with DNA phosphates, such as the minor groove width and depth, sequence-dependent flexibility and bendability of the double helix, are important determinants of sequence-dependent binding to and cutting of DNA by DNase I. It was shown that various conformational perturbations induced by platinum binding in the major groove translated into the minor groove, allowing their detection by DNase I probing. The results also demonstrate the very high sensitivity of DNase I to DNA conformational alterations induced by platinum complexes so that the platinum adducts which induce specific local conformational alterations in DNA are differently recognized by DNase I.     

Use of DNA adducts to identify human health risk from exposure to hazardous environmental pollutants: the increasing role of mass spectrometry in assessing biologically effective doses of genotoxic carcinogens

The carcinogens to which humans are exposed are normally in the form of complex mixtures, and much effort has gone into determining the nature of the most significant carcinogenic components in these mixtures and their mechanisms of action. Essential to achieving this aim in exposed populations is the use of biomarkers, which can characterize the chemical nature of the carcinogens involved and identify key biological effects that result from the exposure. DNA adducts are particularly appropriate as biomarkers in the case of genotoxic carcinogens as they indicate the biologically effective dose of the genotoxin in the target tissue under study. This review considers in particular the use of mass spectrometry (MS), which is having an increasing role in the determination of DNA adducts. Compared to other existing DNA damage detection methods, such as 32P-postlabeling, HPLC-fluorescence or electrochemical detection, immunoassay-based techniques and modified Comet assays, MS provides improved structural characterization of adducts. Greater selectivity in the analyses is achieved by the use of tandem MS with selected reaction monitoring or constant neutral loss of ions. Use of capillary/nano liquid chromatography and micro/nano electrospray ionization improves the analytical sensitivity and higher throughput may be obtained by the use of online-column switching. The application of microfluidics technology offers exciting new possibilities for interfacing sample preparation to the mass spectrometer. Despite these improvements in the use of MS for adduct detection, the main current requirement is to validate these methods both analytically and in molecular epidemiology studies. More knowledge of the stability of stored samples is required. Development of sensitive mass spectrometric DNA adductomic screening systems, and of long-term biomarkers (e.g., phosphotriester adducts that are not repaired efficiently) seems important areas for the future assessment of the effects of human exposure to environmental genotoxins, together with studies of dose-response relationships at low doses.     

DNA damage caused by lipid peroxidation products

Lipid peroxidation is a process involving the oxidation of polyunsaturated fatty acids (PUFAs), which are basic components of biological membranes. Reactive electrophilic compounds are formed during lipid peroxidation, mainly alpha, beta-unsaturated aldehydes. These compounds yield a number of adducts with DNA. Among them, propeno and substituted propano adducts of deoxyguanosine with malondialdehyde (MDA), acrolein, crotonaldehyde and etheno adducts, resulting from the reactions of DNA bases with epoxy aldehydes, are a very important group of adducts. The epoxy aldehydes are more reactive towards DNA than the parent unsaturated aldehydes. The compounds resulting from lipid peroxidation mostly react with DNA showing both genotoxic and mutagenic action; among them, 4-hydroxynonenal is the most genotoxic, while MDA is the most mutagenic. DNA damage caused by the adducts of lipid peroxidation products with DNA can be removed by the repairing action of glycosylases. The formed adducts have been hitherto analyzed using the IPPA (Imunopurification-(32)P-postlabelling assay) method and via gas chromatography/electron capture negtive chemical ionization/mass spectrometry (GC/EC NCI/MS). A combination of liquid chromatography with electrospray tandem mass spectrometry (LC/ES-MSMS) with labelled inner standard has mainly been used in recent years.     

Benzoapyrene-induced DNA damage in Mytilus galloprovincialis: measurement of bulky DNA adducts and DNA oxidative damage in terms of 8-oxo-7,8-dihydro-2´-deoxyguanosine formation

Bulky DNA adducts and 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodGuo) were measured in gill DNA of benzo[a]pyrene (B[a]P)-exposed mussels (50 mg kg^-1 dw day^-1), respectively by the ³²P-post-labelling technique and high performance liquid chromatography coupled to electrochemical detection assay. A time-course study was performed for both biomarkers and their potential use for marine biomonitoring discussed for the sentinel species studied. In gills, B[a]P-related DNA adducts were positively correlated with B[a     

Detection of platinum-DNA adducts by 32P-postlabelling.

We developed a sensitive 32P-postlabeling method for the detection of bifunctional intrastrand crosslinks d(Pt-GpG) and d(Pt-ApG) in DNA in vitro and in vivo. After enzymatic digestion of DNA the positively charged platinum adducts were purified from unplatinated products, using strong cation exchange chromatography. Subsequently the samples were deplatinated with cyanide, because platinated dinucleotides are very poor substrates for polynucleotide kinase. The excess of cyanide was removed using Sep-pak C18 cartridges, and the resulting dinucleoside monophosphates d(GpG) and d(ApG) were subsequently postlabelled. Analysis of the postlabelling mixture was performed by a combined TLC and HPLC-procedure. Good correlations with existing methods (AAS, immunocytochemistry and ELISA) were found in DNA samples treated in vitro and in vivo with cis- or carboplatin. The detection limit of the assay was 1 adduct/10(7) nucleotides in a 10 micrograms DNA sample.     

Synthesis of Mitomycin C and decarbamoylmitomycin C N6 deoxyadenosine-adducts

Mitomycin C (MC), an anti-cancer drug, and its analog, decarbamoylmitomycin C (DMC), are DNA-alkylating agents. MC is currently used in the clinics and its cytotoxicity is mainly due to its ability to form Interstrand Crosslinks (ICLs) which impede DNA replication and, thereby, block cancer cells proliferation. However, both MC and DMC are also able to generate monoadducts with DNA. In particular, we recently discovered that DMC, like MC, can form deoxyadenosine (dA) monoadducts with DNA. The biological role played by these monoadducts is worthy of investigation. To probe the role of these adducts and to detect them in enzymatic digests of DNA extracted from culture cells treated by both drugs, we need access to reference compounds i.e. MC and DMC dA-mononucleoside adducts. Previous biomimetic methods used to generate MC and DMC mononucleoside adducts are cumbersome and very low yielding. Here, we describe the diastereospecific chemical synthesis of both C-1 epimers of MC and DMC deoxyadenosine adducts. The key step of the synthesis involves an aromatic substitution reaction between a 6-fluoropurine 2′-deoxyribonucleoside and appropriately protected stereoisomeric triaminomitosenes to form protected-MC-dA adducts with either an S or R stereochemical configuration at the adenine-mitosene linkage. Fluoride-based deprotection methods generated the final four reference compounds: the two stereoisomeric MC-dA adducts and the two stereoisomeric DMC-dA adducts. The MC and DMC-dA adducts synthesized here will serve as standards for the detection and identification of such adducts formed in the DNA of culture cells treated with both drugs.     

In vitro evolution of RNA aptamers recognizing carcinogenic aromatic amines

The modification of cellular DNA by environmental substances is thought to be a crucial event in chemical induced carcinogenesis. Among the environmental carcinogens, aromatic amines are known for the fact that they can induce several types of cancers through the formation of so-called DNA adducts. We took advantage of the potential of the SELEX method to select for highly specific RNA ligands that recognize specific genotoxic aromatic amines. The aromatic amine 4,4'-methylenedianiline (MDA) was used as a target. Following in vitro selection, we obtained specific MDA-binding RNA molecules based on an affinity chromatography assay. These results open the possibility of using the SELEX technique to generate RNA molecules as diagnostic tools for the detection of DNA damaging compounds and ultimately DNA adducts.