Smoking-related DNA adducts in anal epithelium

Several studies have identified tobacco smoking as a risk factor for anal cancer in both women and men. Samples of anal epithelium from haemorrhoidectomy specimens from current smokers (n=20) and age-matched life-long non-smokers (n=16) were analysed for DNA adducts by the nuclease P₁ digestion enhancement procedure of ³²P-postlabelling analysis. The study included 14 men and 22 women. Both qualitative and quantitative differences in the adduct profiles were observed between the smokers and non-smokers. The mean adduct level was significantly higher in the smokers than in the non-smokers (1.88±0.71 (S.D.) versus 1.36±0.60 adducts per 10⁸ nucleotides, P=0.02, two-tailed unpaired t-test with Welch’s correction); furthermore, the adduct pattern seen in two-dimensional chromatograms revealed the smoking-related diagonal radioactive zone in 17/20 smokers, but not in any of the non-smokers (P<0.00001, Fisher’s exact test). These results indicate that components of tobacco smoke inflict genotoxic damage in the anal epithelium of smokers and provide a plausible mechanism for a causal association between smoking and anal cancer.     

Endocrine cells and melanin-containing cells in the anal canal epithelium

Summary The epithelium of the anal canal from 22 humans was studied in order to demonstrate the possible presence of endocrine cells and melanin-containing cells. Histochemical methods aimed at demonstrating reducing substances, biogenic amines, argyrophilia and melanin, were used. Enterochromaffin cells, and possibly other types of endocrine cells, were demonstrated above the dentate line both in colo-rectal type epithelium and in the anal transitional zone. Melanin-containing cells could also occasionally be found in the anal transitional zone. The presence of endocrine cells in the anal canal epithelium opens up the possibility that carcinoids can originate in this region. Further, the presence of melanin-containing cells might explain the occurrence of malignant melanomas arising above the dentate line.     

DNA adducts of ortho-toluidine in human bladder

Background: 4-Aminobiphenyl (4-ABP) and o-toluidine are known human bladder carcinogens, but only 4-ABP-releasing DNA adducts are known.

Methods: Determination of 4-ABP and o-toluidine-releasing DNA adducts in epithelial and submucosal bladder tissues of sudden death victims (SDV: n?=?46), and bladder tumours (n?=?12) by gas chromatography/mass spectrometry.

Results: Above background, 4 and 11 of 12 tumour samples contained adducts of 4-ABP (0.057?±?0.125?fmol/µg DNA) and o-toluidine (8.72?±?4.49?fmol/µg DNA), respectively. Lower adduct levels were present in both epithelial and submucosal bladder tissues of SDV (4-ABP: 0.011?±?0.022 and 0.019?±?0.047?fmol/µg DNA; o-toluidine: 0.24?±?0.63 and 0.27?±?0.70?fmol/µg DNA).

Conclusion: Detection of o-toluidine-releasing DNA adducts support the carcinogenicity of o-toluidine in the human bladder.     

Repair of DNA alkylation adducts in mammalian cells

Carcinogenic alkylating agents, including nitrosamines, are able to alkylate DNA at various sites. This review presents evidence of the high degree of specificity in the type of DNA damage induced by various N-nitroso compounds and in the DNA repair processes among tissues or cells of different species. The O6-alkylguanine DNA alkyltransferase activity in various human and rodent tissues is discussed as well as the detection of O6-methylguanine in human DNA, using monoclonal antibodies and radioimmunoassay. The relevance of these findings to the mechanisms of cancer induction by nitrosamines is discussed.     

The role of DNA adducts in smoking-related carcinogenesis

Epidemiological studies indicate a close association between smoking and cancer. Biological activity of many chemical carcinogens and of their metabolites is induced by covalent binding of their reactive derivatives to DNA, which consequently causes mutations in critical genes. Carcinogen-DNA adducts formed by exposure to tobacco smoke have a key role in the initiation of various types of cancer including lung cancer. Presence of tobacco smoke-related carcinogen-DNA adducts in various tissues of smokers proves the DNA damaging effect of smoking. DNA adducts are important biomarkers for the biomonitoring of human genotoxic exposures to tobacco smoke. The paper gives a short overview on the role of smoking-related DNA adducts in carcinogenesis.     

Lymphocyte DNA adducts and polymorphism in the DNA repair enzyme XPD

The effect of genetic polymorphism of DNA repair enzyme on the DNA adduct levels was evaluated in this study. We explored the relationship between polymorphism in the nucleotide excision repair enzyme XPD and DNA adduct levels in lymphocytes. Lymphocyte DNA adducts were measured by a ³²     

Lymphocyte DNA adducts and polymorphism in the DNA repair enzyme XPD

The effect of genetic polymorphism of DNA repair enzyme on the DNA adduct levels was evaluated in this study. We explored the relationship between polymorphism in the nucleotide excision repair enzyme XPD and DNA adduct levels in lymphocytes. Lymphocyte DNA adducts were measured by a ³²     

Cisplatin adducts in DNA: distortion and recognition

 cis-Diamminedichloroplatinum (II) (cisplatin) and derivatives are very successful anticancer chemotherapeutic agents. They crosslink cellular DNA, forming bifunctional adducts with the N7 of guanine bases. In this review, recent structures of cisplatin adducts are summarised, and the significance for the recognition of DNA structure by proteins is discussed. Two new structures of intrastrand GpG adducts have been presented, showing a significant kinking of the helix axis and a novel hybrid A-B helical geometry. The relevance of this structure to the recognition of HMG-box and related proteins is discussed. A new structure of a cross-strand cisplatin adduct reveals a major disruption of the local DNA structure. The basepairs containing the modified guanine bases are broken, with extrusion of the cytosine bases into the solvent. The backbone reverses direction locally, with the result that the platinum adduct is located in what is the minor groove of the DNA overall. The extrusion of single bases out of the helix is strongly reminiscent of the effect of certain methylases on their DNA targets. Received: 30 January 1996 / Accepted: 21 February 1996     

Equilenin-derived DNA adducts to cytosine in DNA duplexes: structures and thermodynamics

The drug Premarin is the most widely used formula for hormone replacement therapy. However, long-term exposure to estrogens from the Premarin drug increases the risk of breast cancer. Equilin and equilenin, major components of Premarin, are predominantly metabolized to 4-hydroxyequilenin (4-OHEN). The quinoids produced by 4-OHEN oxidation react with dG, dA, and dC to form unusual stable cyclic bulky adducts, with four stereoisomers identified for each base adduct. The 4-OHEN-dC adducts are most predominant. They are mutagenic in vitro and have been found in human tumor tissue. We have carried out molecular modeling and molecular dynamics simulations to investigate structures and thermodynamics of the four 4-OHEN-dC stereoisomeric adducts in DNA duplexes. Our results show that the structure of each stereoisomer adduct in duplex DNA is specifically governed by its unique stereochemistry. The bulky adducts, with an obstructed Watson-Crick edge and an equilenin ring system near perpendicular to the damaged cytosine, are located in the B-DNA major or minor groove, with the modified cytosine in the syn or anti conformation, respectively. The DNA duplex structures are distorted, in terms of Watson-Crick pairing at and near the lesion, stacking interactions, and groove dimensions. Stereochemistry determines the orientation of the equilenin rings with respect to the 5'- to 3'-direction of the modified strand, as well as the positioning of the equilenin moiety's methyl and hydroxyl groups for each stereoisomer. The unusual structures and the stereochemical effects underlie their biological processing as miscoding DNA lesions whose mutagenic properties may contribute to breast cancer.     

DNA ligase-AMP adducts: Identification of yeast DNA ligase polypeptides

Yeast DNA ligase is radioactively labelled in vitro by incubating a crude cell extract with [α-³²P]ATP. The product of this reaction is the stable covalent ligase-AMP adduct, which can be characterized by its reactivity with either pyrophosphate or nicked DNA and visualized by gel electrophoresis and autoradiography. The Saccharomyces cerevisiae DNA ligase was identified as an 89 kDa polypeptide by exploiting the fact that transformants with multiple copies of the plasmid-encoded DNA ligase (CDC9) gene overproduce the enzyme by two orders of magnitude. A similar strategy has been used to identify the Schizosaccharomyces pombe DNA ligase as an 87 kDa polypeptide. Both values agree well with the coding capacities of the respective cloned gene sequences. When the S. cerevisiae ligase is greatly overproduced with respect to wild-type levels, a second polypeptide of 78.5 kDa is also labelled and has the same properties as the 89 kDa adduct. We suggest that this polypeptide is generated by proteolysis.     

Termination of vitro DNA synthesis at AAF adducts in the DNA.

DNA synthesis catalyzed by E. coli polymerases I or III is inhibited on templates containing N-acetoxy-acetylaminofluorene-reacted adducts. Termination of synthesis occurs just before the site of the adduct. Synthesis on 0X174 templates primed with restriction fragments and treated with AAAF can be visualized on DNA sequencing gels. Comparison of the amounts of the different newly synthesized fragments with those calculated from the probability of termination as determined from the average number of adducts per molecule shows that synthesis terminates, rather than stutters, at each adduct. This method may be useful for detecting the bypass of lesions.     

DNA ligase-AMP adducts: identification of yeast DNA ligase polypeptides

Yeast DNA ligase is radioactively labelled in vitro by incubating a crude cell extract with [alpha-32P]ATP. The product of this reaction is the stable covalent ligase-AMP adduct, which can be characterized by its reactivity with either pyrophosphate or nicked DNA and visualized by gel electrophoresis and autoradiography. The Saccharomyces cerevisiae DNA ligase was identified as an 89 kDa polypeptide by exploiting the fact that transformants with multiple copies of the plasmid-encoded DNA ligase (CDC9) gene overproduce the enzyme by two orders of magnitude. A similar strategy has been used to identify the Schizosaccharomyces pombe DNA ligase as an 87 kDa polypeptide. Both values agree well with the coding capacities of the respective cloned gene sequences. When the S. cerevisiae ligase is greatly overproduced with respect to wild-type levels, a second polypeptide of 78.5 kDa is also labelled and has the same properties as the 89 kDa adduct. We suggest that this polypeptide is generated by proteolysis.     

Mucin histochemistry of the anal canal epithelium. Studies of normal anal mucosa and mucosa adjacent to carcinoma

Summary The epithelial lining of the anal canal is of colo-rectal type in the upper part and squamous in the lower part, while the middle zone is called the anal transitional zone (ATZ). This zone is characterized by an epithelium which bears a resemblance to that of the anal glands and shows little mucus secretion. The histochemical properties of the mucins in the epithelium of 39 anal canals, resected for ano-rectal adenocarcinoma, basaloid carcinoma, squamous carcinoma and malignant melanoma were investigated. The study reveals that (1) the mucin composition of the ATZ epithelium corresponds to that of the anal glands, being characterized by a mixture of sulpho- and sialomucins with scarcity or absence ofO-acylated sialic acids; and (2) cases with carcinomas located near the dentate line show changes in the mucin composition of the adjoining anal canal epithelium, regardless of tumour type. In colo-rectal type mucosa, these mucins consist of increasing amounts of sialomucins with a predominance ofN-acyl derivatives, and in the anal could be detected in the ATZ epithelium. It is concluded that rectal and anal glands in the anal canal are exposed to stimuli which alter the normal process of glycoprotein synthesis and secretion. The changes seem to be secondary to tumour growth and independent of the histological type of tumour.     

The 32P-postlabeling assay for DNA adducts

32P-postlabeling analysis is an ultrasensitive method for the detection and quantitation of carcinogen-DNA adducts. It consists of four principal steps: (i) enzymatic digestion of DNA to nucleoside 3'-monophosphates; (ii) enrichment of the adduct fraction of the DNA digest; (iii) 5'-labeling of the adducts by transfer of 32P-orthophosphate from [gamma-32P]ATP mediated by polynucleotide kinase (PNK); (iv) chromatographic or electrophoretic separation of the labeled adducts or modified nucleotides and quantitation by measurement of their radioactive decay. The assay requires only microgram quantities of DNA and is capable of detecting adducts at frequencies as low as 1 in 10(10) nt, making it applicable to the detection of events resulting from environmental exposures, or experiments using physiological concentrations of agents. It has a wide range of applications in human, animal and in vitro studies, and can be used for a wide variety of classes of compound and for the detection of adducts formed by complex mixtures. This protocol can be completed in 3 d.     

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.     

Distortions induced in DNA by cis-platinum interstrand adducts.

A 22 base pair double-stranded oligonucleotide containing a unique interstrand adduct resulting from chelation of the two guanine residues within the central sequence d(TGCT/AGCA) by a cis-platinum residue has been studied by means of gel electrophoresis, chemical probes, and molecular mechanics. The anomalously slow electrophoretic mobility of the multimers of the platinated and ligated oligomers suggests that the platinated oligonucleotide is bent. The two cytosine residues (complementary to the platinated guanines) are hyperreactive to hydroxylamine, indicating a large exposure of the two bases to the solvent. The adduct does not induce a local denaturation within the flanking sequences since the adenine residues are not reactive with diethyl pyrocarbonate. This is confirmed by the nonreactivity of the complementary T residues with osmium tetraoxide. These results and the molecular mechanics modeling suggest that the interstrand adduct bends the double helix by approximately 55 degrees toward the major groove, that the double helix conserves its average twist angle, and that the distortion induced by the adduct is localized at the platinated sequence d(GC/CG).     

Exocyclic malondialdehyde and aromatic DNA adducts in larynx tissues

Cigarette smoking and alcohol consumption, known to cause free radical generation and lipid peroxidation, are established risk factors for larynx cancer. Malondialdehyde (MDA) is a naturally occurring product of lipid peroxidation, capable of interacting with DNA to form exocyclic MDA-DNA adducts. In the present study, we investigated if the production of MDA-DNA adducts was increased in larynx cancer patients with respect to controls using (32)P-DNA postlabeling techniques. Moreover, we examined the potential effects of cigarette smoking and alcohol consumption on endogenous DNA adducts. We then analyzed the same set of larynx tissues for the presence of (32)P-postlabeled aromatic DNA adducts to determine more about the levels and types of adducts formed in the larynx. We observed that cancer patients tended to have increased levels of MDA and aromatic DNA adducts with respect to controls. In addition, smoking and alcohol were found to influence the formation of endogenous adducts in the larynx tissues. Finally, the amounts of endogenous adducts were found to be comparable to those observed for aromatic DNA adducts in the same set of larynx tissues. These findings imply that endogenous lesions, if not repaired, may contribute to larynx cancer development.